U.S. patent number 6,627,738 [Application Number 09/369,966] was granted by the patent office on 2003-09-30 for no-modified hemoglobins and uses therefor.
This patent grant is currently assigned to Duke University. Invention is credited to Andrew J. Gow, Jonathan S. Stamler.
United States Patent |
6,627,738 |
Stamler , et al. |
September 30, 2003 |
No-modified hemoglobins and uses therefor
Abstract
Nitrosylhemoglobin can be produced by introducing gaseous NO
into an aqueous solution of hemoglobin. It has been demonstrated
that nitrosylhemoglobin in aqueous solution can be converted to
SNO-hemoglobin upon introduction of oxygen to the solution, as is
postulated to occur in the lungs. Nitrosylhemoglobin can be used in
methods to produce the physiological effects of NO, for example, to
reduce vasoconstriction and to inhibit platelet aggregation.
Inventors: |
Stamler; Jonathan S. (Chapel
Hill, NC), Gow; Andrew J. (Durham, NC) |
Assignee: |
Duke University (Durham,
NC)
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Family
ID: |
46276449 |
Appl.
No.: |
09/369,966 |
Filed: |
August 6, 1999 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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PCTUS9802383 |
Feb 5, 1998 |
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874992 |
Jun 12, 1997 |
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796164 |
Feb 6, 1997 |
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PCTUS9614659 |
Sep 13, 1996 |
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667003 |
Jun 20, 1996 |
6197745 |
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616371 |
Mar 15, 1996 |
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Current U.S.
Class: |
530/385; 436/15;
436/66 |
Current CPC
Class: |
A61K
31/00 (20130101); A61K 47/6901 (20170801); A61K
31/095 (20130101); A61K 31/195 (20130101); A61K
35/18 (20130101); C07K 14/00 (20130101); C07K
14/805 (20130101); G01N 21/631 (20130101); G01N
21/76 (20130101); G01N 33/721 (20130101); A61K
38/44 (20130101); A61K 47/6445 (20170801); A61K
31/04 (20130101); A61K 38/00 (20130101); Y10T
436/105831 (20150115) |
Current International
Class: |
A61K
38/42 (20060101); A61K 31/00 (20060101); A61K
31/095 (20060101); A61K 38/41 (20060101); A61K
31/04 (20060101); A61K 31/195 (20060101); A61K
31/185 (20060101); C07K 14/805 (20060101); C07K
14/795 (20060101); A61K 38/00 (20060101); C07K
014/805 () |
Field of
Search: |
;514/6 ;530/385
;436/15,66 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO 93/09806 |
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May 1993 |
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WO |
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WO 93/21525 |
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WO 94/22306 |
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WO 94/22482 |
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WO 94/22499 |
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Oct 1994 |
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WO |
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WO 95/05397 |
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Feb 1995 |
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WO |
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WO 95/07691 |
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Mar 1995 |
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WO |
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WO 96/03139 |
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Feb 1996 |
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WO |
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WO 96/15797 |
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May 1996 |
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WO |
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WO 96/16645 |
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Jun 1996 |
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WO |
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WO 96/17604 |
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Jun 1996 |
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WO |
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WO 96/30006 |
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Oct 1996 |
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WO |
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WO 97/18000 |
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May 1997 |
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WO |
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WO 97/37644 |
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Oct 1997 |
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WO |
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Other References
Jia, L., et al., "S-Nitrosohaemoglobin: A Dynamic Activity of Blood
Involved in Vascular Control," Nature, 380:221-226 (1996). .
Ignarro, L.J., et al., "Mechanism of Vascular Smooth Muscle
Relaxation by Organic Nitrates, Nitrites, Nitroprusside and Nitric
Oxide: Evidence for the Involvement of S-Nitrosothiols as Active
Intermediates," The Journal of Pharmacology and Experimental
Therapeutics, 218(3):739-749 (1981). .
Khartitonov, V.G., et al., "Interactions of Nitric Oxide with Heme
Proteins Using UV-VIS Spectroscopy," Methods in Nitric Oxide
Research, pp. 39-45, Edited by Martin Feelisch and Jonathan S.
Stamler, John Wiley & Sons Ltd. (1996)..
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Primary Examiner: Low; Christopher S. F.
Assistant Examiner: Lukton; David
Attorney, Agent or Firm: Hamilton, Brook, Smith &
Reynolds, P.C.
Government Interests
GOVERNMENT SUPPORT
This invention was made with government support under Grant Nos.
HL52529 and HR59130 awarded by the National Institutes of Health.
The government has certain rights in the invention.
Parent Case Text
RELATED APPLICATIONS
This application is a continuation of PCT/US98/02383 filed on Feb.
5, 1998, which is a continuation-in-part of U.S. application Ser.
No. 08/874,992 filed on Jun. 12, 1997, which is a
continuation-in-part of U.S. application Ser. No. 08/796,164 filed
on Feb. 6, 1997, which is a continuation-in-part of PCT/US96/14659
filed on Sep. 13, 1996, which is a continuation of U.S. application
Ser. No. 08/667,003 filed on Jun. 20, 1996, now U.S. Pat. No.
6,197,745 which is a continuation-in-part of U.S. application Ser.
No. 08/616,371 filed on Mar. 15, 1996, which claims the benefit of
U.S. Provisional Application No. 60/003,801 filed on Sep. 15, 1995.
The teachings of all of the above applications are each
incorporated herein by reference in their entirety.
Claims
What is claimed is:
1. A method of inhibiting vasoconstriction, said method comprising
administering to a mammal in need thereof a composition comprising
nitrosylhemoglobin for a time and under conditions to inhibit
vasoconstriction.
Description
BACKGROUND OF THE INVENTION
Interactions of hemoglobin (Hb) with small diffusible ligands, such
as O.sub.2, CO.sub.2 and NO, are known to occur at its metal
centers and amino termini. The O.sub.2 /CO.sub.2 delivery
functions, which arise in the lung and systemic microvasculature,
are allosterically controlled. Such responsiveness to the
environment has not been known to apply in the case of NO.
Specifically, it has been thought previously that NO does not
modify the functional properties of Hb to any physiologically
significant degree. Kinetic modeling predicts that the vast
majority of free NO in the vasculature should be scavenged by Hb
(Lancaster 1994). Accordingly, the steady-state level of NO may
fall below the K.sub.m for target enzymes such as guanylate cyclase
(Lancaster 1994), if not in the unperturbed organism, then with
oxidant stress such as that found in atherosclerosis. These
considerations raise the fundamental question of how NO exerts its
biological activity.
One answer to this question is found in the propensity of nitric
oxide to form S-nitrosothiols (RSNOS) (Gaston, B. et al., Proc.
Natl. Acad. Sci. USA 90:10957-10961 (1993)), which retain NO-like
vasorelaxant activity (Stamler, J. S., et al., Proc. Natl. Acad.
Sci. USA 89:444-448 (1992)), but which can pass freely in and out
of cells, unlike Hb. In particular, the NO group of RSNOs possesses
nitrosonium (NO.sup.+) character that distinguishes it from NO
itself. It is increasingly appreciated that RSNOs have the capacity
to elicit certain functions that NO is incapable of (DeGroote, M.
A. et al., Proc. Natl. Acad. Sci. USA 92:6399-6403 (1995); Stamler,
J. S., Cell 78:931-936 (1994)). Moreover, consideration has been
given to the possibility that --SNO groups in proteins serve a
signaling function, perhaps analogous to phosphorylation (Stamler,
J. S. et al., Proc. Natl. Acad. Sci. USA 89:444-448 (1992);
Stamler, J. S. Cell, 78:931-926 (1994)). Although S-nitrosylation
of proteins can regulate protein function (Stamler, J. S. et al.,
Proc. Natl. Acad. Sci. USA 89:444-448 (1992); Stamler, J. S., Cell,
78:931-936 (1994)), intracellular S-nitrosoproteins--the sine qua
non of a regulatory posttranslational modification--has heretofore
not been demonstrated.
Hemoglobin is a tetramer composed of two alpha and two beta
subunits. In human Hb, each subunit contains one heme, while the
beta (.beta.) subunits also contain highly reactive SH groups
(cys.beta.93) (Olson, J. S., Methods in Enzymology 76:631-651
(1981); Antonini, E. & Brunori, M. In Hemoglobin and Myoglobin
in Their Reactions with Ligands, American Elsevier Publishing Co.,
Inc., New York, pp. 29-31 (1971)). These cysteine residues are
highly conserved among species although their function has remained
elusive.
NO (nitric oxide) is a biological "messenger molecule" which
decreases blood pressure and inhibits platelet function, among
other functions. NO freely diffuses from endothelium to vascular
smooth muscle and platelet and across neuronal synapses to evoke
biological responses. Under some conditions, reactions of NO with
other components present in cells and in serum can generate toxic
intermediates and products at local concentrations in tissues which
are effective at inhibiting the growth of infectious organisms.
Thus, it can be seen that a method of administering an effective
concentration of NO or biologically active forms thereof would be
beneficial in certain medical disorders.
Platelet activation is an essential component of blood coagulation
and thrombotic diathesis. Activation of platelets is also seen in
hematologic disorders such as sickle cell disease, in which local
thrombosis is thought to be central to the painful crisis.
Inhibition of platelet aggregation is therefore an important
therapeutic goal in heart attacks, stroke, and shock (disseminated
intravascular coagulation) and in chronic conditions such as
peripheral vascular disease, heart disease, brain disease, lung
disease and atherosclerosis. Researchers have attempted to give
artificial hemoglobins to enhance oxygen delivery in all of the
above disease states. However, as recently pointed out by Olsen and
coworkers, administration of underivatized hemoglobin leads to
platelet activation at sites of vascular injury (Olsen S. B. et
al., Circulation 93:327-332 (1996)). This major problem has led
experts to conclude that cell-free underivatized hemoglobins pose a
significant risk of causing blood clots in the patient with
vascular disease or a clotting disorder (Marcus, A. J. and J. B.
Broekman, Circulation 93:208-209 (1996)). New methods of providing
for an oxygen carrier and/or a method of inhibiting platelet
activation would be of benefit to patients with vascular disease or
who are otherwise at risk for thrombosis.
SUMMARY OF THE INVENTION
The invention relates to methods of producing and isolating SNO-Hb
(S-nitrosohemoglobin, which includes for instance, oxy-, deoxy-, or
met- hemoglobin for use in therapy) by reaction of Hb with
S-nitrosothiol in procedures which avoid oxidation of the heme. The
invention also includes methods of producing isolated, nitrosated
(including nitrosylated at thiols or metals) and nitrated
derivatives of hemoglobins in which the heme Fe can be oxidized or
not oxidized, depending on the steps of the method. The invention
also relates to a method of therapy for a condition in which it is
desired to oxygenate, to scavenge free radicals, or to release
NO.sup.+ groups or other forms of biologically active NO to
tissues. A composition comprising SNO-Hb in its various forms and
combinations thereof (oxy, deoxy, met; specifically S-nitrosylated,
or nitrosated or nitrated to various extents) can be administered
to an animal or human in these methods. Compositions comprising
thiols and/or NO donating agents can also be administered to
enhance the transfer of NO.sup.+ groups. Examples of conditions to
be treated by nitrosated or nitrated forms of hemoglobin include
ischemic injury, hypertension, angina, reperfusion injury and
inflammation, and diseases characterized by thrombosis. Further
embodiments of the invention are methods for assessing oxygen
delivery to the tissues of a mammal by measuring SNO-Hb and
nitrosylhemoglobin in blood.
BRIEF DESCRIPTIONS OF THE DRAWINGS
FIGS. 1A-1D are spectrographs of different forms of Hb as described
in Example 1.
FIG. 2A is a graph showing formation, with time, of SNO-Hb by
S-nitrosylation.
FIG. 2B is a graph showing the decomposition, with time, of oxy and
deoxy forms of SNO-Hb.
FIG. 3A is a graph showing the loading of red blood cells
(erythrocytes) with S-nitrosocysteine, over time. The inset is a
series of spectrographs of forms of Hb as described in Example
3.
FIG. 3B is a series of tracings recording isometric tone of a
rabbit aortic ring following treatment of the aortic ring with
various agents as described in Example 3.
FIG. 4A is a graph of change in tension of a rabbit aortic ring
versus concentration of the Hb used in the experiment.
FIG. 4B is a graph of change in tension of a rabbit aortic ring
versus concentration of the Hb used in the experiment, where
glutathione was also added to test the effect as compared to FIG.
4A.
FIG. 4C is a graph of the ratio of S-nitrosoglutathione
formed/starting SNO-Hb concentration versus time, showing rates of
NO group transfer from oxy and met forms of Hb to glutiathione.
FIG. 4D is a graph of S-nitrosothiols exported from loaded red
blood cells over time.
FIG. 5 is a graph showing the mean arterial blood pressure in rats
after they received various doses of oxyHb (.tangle-solidup.),
SNO-oxyHb (.box-solid.), or SNO-metHb (.circle-solid.).
FIGS. 6A-6F are a series of tracings recording blood pressure
(FIGS. 6A and 6B), coronary artery diameter (FIGS. 6C and 6D) and
coronary artery flow (FIGS. 6E and 6F), after administration of
S-nitrosohemoglobin to anesthetized dogs.
FIG. 7A is a graph illustrating the effect of unmodified HbA.sub.0
on platelet aggregation. The maximal extent of aggregation of
platelets is plotted against the concentration of HbA (10 nM to 100
.mu.m) preincubated with platelets. Experiments were performed as
in Example 9. Vertical bars plotted with each data point indicate
the standard deviation.
FIG. 7B is a graph illustrating the effect of
S-nitroso(oxy)hemoglobin on platelet aggregation. The normalized
maximal extent of aggregation of platelets is plotted against the
concentration of HbA (10 nM to 100 .mu.m) preincubated with
platelets.
FIG. 7C is a graph illustrating the antiaggregation effects on
platelets by S-nitroso(met)hemoglobin.
FIG. 8 is a bar graph showing the amount of cGMP (guanosine
3',5'-cyclic phosphoric acid), assayed as in Example 10, for 1, 10
and 100 .mu.M concentrations of native Hb, SNO-oxyHb or SNO-metHb
interacting with 10.sup.8 platelets.
FIG. 9A is a graph which shows the spectra (absorbance versus
wavelength in nanometers) of HbA.sub.0 treated as described in
Example 11. The shift in the wavelength of maximum absorbance of
spectrum B relative to spectrum A illustrates the extent of
addition of NO groups to HbA.sub.0.
FIG. 9B is a graph which shows the spectra of Hb treated with
100-fold excess S-nitrosoglutathione as described in Example
11.
FIG. 9C is a graph which shows the spectra of HbA.sub.0 treated
with excess S-nitrosocysteine as described in Example 11.
FIG. 9D is a graph which shows the spectra of rat Hb treated with
100-fold excess S-nitrosocysteine. Spectrum A shows nitrosated Hb
not further treated with dithionite; spectrum B shows nitrosated Hb
further treated with dithionite.
FIG. 9E is a graph illustrating the increase in nitrosated Hb
product with time by reacting HbA.sub.0 with either 100.times.
excess S-nitrosocysteine (top curve) or 10.times. excess
S-nitrosocysteine (middle curve). HbA.sub.0 was preincubated with
100 .mu.M inositol hexaphosphate before reacting with 10.times.
excess S-nitrosocysteine (bottom curve; triangle points). (See
Example 11.)
FIG. 10 is a graph illustrating the percent change, with time, in
blood flow measured in caudatoputamen nucleus of rats after
injection of the rats with: .largecircle., 100 nmol/kg SNO-Hb;
.circle-solid., 1000 nmol/kg SNO-Hb; or .box-solid., 1000 nmol/kg
underivatized Hb (see Example 12).
FIG. 11 is a graph illustrating the percent change in tension of a
ring of aorta from rabbit, plotted as a function of the log of the
molar concentration of hemoglobin tested (see Example 13).
.circle-solid., Hb treated with S-nitrosocysteine at a ratio of 1:1
CYSNO/Hb; .largecircle., Hb treated with CYSNO at a ratio of 10:1
CYSNO/Hb; .diamond-solid., Hb treated with CYSNO at a ratio of
100:1.
FIG. 12 is a graph of the absorbance versus the wavelength of light
(nm), for aqueous solutions of 17 .mu.M deoxyhemoglobin, 1 .mu.M
NO, and varying amounts of dissolved oxygen added by sequential
injections of room air. The absorbance of the initial solution (no
added air) is shown by the curve with the highest peak at
approximately 430 nm. Sequential additions of 50 .mu.l of air shift
the curve leftwards on the graph. See Example 14.
FIG. 13 is a graph showing the yield of SNO-Hb as micromolar
concentration (left axis, diamonds) and as % of NO added (right
axis, squares), plotted against the heme:NO ratio, when
nitrosyl-deoxyHb made at various ratios of heme:NO was exposed to
oxygen. See Example 15.
FIG. 14A is a graph showing difference spectra (each a spectrum of
the NO and Hb mixture minus spectrum of the starting deoxyHb), for
17 .mu.M hemoglobin and NO mixtures, for the concentrations of NO
shown. See Example 16.
FIG. 14B is a graph showing the peak wavelength of the difference
spectra plotted against the concentration of nitric oxide added to
the solution as in FIG. 14B.
FIG. 15A is a graph showing difference spectra (deoxyhemoglobin and
air mixtures minus initial deoxyhemoglobin spectrum), for
successive additions of air.
FIG. 15B is a graph showing difference spectra (20 .mu.M
deoxyhemoglobin and 1 .mu.M NO mixture, with successive additions
of air, minus initial deoxyhemoglobin spectrum). See Example
17.
FIG. 16 is a graph showing two difference spectra (A.sub.418 of
hemoglobin and NO solution at heme:NO 20:1 minus initial
deoxyhemoglobin A.sub.418) for the mutant .beta.93Ala Hb and wild
type .beta.93Cys Hb. See Example 18.
FIG. 17 is a graph showing the yield of SNO-Hb as micromolar
concentration (left axis, diamonds) and as % of NO added (right
axis, squares), plotted against the heme:NO ratio, when
nitrosyl-deoxyHb made at various ratios of heme:NO was exposed to
oxygen. See Example 19.
FIG. 18A is a graph showing the percentage content of oxidized
hemoglobin (metHb) for different concentrations of Hb (symbols
below) to which NO was added to reach varying final concentrations
(horizontal axis). .diamond-solid. represents 1.26 .mu.M
hemoglobin, .box-solid. represents 5.6 .mu.M hemoglobin,
.tangle-solidup. represents 7.0 .mu.M hemoglobin, X represents 10.3
.mu.M hemoglobin, {character pullout}represents 13.3 .mu.M
hemoglobin, and .circle-solid. represents 18.3 .mu.M hemoglobin.
See Example 20.
FIG. 18B is a graph showing the yield of oxidized hemoglobin
(.mu.M) plotted against the final concentration of NO added to
solutions of Hb at the concentrations indicated by the symbols as
for FIG. 18A.
FIG. 19 is a graph showing the concentration of oxidized Hb (metHb)
plotted against the NO concentration, in experiments performed as
described in Example 21 in 10 mM (.diamond-solid.), 100 mM
(.DELTA.), or 1 M ({character pullout}) sodium phosphate buffer, pH
7.4.
FIGS. 20A and 20B are graphs showing the contractile effects of
oxyHb, SNO-oxyHb, deoxy-Hb and SNO-deoxy-Hb on thoracic aortic ring
isolated from rabbit. Measurements are percent increase in tension
of aortic ring as a function of the log of the concentration of
hemoglobin or SNO-hemoglobin. Measurements are made after the
tension has stabilized.
FIG. 20C is a graph showing the percent change in tension of
contracted aortic ring as a function of the log concentration of
SNO-hemoglobin at the concentrations of O.sub.2 indicated, in
addition to 10 .mu.M glutathione.
FIG. 20D is a graph showing the percent change in tension of
contracted aortic ring as a function of the log concentration of
SNO-glutathione, in the concentrations of O.sub.2 indicated.
FIG. 21A and FIG. 21B are each a series of four graphs illustrating
the change with time in tension of rabbit aortic ring upon the
addition of red blood cells treated with S-nitrosocysteine ("red
blood cells loaded with nitric oxide"), or untreated red blood
cells, as indicated, in the concentration of O.sub.2 indicated.
FIG. 21C is a graph illustrating the change with time in tension of
rabbit aortic ring contracted with phenylephrine under hypoxic
conditions (6-7 torr) and then exposed to either 1 .mu.M Hb or
SNO-Hb.
FIG. 22 is a bar graph depicting the concentrations of FeNO/Hb and
SNO/Hb in venous or arterial blood as measured in Example 24.
ATA=atmospheres of absolute pressure.
FIGS. 23A-23I are each a graph showing the effects of SNO-Hb
(.circle-solid.) and Hb (.box-solid.) (1 .mu.mol/kg infused over 3
minutes) on local blood flow in substantia nigra (SN), caudate
putamen nucleus, and parietal cortex of rats, in 21% O.sub.2 (FIGS.
23A, 23B and 23C), in 100% O.sub.2 (FIGS. 23D, 23E and 23F), and in
100% O.sub.2 at 3 atmospheres absolute pressure (FIGS. 23G, 23H and
23I) as measured in Example 25.
FIG. 24A is a bar graph showing the percent change in blood
pressure of rats, during exposure to three different conditions
(inspired O.sub.2 concentrations of 21%, 100%, or 100% O.sub.2 at 3
ATA) upon infusion of GSNO, SNO-Hb, or Hb, as tested in Example
26.
FIG. 24B is a bar graph showing the percent change in blood
pressure of rats [pre-administered (+ L-NMMA), or not
preadministered (-L-NMMA), N.sup.G -monomethyl-L-arginine]upon
infusion of SNO-RBCs (RBCs=red blood cells), as tested in Example
26.
FIG. 25 is a bar graph showing the results of
photolysis-chemiluminescence assays to measure NO bound in the form
of S-nitrosothiol and NO bound at the heme, on SNO-Hb(FeII).sub.2
and deoxyHb(FeII)NO prepared as described in Example 27.
FIG. 26 is a diagram illustrating (upper panel) alternative
reactions proposed for .beta.-chain nitrosyl hemes in the T
structure and (lower panel) a model of NO binding to hemes and
thiols of hemoglobin, in the circulation of a mammal or bird, for
example.
DETAILED DESCRIPTION OF THE INVENTION
Roles for Hemoglobin in Physiology
The increase in SNO-Hb content of red cells across the pulmonary
circuit (right ventricular inport-left ventricle) suggests that the
Hb molecule is S-nitrosylated in the lung. Selective transfer of
the NO group from endogenous RSNOs found in lung (Gaston, et al.
(1993)) and blood (Scharfstein, J. S. et al., J. Clin. Invest.
94:1432-1439 (1995)) to SH groups of Hb, substantiate these
findings. The corresponding decline in Hb(FeII)NO levels across the
pulmonary bed reveals a role for the lung either in the elimination
of NO or in its intramolecular transfer from heme to cys.beta.93.
Taken in aggregate, these data extend the list of
function-regulating interactions of Hb with small molecules within
the respiratory system, previously known to include the elimination
of CO and CO.sub.2, and uptake of O.sub.2. Since, as demonstrated
herein, oxygenation of Hb leads to structural changes that increase
the NO-related reactivity of cys.beta.93, O.sub.2 can now be
regarded as an allosteric effector of Hb S-nitrosylation.
The arterial-venous difference in SNO-Hb concentration suggests
that the protein acts as an NO group donor in the systemic
circulation. There is good indication that SNO-Hb functions in
regulation of vasomotor tone. In the microcirculation, where
control of blood pressure is achieved, erythrocytes come in
intimate contact with endothelial surfaces. Under these conditions,
Hb can contract the vasculature by sharply decreasing the steady
state level of free NO (Lancaster, J. R., (1994)). This is believed
to contribute to the increases in blood pressure that occur with
infusion of cell-free Hbs (Vogel, W. M., et al., Am. J. Physiol.,
251:H413-H420 (1986); Olsen, S. B., et al., Circulation 93:329-332
(1996)). The transient nature of such hypertensive responses,
however, is consistent with the subsequent formation of SNO-Hb
which counteracts this effect, evidenced by its lowering of blood
pressure at naturally occurring concentrations. Thus, the capacity
of the erythrocyte to support the synthesis and metabolism of
SNO-Hb is important for normal blood flow.
Mammals must have adopted unique molecular mechanisms to ensure
adequate NO delivery in the microcirculation. Results herein
suggest that Hb has evolved both electronic and conformational
switching mechanisms to achieve NO homeostasis. Specifically, NO
scavenging by the metal center(s) of SNO-Hb(FeII)O.sub.2 is sensed
through its conversion to met(FeIII) (FIG. 1B). This electronic
switch effects decomposition of SNO-Hb with NO group release (FIGS.
3A, 3B, 4A). In this manner, the NO-related activity of SNO-Hb is
partly determined by the amount of NO scavenged. Changes in O.sub.2
tension also function to regulate NO delivery, as it is observed
herein that NO release is facilitated by deoxygenation. This
allosteric effect promotes the efficient utilization of O.sub.2, as
NO controls mitochondrial respiration (Shen, W., et al.,
Circulation 92:3505-3512 (1995)).
S-nitrosothiol groups in proteins have been implicated in NO
metabolism and in regulation of cellular functions (Stamler, J. S.,
et al., Proc. Natl. Acad. Sci. USA 89:444-448 (1992); Stamler, J.
S., Cell 78:931-936 (1994)). The identification of SNO-Hb in
erythrocytes is the first demonstration of an intracellular
S-nitrosoprotein and gives further credence to the role of such
proteins in cellular regulation. The question arises as to how
SNO-Hb relaxes blood vessels when any free NO released would be
scavenged instantaneously by Hb itself according to previous
theories (Lancaster, J. R., (1994)). Noteworthy in this regard are
studies showing that RSNO activity involves nitrosyl (NO.sup.+)
transfer to thiol acceptors (Scharfstein, J. S., et al., (1994);
Arnelle, D. R. and Stamler, J. S., Arch. Biochem. Biophys.
318:279-285 (1995); Stamler, J. S., et al., Proc. Natl. Acad. Sci.
USA 89:7674-7677 (1992)), which serve to protect the NO-related
activity from inactivation at metal centers. Findings presented
herein indicate that S-nitrosothiol/thiol exchange with glutathione
(forming GSNO) occurs within erythrocytes, and is influenced by the
oxidation state of heme and its occupation by ligand. Certain
activities of GSNO in bacteria require transport of intact
dipeptide (i.e., S-nitrosocysteinylglycine) across the cell
membrane (DeGroote, M. A., et al., Proc. Natl. Acad. Sci. USA
92:6399-6403 (1995)). The data presented below in the Examples show
that S-nitrosothiol transport occurs also in eukaryotic cells.
GSNO, or related thiol carriers exported by erythrocytes (Kondo,
T., et al., Methods in Enzymology, Packer, L., ed., Academic Press,
252:72-83 (1995)), might also initiate signaling in or at the
plasmalemma (Stamler, J. S., Cell 78:931-936 (1994)), given reports
of thiol-dependent activation of potassium channels by EDRF
(Bolotina, V. M., et al., Nature 368:850-853 (1994)). Alternative
possibilities also merit consideration. In particular, reports that
Hb associates with red cell membranes via cys.beta.93 (Salhany, J.
M. and Gaines, K. C., Trends in Biochem. Sci., pp. 13-15 (Jan.
1981)) places Hb in a position to donate the NO group directly to
contacting endothelial surfaces, perhaps via SNO/SH exchange. Cell
surface interactions appear to be operative in signaling mediated
by other S-nitrosoproteins (Stamler, J. S., et al., Proc. Natl.
Acad. Sci. USA, 89:444-448 (1992); Stamler, J. S., Cell, 78:931-936
(1994)).
The highly conserved Cys.beta.93 residues in Hb influence the
oxygen affinity and redox potential of the heme iron and its
physiochemical properties (Garel, C., et al., Biochem. 123:513-519
(1982); Jocelyn, P. C., et al., Biochemistry of the SH Group, p.
243, Academic Press, London; (1972); Craescu, C. T., J. Biol. Chem.
261:14710-14716 (1986); Mansouri, A., Biochem. Biophys. Res.
Commun., 89:441-447 (1979)). Nonetheless, their long sought-after
physiological function has remained a mystery. The studies herein
suggest new sensory and regulatory roles for Hb, in which
Cys.beta.93 functions in transducing NO-related signals to the
vessel wall. In particular, the physiological function of
Cys.beta.93, which is invariant in all mammals and birds, is to
deliver under allosteric control, NO-related biological activity
that cannot be scavenged by heme. Thus, these data bring to light a
dynamic circuit for the NO group in which intraerythrocytic Hb
participates as both a sink and a donor, depending on its
microenvironment. Such observations provide answers to paradoxes
that arise from conceptual frameworks based solely on diffusional
spread and reaction of free NO (Lancaster, J. R., (1994); Wood and
Garthwaite, J. Neuropharmacology 33:1235-1244 (1994)); and has
implications that extend to other thiol- and metal-containing
(heme) proteins, such as nitric oxide synthase and guanylate
cyclase.
The discoveries reported here have direct therapeutic implications.
Specifically, concerns over loss of NO-related activity due to
inactivation by blood Hb (Lancaster, J. R., (1994)) are obviated by
the presence of an RSNO subject to allosteric control. Forms of
SNO-Hb can be free of the adverse hypertensive properties of
cell-free Hb preparations that result from NO scavenging at the
metal centers. A composition comprising one or more of the various
forms of cell-free SNO-Hb (e.g., SNO-Hb[FeII]O.sub.2,
SNO-Hb[FeIII], SNO-Hb[FeII]CO) can be administered in a
pharmaceutically acceptable vehicle to a human or other mammal to
act as a blood substitute.
Blood Flow Regulation by S-Nitrosohemoglobin is Controlled by the
Physiological Oxygen Gradient
In the classical allosteric model, Hb exists in two alternative
structures, named R (for relaxed, high O.sub.2 affinity) and T (for
tense, low O.sub.2 affinity). The rapid transit time of blood
through the capillaries requires that Hb assume the T-structure to
efficiently deliver O.sub.2 (M. F. Perutz, pp. 127-178 in Molecular
Basis of Blood Diseases, G. Stammatayanopoulos, Ed. (W. B. Saunders
Co., Philadelphia, 1987); Voet, D. and Voet, J. G., pp. 215-235
(John Wiley & Sons Inc., New York, 1995). The switch from R to
T in red blood cells normally takes place when the second molecule
of O.sub.2 is liberated. This allosteric transition also controls
the reactivity of two highly conserved cysteine.beta.93 residues
that can react with `NO`. Thiol affinity for NO is high in the R or
oxy structure and low in T or deoxy structure. This means that the
NO group is released from thiols of Hb in low PO.sub.2 and explains
the arterial-venous (A-V) difference in the S-nitrosohemoglobin
(SNO-Hb) level of blood (see Table 2, Example 8). A major function
of (S)NO in the vasculature is to regulate blood flow, which is
controlled by the resistance arterioles (Guyton, A. C., in Textbook
of Medical Physiology (W. B. Saunders Co., Philadelphia, 1981) pp.
504-513). It is shown from the Examples herein that (partial)
deoxygenation of SNO-Hb in these vessels (Duling, B. and Berne, R.
M. Circulation Research, 27:669 (1970); Popel, A. S., et al.,
(erratum Am. J. Physiol. 26(3) pt. 2). Am. J. Physiol. 256, H921
(1989); Swain, D. P. and Pittman, R. N. Am. J. Physiol. 256,
H247-H255 (1989); Torres, I. et al., Microvasc. Res., 51:202-212
(1996); Buerk, D. et al., Microvasc. Res., 45:134-148 (1993))
actually promotes O.sub.2 delivery by liberating (S)NO. That is,
the allosteric transition in Hb functions to release (S)NO in order
to increase blood flow.
O.sub.2 delivery to tissues is a function of the O.sub.2 content of
blood and blood flow (Dewhirst, M. W. et al., Cancer Res.,
54:3333-3336 (1994); Kerger, H. et al., Am. J. Physiol.,
268:H802-H810 (1995)). Blood oxygen content is largely determined
by Hb, which undergoes allosteric transitions in the lung and
systemic microvasculature that promote the binding and release of
O.sub.2 (L. Stryer, in Biochemistry L. Stryer, Ed. (W. H. Freeman
& Co., San Francisco, 1981) pp. 43-82; Guyton, A. C. in
Textbook of Medical Physiology (W. B. Saunders Co., Philadelphia,
1981); Perutz, M. F., pp. 127-178 in Molecular Basis of Blood
Diseases, G. Stammatayanopoulos, Ed. (W. B. Saunders Co.,
Philadelphia, 1987); Voet, D. and Voet, J. G. (John Wiley &
Sons Inc., New York, 1995) pp. 215-235 pp. 208-215, 224-225,
230-245, 344-355)). Intimate contact between erythrocyte and
endothelium is believed to facilitate O.sub.2 delivery by
minimizing the distance for O.sub.2 diffusion into surrounding
tissues (Caro, C. G. et al., Oxford University Press, Oxford, 363
(1978)). On the other hand, regional blood flow is regulated by
metabolic requirements of the tissue: blood flow is increased by
hypoxia and decreased when O.sub.2 supply exceeds demand (Guyton,
A. C., in Textbook of Medical Physiology (W. B. Saunders Co.,
Philadelphia, 1981) pp. 504-513)). These classical physiological
responses are thought to be partly mediated by changes in the level
of endothelial-derived NO and its biological equivalents (Park, K.
H. et al., Circ. Res, 71:992-1001 (1992); Hampl, V. et al., J.
Appl. Physiol. 75(4):1748-1757 (1993)).
This standard picture has its problems. First, it is puzzling that
significant O.sub.2 exchange occurs in the precapillary resistance
vessels (evidenced by the periarteriolar O.sub.2 gradient; Duling,
B. and Berne, R. M. Circulation Research, 27:669 (1970); Popel, A.
S., et al., (erratum Am. J. Physiol. 26(3) pt. 2). Am. J. Physiol.
256, H921 (1989); Swain, D. P. and Pittman, R. N. Am. J. Physiol.
256, H247-H255 (1989); Torres, I. et al., Microvasc. Res.,
51:202-212 (1996); Buerk, D. et al., Microvasc. Res., 45:134-148
(1993)). Why is O.sub.2 lost to counter-current venous exchange
prior to reaching the tissues? Second, close contact between
endothelial surfaces and erythrocytes leads to sequestration of NO
by Hb (Stamler, J. S., Nature, 380:108-111 (1996); Perutz, M. F.,
Nature, 380:205-206 (1996)). Decreases in the steady-state levels
of NO in terminal arterioles (King, C. E. et al., J. Appl.
Physiol., 76(3):1166-1171 (1994); Shen, W. et al., Circulation,
92:3505-3512 (1995); Kobzik, L. et al., Biochem. Biophys. Res.
Comm., 211(2):375-381 (1995); Persson, M. G., et al., Br. J.
Pharmacol., 100:463-466 (1990) and capillaries (Mitchell, D., and
Tyml, K., Am. J. Physiol., 270 Heart Circ. Physiol., 39),
H1696-H1703 (1996)) contract blood vessels, blunt hypoxic
vasodilation and reduce red cell velocity. This line of reasoning
leads to the paradox: the red blood cell seems to oppose its own
O.sub.2 delivery function (note in vivo effects of Hb in FIGS.
10A-10I).
The finding that the O.sub.2 gradient in precapillary resistance
vessels promotes NO group release from SNO-Hb appears to solve
these problems. SNO-Hb compensates for NO scavenging at the heme
iron by assuming the T-structure which liberates SNO. Specifically,
Cys93 donates the NO group in deoxy structure whereas it cannot do
so in the oxy conformation. Accordingly, the O.sub.2 gradient
determines whether SNO-Hb dilates or constricts blood vessels.
Stated another way, SNO-Hb senses the tissue PO.sub.2 (i.e., the
periarteriolar O.sub.2 gradient) and then utilizes the allosteric
transition as a means to control arteriolar tone. If the tissue is
hypoxic (i.e., the O.sub.2 gradient is high), SNO is released to
improve blood flow. However, if O.sub.2 supply exceeds demand
(i.e., the O.sub.2 gradient is small), SNO-Hb holds on to the NO
group by maintaining the R-structure--with the net effect of
reducing blood flow in line with demand. SNO-Hb thereby contributes
to the classical physiological responses of hypoxic vasodilation
and hyperoxic vasoconstriction.
Based on studies described herein, especially Examples 22-26, the
following picture emerges. Partially nitrosylated Hb (Hb[FeII]NO)
enters the lung in T-structure (see venous measurements in FIG.
22). There, S-nitrosylation is facilitated by the O.sub.2 -induced
conformational change in Hb. SNO-oxyHb (SNO-Hb[FeII]O.sub.2) enters
the systemic circulation in R-structure (see arterial levels in
FIG. 22). Oxygen losses in precapillary resistance vessels then
effect an allosteric transition (from R to T) in Hb which liberates
`NO` to dilate blood vessels (see especially FIGS. 20D and 23A-C).
NO released from Hb is transferred directly to the endothelium, or
by way of low mass S-nitrosothiols--such as GSNO--which are
exported from RBCs (see FIG. 4D and Example 4; see also FIGS. 20C
and 24A). Thus, the O.sub.2 gradient in arterioles serves to
enhance O.sub.2 delivery: it promotes an allosteric transition in
Hb which releases NO-related activity to improve blood flow.
Assay methods
The invention also relates to a method for determining the
concentration of nitrosyl(FeII)--hemoglobin in a blood sample,
thereby serving as a measure of the level of NO in the animal or
human from which the blood sample has been taken. The method is
related to one used previously for the measurement of
S-nitrosoproteins and smaller molecular weight S-nitrosothiols in
plasma (See U.S. Pat. No. 5,459,076; Oct. 17, 1995. The contents of
this patent are hereby incorporated by reference in their
entirety.) However, the primary focus of the present invention is
on assaying for nitrosyl(FeII)--hemoglobin rather than
S-nitrosothiols.
In contrast to the previous method, in which the red blood cells
were removed and discarded from the sample to be analyzed, the
subject invention method uses the red blood cells. The method
measures NO which has reacted with the thiol groups of hemoglobin
in the form of S-nitroso-hemoglobin (SNO-Hb) as well as NO bound to
the Fe of the heme (nitrosyl(FeII)-hemoglobin or Hb(FeII)NO). As
shown in the table, the level of S-nitroso-hemoglobin in venous
blood is negligible compared to the level of Hb(FeII)NO. Therefore,
to specifically measure the level of Hb(FeII)NO in venous blood, it
is unnecessary to include steps in which Hb samples are divided
into two aliquots which are then either treated or not treated with
a 10-fold excess of HgCl.sub.2 over the protein concentration.
Reaction of Hb with HgCl.sub.2 removes NO from thiol groups
selectively, without disturbing NO bound at the heme. Values for NO
obtained from the HgCl.sub.2 reaction, if significant, should be
subtracted from the total NO obtained for the measurements without
the HgCl.sub.2 reaction, to obtain an accurate value for
Hb(FeII)NO.
In one embodiment of the invention, a blood sample is taken from a
mammal, such as a human, and the solid parts including cells are
isolated away from the remaining fluid. The cells are then lysed by
standard methods, and a protein fraction is prepared from the
lysate. Before quantitating nitric oxide adducts (nitrosonium
adducts, which include low molecular weight S-nitrosothiols (which
are small enough to be freely diffusible through cell membranes,
such as the S-nitrosothiol S-nitrosoglutathione) and high molecular
weight S-nitrosothiols such as S-nitroso-proteins), it is
preferable to first remove low molecular weight S-nitrosothiols
endogenous to the red blood cells, which would also contribute to
the NO value, by a step which separates low molecular weight
molecules away from the red blood cell proteins (referred to as
desalting). This step can include, for example, dialysis or column
chromatography based on separation by size of the molecules. A
further step is to subject the protein fraction to photolysis, as
in a photolysis cell, where it is irradiated with light of the
appropriate wavelength to liberate NO from the various forms of
hemoglobin. The resulting NO is detected by reaction with
ozone.
One embodiment of the invention utilizes a chemiluminescence
apparatus in which a photolysis cell is linked directly to the
reaction chamber and detector portion, thereby bypassing the
pyrolyzer. A sample of the blood protein fraction is injected into
the photolysis cell, either directly, or as chromatographic
effluent from a high-performance liquid or gas chromatography
system which is connected to the photolysis cell.
The sample is then irradiated with a mercury vapor lamp, and
directed through a series of cold traps, where liquid and gaseous
fractions which are less volatile than nitric oxide (such as
nitrite and nitrate) are eliminated, leaving only free nitric oxide
remaining in the cell. The nitric oxide is then transported by a
gaseous stream, preferably helium, into the chemiluminescence
spectrometer. In the alternative, other inert gases may be
used.
Once present in the chemiluminescence spectrometer, the free nitric
oxide is detected by its chemical reaction with ozone, resulting in
the generation of signals that are recorded on a digital
integrator. If desired, flow rates and illumination levels in the
photolysis cell can be adjusted to cause complete photolysis of the
S-nitric oxide bond of the S-nitrosothiol compounds. Flow rates and
illumination levels may be adjusted by routine methods available in
the art, in order to achieve optimal cleavage of the bond between
the particular adduct and nitric oxide, nitrosonium or nitroxyl,
whichever is bound.
In a variation, the invention relates to a method for detecting
S-nitrosothiols, including primarily S-nitroso-hemoglobin (SNO-Hb)
in a blood sample. This method comprises inactivating the
chemiluminescence, signal-generating capability of any nitric oxide
which is associated with a thiol, in the protein fraction derived
from the blood sample, and determining the amount of thiol-bound
nitric oxide by measuring the quantitative difference between total
nitric oxide and nitric oxide remaining after inactivation.
A particular embodiment of this variation relates to a method in
which the protein fraction derived from the blood sample is treated
with a source of mercury ions, mercurous ions being preferred,
followed by air incubation, which oxidizes the nitric oxide and
nitrosonium and renders them undetectable. Compounds suitable for
pretreatment include Hg.sub.2 Cl.sub.2 and other mercurous ion
salts and organic mercurials. The treated sample is then injected
into the photolysis cell, where NO.sup.+ is converted to NO.
(nitric oxide) and the nitric oxide is detected by the
chemiluminescence method described above. The amount of nitric
oxide which is specifically derived from S-nitrosothiols is
determined by comparing the chemiluminescence signal generated by
the mercury ion-treated sample, with a chemiluminescence signal
generated by a sample of the equivalent biological fluid which is
not treated with mercury ion prior to injection into the photolysis
cell.
In a further embodiment of the claimed invention, the methods
described herein can be utilized to determine the presence of a
disease state which involves abnormal levels of nitric oxide and
its biologically active equivalents, by monitoring Hb(FeII)NO and
SNO-Hb levels in blood, and more particularly, Hb(FeII)NO in venous
blood from a patient. The ability to specifically assay for
Hb(FeII)NO in venous blood distinguishes this assay over previously
known methods. Nitric oxide adducts represent a pool of bioactive
nitric oxide in physiological systems. Therefore, in disease states
in which the pathogenesis derives from the effects of abnormal
levels of nitric oxide, these methods provide a means for the
clinician to determine the presence of, and monitor the extent of,
the disease state. Such information enables the clinician to
determine the appropriate pharmacological intervention necessary to
treat the disease state. Such disease states and medical disorders
include, but are not limited to, respiratory distress, septic
shock, cardiogenic shock, hypovolemic shock, atherosclerosis,
hyperhomocysteinemia, venous thrombosis, arterial thrombosis,
coronary occlusion, pulmonary embolism, cerebrovascular accidents,
vascular fibrosis, ectopia lentis, osteoporosis, mental
retardation, skeletal deformities, pulmonary hypertension,
malignancy, infections, inflammation, asthma, tolerance to
narcotics and central nervous system disorders. Furthermore, the
use of these methods is not limited to these diseases. This method
can be of use in assaying biologically active nitric oxide
equivalents in any disease state or medical disorder in which
nitric oxide is implicated.
The data set forth in the Examples below demonstrate that a
determination of NO bound to hemoglobin as nitrosylhemoglobin and
SNO-Hb can be used to assess the efficiency of oxygen delivery to
the tissues of an animal or a human patient. Values for
nitrosylhemoglobin and SNO-Hb in blood can be determined together,
in one method, or they can be determined in separate methods. An
additional determination for oxygen in the blood, as measured by
methods known in the art, can be used in conjunction with
determinations of nitrosylhemoglobin and SNO-Hb concentrations, to
assess oxygen delivery to a site in the body of a human or other
mammal from which a blood sample is taken.
Further Embodiments
The subject invention relates to a method of loading cells with a
nitrosating agent as exemplified for red blood cells as in FIG. 3A,
but which can be accomplished in more ways. Suitable conditions for
pH and for the temperature of incubation are, for example, a range
of pH 7-9, with pH 8 being preferred, and a temperature range of 25
to 37.degree. C. For red blood cells, short incubation times of 1
to 3 minutes are preferred for limiting the formation of
S-nitrosylated forms of Hb. However, intracellular concentrations
of 1 mM nitrosating agent can be reached.
The nitrosating agent should be a good donor of NO.sup.+ and should
be able to diffuse through the cell membrane of the target cell
type. That is, it is preferably of low molecular weight, compared
to the molecular weight of S-nitrosoproteins. Examples are
S-nitroso-N-acetylcysteine, S-nitrosocysteinylglycine,
S-nitrosocysteine, S-nitrosohomocysteine, organic nitrates and
nitrites, metal nitrosyl complexes, S-nitro and S-nitroso
compounds, thionitrites, diazeniumdiolates, and other related
nitrosating agents as defined in Feelisch, M. and Stamler, J. S.,
"Donors of Nitrogen Oxides" chapter 7, pp. 71-115 In Methods in
Nitric Oxide Research (Freelisch, M. and Stamler, J. S., eds.) John
Wiley and Sons, Ltd., Chichester, U.K. (1996), the contents of
which chapter are hereby incorporated by reference in their
entirety. Nitrosating agents have differential activities for
different reactive groups on metal-containing proteins. A
nitrosating agent can be chosen for minimal oxidation of the heme
iron of Hb, and maximum activity in nitosylating thiol groups such
as found on cysteine. Assay methods are available for detection of
nitrosation products, including S-nitrosothiols. See Stamler et
al., U.S. Pat. No. 5,459,076, the contents of which are hereby
incorporated by reference in their entirety. See also, for example,
Keefer, L. K., and Williams, D. L. H., "Detection of Nitric Oxide
Via its Derived Nitrosation Products," chapter 35, pp. 509-519 In
Methods in Nitric Oxide Research (Freelisch, M. and Stamler, J. S.,
eds.) John Wiley and Sons, Ltd., Chichester, U.K., 1996; see also
Stamler, J. S. and Feelisch, M., "Preparation and Detection of
S-Nitrosothiols," chapter 36, pp. 521-539, ibid. Nitrite and
nitrate products can be assayed by methods described, for instance,
in Schmidt, H. H. H. W. and Kelm, M., "Determination of Nitrite and
Nitrate by the Griess Reaction," chapter 33, pp. 491-497, ibid.,
and in Leone, A. M. and Kelm, M., "Capillary Electrophoretic and
Liquid Chromatographic Analysis of Nitrite and Nitrate," chapter
34, pp. 499-507, ibid.
Such low molecular weight nitrosating agents can be used in red
blood cells to deliver NO-related activity to tissues. Treatment of
red blood cells with nitrosating agent further serves to increase
the O.sub.2 delivery capacity of red blood cells. Such treatment of
red blood cells also allows for the scavenging of free radicals,
such as oxygen free radicals, throughout the circulation. It is
possible to load red blood cells with S-nitrosothiol, for example,
by a process of removing whole blood from a patient's body (as a
minimal method of isolating red blood cells), treating the red
blood cells with low molecular weight nitrosating agent, such as by
incubating the red blood cells in a solution of S-nitrosothiol, and
then reintroducing the red blood cells into the same patient,
thereby allowing the treatment of a number of types of diseases and
medical disorders, such as those which are characterized by
abnormal O.sub.2 metabolism of tissues, oxygen-related toxicity,
abnormal vascular tone, abnormal red blood cell adhesion, and/or
abnormal O.sub.2 delivery by red blood cells. Such diseases can
include, but are not limited to, ischemic injury, hypertension,
shock, angina, stroke, reperfusion injury, acute lung injury,
sickle cell anemia, and blood borne infectious diseases such as
schistosomiasis and malaria. The use of such "loaded" red blood
cells also extends to blood substitute therapy and to the
preservation of living organs, such as organs for transplantation.
In some cases, it will be appropriate to treat a patient with
loaded red blood cells originating from a different person.
A particular illustration of the mechanism of the treatment method
is presented here by considering sickle cell anemia. Sickle cell
patients suffer from frequent vascular occlusive crises which
manifest in clinical syndromes such as the acute chest syndrome and
hepatic dysfunction. Both endothelial cell dysfunction, resulting
in a clotting diathesis as well as dysfunction intrinsic to the red
blood cell, are central to disease pathogenesis. At the molecular
level, the increased expression of vascular adhesion molecules such
as VCAM promote the adhesion of sickled red blood cells containing
abnormal hemoglobin. It follows that decreasing cytokine expression
on endothelial cells, promoting endothelial function and
attenuating red cell sickling, are key therapeutic objectives.
However, currently used therapies have been generally
unsuccessful.
In this novel method for loading red blood cells with intracellular
NO-donor S-nitrosothiols, the effect is to increase oxygen
affinity--which in and of itself should attenuate red blood cell
sickling--and to endow the red blood cell with vasodilator and
antiplatelet activity, which should reverse the vasoocclusive
crisis. Moreover, nitric oxide should attenuate the expression of
adhesion molecules on endothelial cell surfaces, thus restoring
endothelial function.
Herein is described a novel therapeutic approach to the treatment
of sickle cell disease which involves loading of red blood cells
with S-nitrosothiols or other nitrosating agents. Two examples of
therapeutic approaches are given. In the first, the patient's own
red blood cells are S-nitrosated extracorporeally (yielding
"loaded" red blood cells) and then given to the patient. The second
approach is to directly administer to a patient an agent such as
S-nitrosocysteine, which is permeable to red blood cells.
For some diseases or disorders, the administration of NO-loaded red
blood cells is especially desirable. Upon a change from the
oxygenated to the deoxygenated state, or upon a change in the
oxidation state of the heme Fe from the reduced state (FeII) to the
oxidized (FeIII) state, NO is released from the thiol groups of
hemoglobin, and is rapidly transferred to glutathione to form
S-nitrosoglutathione. Red blood cells are known to have a high
concentration of glutathione. S-nitrosoglutathione efficiently
delivers NO to tissues.
In another aspect, the invention is a method for the treatment of
infection by administering to an infected mammal an agent which
causes S-nitrosation of thiol groups within the cells which are the
target of such agent. For example, an S-nitrosothiol to which
lymphocytes are highly permeable can be administered to a patient
infected with HIV. Such treatment for HIV can also be used
extracorporeally, to blood isolated from the patient. In another
application, the infection is bacterial, and the S-nitrosothiol to
be used as an anti-bacterial agent is one to which the target
bacterial cells are highly permeable, as compared to the
permeability properties of the host cells. (See, for example De
Groote, M. A., et al., Proc. Natl. Acad. Sci. USA 92:6399-6403
(1995).) Alternatively, nitrosothiols can be used to treat
Plasmodium falciparum within red blood cells.
Another embodiment of the invention is a method for specifically
modifying a protein containing one or more metal atoms so that the
protein becomes S-nitrosylated at one or more thiol groups without
modifying the metal, as by changing the oxidation state or causing
the metal atoms to bind NO. This can be accomplished by the use of
a reagent which possesses NO.sup.+ character, such as a
nitrosothiol (See, for instance, Example 4A.), which reacts
specifically with thiol groups of a protein in which metal is
bound.
An S-nitrosation method has been devised which does not affect the
heme of hemoglobin. SNO-Hb (SNO-Hb(FeII)O.sub.2) can be synthesized
from Hb(FeII)O.sub.2 with up to 2 SNO groups per tetramer without,
or with only minimlal, oxidation of the heme Fe from FeII to FeIII.
Preferably, the proportion of metHb in such a SNO-Hb(FeII)O.sub.2
composition is less than about 10%, more preferably, less than
about 5%, and still more perferably, less than about 2%. In
contrast, when Hb(FeII)O.sub.2 is incubated with excess nitric
oxide or nitrite, methemoglobin (HbFe[III]) forms rapidly (Example
1B) and to a significant extent. When Hb[FeII] is incubated with
nitric oxide, NO binds rapidly to the heme, forming Hb(FeII)NO to a
significant extent (Example 1A).
Although rates of formation of SNO-Hb(FeII)O.sub.2 from
Hb(FeII)O.sub.2 are more rapid (see Example 2A), the corresponding
SNO-deoxyHb form can also be made by incubation of
S-nitrosoglutathione or S-nitrosocysteine, for example, with
Hb(FeII), yielding SNO-Hb(FeII), as in Example 1C. Preferably, the
proportion of metHb found in such a SNO-Hb(FeII) composition is
less than about 10%, more preferably, less than about 5%, and still
more perferably, less than about 2%.
The effects of the various forms of Hb on
vasodilation--constriction, dilation or a neutral effect--depend on
three factors: whether 1) the Fe of the heme is oxidized, 2)
O.sub.2 is bound at the heme (that is, the oxygenation state,
dictated by the conformation of the protein as R state or T state),
and 3) thiol is present in sufficient concentration to facilitate
the transfer of NO.sup.+.
The importance of the first factor is shown in FIG. 4A.
Hb(FeII)O.sub.2 and SNO-Hb[FeII]O.sub.2 act as vasoconstrictors,
but SNO-Hb[FeIII] (metHb form, where FeII has been oxidized to
FeIII) acts as a vasodilator. FIG. 4A shows that
SNO-Hb[FeII]O.sub.2 with oxygen bound at the heme, and with a ratio
of SNO/Hb=2, acts as a powerful vasoconstrictor.
SNO-Hb(FeII) is also a vasodilator. FIG. 2B illustrates the second
factor in demonstrating that rates of RSNO decomposition and
transfer are much faster for SNO-Hb in the deoxy state than for
SNO-Hb in the oxy state.
It can be seen how the NO.sup.+ -donating properties of SNO-Hb
depend on oxygen concentrations. SNO-Hb releases oxygen at sites of
low oxygen concentration or under oxidizing conditions. SNO-Hb
releases its NO group(s) to cause vasodilation either due to 1)
oxidation of the heme Fe to FeIII or 2) loss of the O.sub.2 from
the heme by deoxygenation. It is shown in FIG. 2B that NO is
transferred off SNO-Hb best in the deoxy state. In ischemia, SNO-Hb
deoxygenates, rapidly followed by the loss of NO. It can be seen
from the data that SNO-metHb having a ratio of 1 SNO/SNO-metHb is a
more powerful vasodilator than SNO-oxyHb having a ratio of 2
SNO/SNO-oxyHb. It should be noted that S-nitrosation of Hb induces
the R state (oxy conformation). Thus, it follows that 1 SNO-oxyHb
molecule having a ratio of 1 SNO/SNO-oxyHb is less potent than 10
molecules of SNO-oxyHb having a ratio of 0.1 SNO/SNO-oxyHb.
The third factor is illustrated by the results shown in FIG. 4B.
These results demonstrate potentiation by thiol of the vasodilator
effect of SNO-Hb(FeII)O.sub.2 and SNO-Hb(FeIII). Transfer of
NO.sup.+ from SNO-Hb to low molecular weight nitrosothiols is more
efficient when Hb is in the deoxy state compared to the oxy state
(FIG. 2B) or in the met state compared to the oxy state (FIG.
4C).
NO is released or transferred as NO.sup.+ (nitrosyl cation) from
SNO-Hb. The SNO groups of SNO-Hb have NO.sup.+ character. Transfer
of NO.sup.+ from SNO-Hb occurs most efficiently to low molecular
weight thiols, such as glutathione, and is most efficient when the
heme is oxidized (SNO-metHb) or the SNO-Hb is in the deoxy
state.
A nitrosating agent, especially one which can readily enter cells,
especially red blood cells, can be used to treat a mammal with a
disease or medical disorder which can be alleviated by increased
biologically active NO in the affected tissues, such as sepsis,
shock, angina, stroke, reperfusion injury, acute lung injury,
sickle cell anemia, infection of red blood cells, and organ
transplantation. One embodiment of the invention resulting from the
findings herein is a method of therapy that enhances the transfer
of NO.sup.+ from SNO-Hb to low molecular weight thiols, thereby
delivering NO biological activity to tissues, by the
coadminstration of low molecular weight thiols, along with a form
of SNO-Hb, to a mammal in need of the physiological effects of NO,
such as one suffering from the above medical conditions. To further
increase the effect of NO release it is preferred that the SNO-
forms of metHb or deoxyHb (or an equivalent conformation or spin
state) be administered with the thiol (See FIG. 2B, for example.) A
mixture of SNO-metHb and SNO-oxyHb, and possibly also thiol, or
more specifically, an S-nitrosothiol, can also be used. The
composition and proportion of these components depends on the
disease state. For example, in sickle cell anemia, to achieve both
enhanced O.sub.2 delivery and NO delivery, a composition comprising
SNO-oxyHb can be administered. Where no further delivery of O.sub.2
is desirable, as in acute respiratory distress syndrome, for
example, the SNO- forms of metHb and deoxyHb are especially
preferred. Alternatively, the ratios of SNO/Hb can be regulated to
control O.sub.2 release.
A further invention arising out of the discoveries presented herein
is a method for preserving a living organ ex vivo, for example for
transplantation, comprising perfusing the organ with a composition
comprising nitrosated hemoglobin and low molecular weight thiol or
NO donating agent, wherein SNO-Hb(FeII)O.sub.2 is a preferred
nitrosated hemoglobin.
The vessel ring bioassay data of FIG. 4A agree well with the in
vivo data of FIG. 5. The results of the experiments described in
Example 5 confirm that Hb(FeII)O.sub.2 (oxyHb) causes an increase
in blood pressure in vivo, as it did also in vitro. SNO-Hb(FeIII)
(SNO-metHb) causes a decrease in blood pressure in vivo as well as
in vitro. SNO-Hb(FeII)O.sub.2 (SNO-oxyHb) has a negligible effect
on blood pressure in vivo in contrast to the increase in tension
seen in the corresponding vessel ring bioassay. For SNO-oxyHb the
in vivo effect is neutral. This is explained by the constrictive
effect caused by NO becoming bound to the heme being compensated by
the release of NO upon deoxygenation. Therefore, SNO-oxyHb can
deliver O.sub.2 with minimal effect on blood pressure.
With knowledge of the results herein it is possible to synthesize
Hb proteins with predicted NO releasing properties, which will
constrict, dilate, or have no effect on blood vessels. An
additional option is the choice between making oxygenated or
deoxygenated forms to administer for medical conditions in which
O.sub.2 delivery is desirable, or undesirable, respectively.
It is possible to produce a variety of modified Hbs having specific
desired properties of O.sub.2 and NO delivery. For example, Hb in
the R state or R-structure (oxyHb) can be converted to the T state
or T-structure (deoxyHb) by a number of known methods. This can be
done, for example, by reaction of Hb with inositol hexaphosphate.
It is also known to those skilled in the art that Hb in the R state
can be made, for example, by treating Hb with carboxypeptidase.
Similarly, it is known that metHb can be synthesized using
ferricyanide or nitrite.
Producing Hb molecules which are locked in the T state allows the
synthesis of RSNO-Hb which remains in a form that is a biologically
active donor of NO, rather than a carrier of NO. Hb which is locked
in the R state can be used as a substrate for the synthesis of
RSNO-Hb which carries a maximum amount of NO per molecule.
Another embodiment of the invention is a blood substitute
comprising one or more forms of Hb which have been specifically
S-nitrosated to some extent at one or more thiol groups of the Hb,
in order to regulate O.sub.2 release and NO release. Conditions to
be treated include those in which NO or O.sub.2 delivery is
desired, those in which NO or O.sub.2 utilization is desired, or
those in which NO or O.sub.2 is in excess. For example, in a
medical condition which is characterized by the presence of an
excess of oxygen free radicals and excess NO., both the heme of
SNO-Hb and NO released by SNO-Hb serve to trap oxygen free
radicals. The heme Fe is oxidized in the process of scavenging
oxygen free radicals and NO., and NO is released from the oxidized
Hb by donation to a thiol, in the form of RSNO.sup.+, which is not
toxic. Inflammation and reperfusion injury, for example, are
characterized by excess NO production and an excess of oxygen free
radicals. Forms of Hb scavenge oxygen radicals and free NO,
converting NO to forms that are not toxic.
A further embodiment of the invention is a method of therapy for a
condition that would benefit from the delivery of NO in a
biologically active form or O.sub.2 or both, based on the
administration of a blood substitute comprising a form of
nitrosated Hb, such as S-nitrosohemoglobin, either alone or in
combination with a low molecular weight thiol, for example. For
example, SNO-Hb is useful to treat myocardial infarction. SNO-Hb
has the effect of donating NO, keeping blood vessels open. SNO-Hb
deoxygenates at low oxygen tension, delivering oxygen and releasing
NO at the same site, thereby causing vasodilation. (See Example 7
and FIGS. 6A-6F.) These effects can be augmented by also
administering thiol, either simultaneously with SNO-Hb, or before
or after. For the purpose of treating myocardial infarction, for
example, a high concentration or dose of SNO-Hb that has a low
ratio of SNO/SNO-Hb is appropriate. Alternatively, SNO-metHb can be
used for this purpose. A further application of these principals is
a method for increasing cerebral blood flow in a mammal comprising
administrating to the mammal a composition comprising
S-nitrosohemoglobin, as illustrated in FIGS. 23A-23I.
In another aspect, the invention is a method of enhancing NO-donor
therapy by coadministering a composition comprising SNO-Hb or other
forms of nitrosated Hb together with a nitroso-vasodilator
(nitroglycerin, for example) which would be otherwise consumed by
the conversion of oxyHb to metHb in Hb which has not been
S-nitrosated. A composition comprising a low molecular weight thiol
can have the effect of producing a vasorelaxant response in a
mammal (see Example 22 and FIG. 20D). Any of the forms of isolated
Hb described herein can be used in the manufacture of a medicament
for the treatment of medical conditions characterized by
abnormalities of nitric oxide and/or oxygen metabolism, as
appropriate from the effects of the particular form or forms of Hb
included in the medicament.
Platelet activation is manifested by a number of events and
reactions which occur in response to adhesion of platelets to a
nonplatelet surface such as subendothelium. Binding of agonists
such as thrombin, epinephrine, or collagen sets in motion a chain
of events which hydrolyzes membrane phospholipids, inhibits
adenylate cyclase, mobilizes intracellular calcium, and
phosphorylates critical intracellular proteins. Following
activation, platelets secrete their granule contents into plasma,
which then allow the linking of adjacent platelets into a
hemostatic plug. (See pages 348-351 in Harrison's Principles of
Internal Medicine, 12th edition, eds. J. D. Wilson et al.,
McGraw-Hill, Inc., New York, 1991).
A thrombus is a pathological clot of blood formed within the
circulatory system. It can remain attached to its place of origin
or become dislodged and move to a new site within the circulatory
system. Thromboembolism occurs when a dislodged thrombus or part of
a thrombus partially or completely occludes a blood vessel and
prevents oxygen transport to the affected tissues, ultimately
resulting in tissue necrosis.
Sites where damage has occurred to the vascular surface are
especially susceptible to the adherence of platelets and the
formation of thrombi. These sites include those on the interior
surface of a blood vessel in which damage to the endothelium,
narrowing or stenosis of the vessel, or atherosclerotic plaque
accumulation has occurred.
NO is one of several endothelium-derived thromboregulators, which
are defined as physiological substances that modulate the early
phases of thrombus formation. In particular, NO reduces platelet
adhesion, activation and recruitment on the endothelial cell
surface, and achieves this, it is thought, by activating platelet
guanylate cyclase, thereby increasing platelet intracellular CGMP
(Stamler, J. S. et al, Circ. Res. 65:789-795 (1989)), and
decreasing intraplatelet Ca.sup.2+ levels. NO and the prostacylcin
prostaglandin (PG) I.sub.2 act synergistically to inhibit and
actively mediate platelet disaggregation from the collagen fibers
of the subendothelial matrix. Unlike prostacyclin, NO also inhibits
platelet adhesion. Furthermore, platelets synthesize NO, and the
L-arginine-NO pathway acts as an intrinsic negative feedback
mechanism to regulate platelet reactivity. NO is involved in
leukocyte interactions with the vessel wall and can inhibit
neutrophil aggregation. (See review article, Davies, M. G. et al.,
British Journal of Surgery 82:1598-1610, 1995.)
NO is antiathrogenic in a number of ways. (See, for example,
Candipan, R. C. et al., Arterioscler. Thromb. Vasc. Biol. 16:44-50,
1996.) NO inhibits smooth muscle proliferation and attenuates LDL
(low density lipoprotein) oxidation and other oxidant-related
processes.
Hemoglobin may promote atherosclerosis as well as thrombosis as a
consequence of its NO-scavenging property. This limitation of
hemoglobin derives from its high affinity for nitric oxide. In
vitro, NO is a potent inhibitor of platelet aggregation and
adhesion to collagen fibrils, the endothelial cell matrix and
monolayers (Radomski, M. W. et al., Br. J. Pharmacol. 92:181-187
(1987); Radomski, M. W. et al., Lancet 2:1057-1058 (1987); Radomski
M. W. et al., Biochem. Biophys. Res. Commun. 148:1482-1489 (1987)).
NO elevates CGMP levels in platelets, thereby decreasing the number
of platelet-bound fibrinogen molecules and inhibiting intracellular
Ca.sup.++ flux and platelet secretion (Mellion, B. T. et al., Blood
57:946-955 (1981); Mendelson, M. E. et al., J. Biol. Chem.
165:19028-19034 (1990); Lieberman, E. et al., Circ. Res.
68:1722-1728 (1991)). Scavenging of nitric oxide by Hb prevents the
molecule from inhibiting platelets. This explanation has been given
support by in vivo studies (Krejcy, K. et al., Arterioscler.
Thromb. Vasc. Biol. 15:2063-2067 (1995)).
The results shown in FIGS. 7A-7C (see Example 9) show that
nitrosated hemoglobins, including SNO-Hb, can be used in a
therapeutically effective amount, in the treatment of acute blood
clotting events that occur as a result of increased platelet
deposition, activation and thrombus formation or consumption of
platelets and coagulation proteins. Such complications are known to
those of skill in the art, and include, but are not limited to
myocardial infarction, pulmonary thromboembolism, cerebral
thromboembolism, thrombophlebitis, sepsis and unstable angina, and
any additional complication which occurs either directly or
indirectly as a result of the foregoing disorders.
SNO-Hb and other nitrosated hemoglobins can also be used
prophylactically, for example, to prevent the incidence of thrombi
in patients at risk for recurrent thrombosis, such as those
patients with a personal history or family history of thrombosis,
with atherosclerotic vascular disease, with chronic congestive
heart failure, with malignancy, or patients who are pregnant or who
are immobilized following surgery.
NO is known to activate soluble guanylate cyclase, which produces
cGMP. cGMP mediates inhibition of platelet aggregation. Results in
Example 10 demonstrate that this inhibition of platelet aggregation
may be mediated not by cGMP alone, but by some other mechanism as
well.
Certain compounds or conditions are known to cause a shift in the
allosteric equilibrium transition of Hb towards either of the two
alternative quaternary structures of the tetramer, the T- or
R-structures. (See, for example, pages 7-28 in Perutz, M.,
Mechanisms of Cooperativity and Allosteric Regulation in Proteins,
Cambridge University Press, Cambridge, U.K., 1990.) These are, for
instance, the heterotropic ligands H.sup.+, CO.sub.2,
2,3-diphosphoglycerate (2,3-DPG) and Cl.sup.-, the concentrations
of which modulate oxygen affinity. The heterotropic ligands lower
the oxygen affinity by forming additional hydrogen bonds that
specifically stabilize and constrain the T-structure. Other
compounds affecting the allosteric equilibrium include inositol
hexaphosphate (IHP) and the fibric acid derivatives such as
bezafibrate and clofibrate. The fibric acid derivatives,
antilipidemic drugs, have been found to combine with deoxy-, but
not with oxyhemoglobin. They stabilize the T-structure by combining
with sites in the central cavity that are different from the DPG
binding sites. Other allosteric effectors have been synthesized
which are related to bezafibrate. A ligand that stabilizes
specifically the R-structure increases the oxygen affinity, and a
ligand that stabilizes the T-structure does the reverse. Other
ligands can affect the spin state of the heme. For example, in
deoxyhemoglobin and in methemoglobin the Fe is high-spin ferrous
(S=2) and 5-coordinated; in oxyhemoglobin and in cyan-metHb the Fe
is low-spin ferrous (S=0) and 6-coordinated; when H.sub.2 O is the
sixth ligand, methemoglobin is also high-spin. The inhibition of
platelet aggregation by S-nitroso-methemoglobin seen in FIG. 7C is
consistent with enhanced potency in the high spin conformation.
Substances which control the allosteric equilibrium or spin state
of hemoglobin can be administered in a pharmaceutical composition
to a human or other mammal, in a therapeutically effective amount,
to promote the formation of, or to stabilize, a particular
allosteric structure and/or spin state of hemoglobin, thereby
regulating platelet activation, e.g., by converting hemoglobin from
R-structure to T-structure.
The dosage of Hb required to deliver NO for the purpose of platelet
inhibition can be titrated to provide effective amounts of NO
without causing drastic changes in blood pressure. If the goal of
the therapy is to deliver oxygen, the Hb can be administered in a
unit of blood to avoid a drop in blood pressure. If the goal is to
alleviate shock, very little Hb can be administered compared to the
amount to be given for myocardial infarction. For shock, the more
important goal is to deliver NO rather than to deliver oxygen. For
this objective, it can be preferable to use continuous infusion or
several infusions per day. Example 12 (see FIG. 10) shows that the
effects of SNO-Hb(FeII)O.sub.2 on blood flow in rat brain last over
20 minutes; in other experiments an effect has been seen for up to
an hour. There is a correlation between blood pressure effects and
platelet inhibition effects, but platelet inhibition occurs at a
lower NO concentration than that which is required to produce blood
pressure effects, and generally lasts longer.
Example 11 shows that S-nitrosothiols can be used to add NO groups
not only on the thiol groups of cysteine residues in hemoglobin,
but also on other reactive sites of the hemoglobin molecule. The
products of the nitrosation reactions in Example 11 were hemoglobin
molecules with more than 2 NO groups per Hb tetramer. The exact
sites of the addition of NO have not been confirmed, but it is
expected that NO addition occurs at thiol groups and various other
nucleophilic sites within Hb, including metals. Reactive sites,
after the thiol groups, are tyrosine residues and amines, and other
nucleophilic centers.
Nitrosation reactions on other proteins have been investigated
previously (Simon, D. I. et al., Proc. Natl. Acad. Sci. USA
93:4736-4741 (1996)). Methods of modifying proteins to produce
nitrosoproteins are known in the art, and include, for example,
exposing the protein to NaNO.sub.2 in 0.5 M HCl (acidified
NO.sub.2.sup.-) for 15 minutes at 37.degree. C. An alternative
method is to place a helium-deoxygenated solution of protein in 100
mM sodium phosphate, pH 7.4, inside dialysis tubing and expose the
protein to NO gas bubbled into the dialysate for 15 minutes.
(Stamler, J. S. et al., Proc. Natl. Acad. Sci. USA 89:444-448
(1992); see also Williams, D. L. H. Nitrosation, Cambridge
University Press, New York (1988), which gives further methods of
nitrosation).
By these methods, multiple NO-related modifications ("NO groups" or
37 NO biological equivalents" resulting from nitrosations,
nitrosylations or nitrations) can be made on Hb at nucleophilic
sites, which can include thiols, nucleophilic oxygen atoms as found
in alcohols, nucleophilic nitrogen atoms as found in amines, or the
heme iron. Agents which can be contacted with hemoglobin to
facilitate nitrosations, nitrosylations or nitrations of Hb can be
thought of as "NO or NO.sup.+ donating agents." The products of
such modifications may have such groups, for example, as --SNO,
--SNO.sub.2, --ONO, ONO.sub.2, --CNO, --CNO.sub.2, --NNO,
--NNO.sub.2, --FeNO, --CuNO, --SCuNO, SFeNO and the different
ionized forms and oxidation variants thereof. (See, regarding
oxidation of hemoglobin by Cu.sup.++, Winterboume, C., Biochemistry
J. 165:141-148 (1977)). The covalent attachment of the NO group to
sulfhydryl residues in proteins is defined as S-nitrosation; the
covalent attachment of the NO group to a metal, such as Fe, can be
called nitrosylation, yielding a metal-nitrosyl complex. General NO
attachment to nucleophilic centers is referred to herein as
nitrosation. Thus, the term nitrosated hemoglobin as used herein
includes SNO-Hb and Hb(FeII)NO as well as other forms of hemoglobin
nitrosated at other sites in addition to thiols and metals. In
addition, Hb can be nitrated. Compositions comprising Hbs which
have been nitrosated and/or nitrated by a nitric oxide donating
compound at multiple different types of nucleophilic sites (termed
polynitrosated, that is, having NO equivalents added to other
nucleophilic sites as well as to thiols; or polynitrated,
respectively) will permit transnitrosation reactions and the
release of NO and its biological equivalents in the circulatory
system at different rates and engendering different potencies.
Polynitrosated or polynitrated hemoglobins can be reacted with a
reagent which selectively reduces FeIII to FeII (for example,
cyanoborohydride or methemoglobin reductase), if it is desired to
reduce heme Fe that may have been oxidized.
These and other nitrosation and nitration reactions can cause
oxidation of the heme Fe to some extent, under some conditions.
However, some minor degree of oxidation is acceptable. The
nitrosated Hb is still be useful as a therapeutic agent if oxidized
to a minor extent. For applications where the NO-delivering
function, rather than the O.sub.2 -delivering function of
nitrosated Hb, is more desirable, extensive oxidation of the heme
Fe is acceptable.
If it is desired to avoid oxidation of the heme Fe, it is possible
to remove the heme, perform the necessary chemical reactions upon
the protein to nitrosate to the extent desired, and replace the
heme into the modified hemoglobin product. (See, for removing and
replacing the heme, Antonini, E. and Brunori, M., Hemoglobin and
Myoglobin in their Reactions with Ligands, Elsevier, New York,
1971.)
In addition to the nitrosating under conditions that do not oxidize
the heme, such as brief exposure to low molecular weight RSNOs, as
illustrated in Examples 1 and 2, alternative methods can be used to
produce nitrosated hemoglobin in which the heme Fe is not oxidized.
For instance, it is possible to produce by recombinant methods
.alpha. and .beta. globin chains, nitrosate them to the extent
desired, then assemble the chains with heme to form a functional,
nitrosated tetramer. (See, for example, European Patent Application
EPO 700997, published Mar. 13, 1996, "Production in bacteria and
yeast of hemoglobin and analogues thereof.")
Another alternative method to nitrosate the .alpha. and .beta.
globin chains without producing a form of metHb as the end product,
is to nitrosate the intact Hb molecule to the extent desired,
thereby allowing the heme Fe to be oxidized, then reduce the heme
Fe from FeIII to FeII by treating the nitrosated Hb with either
methemoglobin reductase or a cyanoborohydride such as sodium
cyanoborhydride.
It has been generally thought that nitric oxide as NO gas in
solution reacts with hemoglobin (Hb) in two major ways: 1) with the
deoxyHb to form a stable nitrosyl (FeII) heme adduct; and 2) with
oxyHb to form nitrate and metHb--a reaction that inactivates NO.
These two reactions contributed to the idea that Hb is a scavenger
of NO. In both of these reactions, NO biological activity is lost.
The results described herein demonstrate that, in fact, neither
reaction occurs under physiological conditions. Rather, the
products of the NO/Hb reaction are dictated by the ratio of NO to
Hb, and by the conformation of Hb--R(oxy) vs. T(deoxy).
At low ratios of NO to deoxyHb (e.g., 1:100 or less), the Hb
molecule is in T-structure. Under this condition, NO introduced as
gas to a Hb solution binds to the .alpha.-hemes, as has been seen
by EPR. Upon introduction of oxygen, with conversion to the R
state, NO is transferred to a thiol of cysteine to yield
S-nitrosohemoglobin with close to 100% efficiency. At ratios of
NO/Hb of 1:25-1:50, the efficiency of formation of SNO-Hb is
.about.35% (decreasing with increasing NO/Hb ratio). The reaction
appears to involve migration of NO from a heme to .beta. heme and
then to the .beta. thiol. In going from the heme to a thiol, the
heme or nitrosothiol needs to lose an electron by oxidation
(NO.fwdarw.NO.sup.+ or RSNO..fwdarw.RSNO). Oxygen serves as an
electron acceptor in the system, driving the reaction
thermodynamically, as well as causing a conformational change by
its binding at the heme, which exposes the thiol groups. At higher
ratios of NO to Hb (1:20-1:2), with the protein still in
T-structure, the protein liberates NO.sup.- from the .beta. hemes
with production of metHb. This occurs in the absence of O.sub.2 and
provides another indication that the NO bound to .beta.-hemes is
unstable. Once O.sub.2 is introduced, S-nitrosothiol (SNO) forms,
but the relative yield is very low because of loss to NO.sup.-. The
yield of SNO-Hb approaches zero at NO/Hb ratios of 1:2, upon
introduction of oxygen.
At the higher ratios of NO to Hb (i.e., >0.75-1), NO itself
maintains the R-structure. Under this condition, the NO is more
stable because of an unusual constraint on the molecule.
Specifically, loss of NO from the .beta.-hemes promotes the
T-structure, whereas formation of SNO-Hb selects for the
R-structure. This is not a favored reaction. The consequence is
that small amounts of S-nitrosohemoglobin are formed, but the
yields are low (.about.5%). This does not exclude the possibility
that the molecule has therapeutic value.
The reaction of NO with oxyHb is also dependent on the ratios of NO
to oxyHb. Under conditions of relatively high (non-physiological)
ratios of NO to Hb, (NO/oxyHb >1:20), NO appears to destabilize
the hydrogen bond between the O.sub.2 and the proximal histidine
(by competing for it) yielding some metHb. By changing the ionic
composition of the solvent buffer (e.g., borate 0.2 M, pH 7.4),
formation of metHb can be significantly reduced even with excess NO
(NO/Hb=3:1). On the other hand, metHb formation is facilitated in
acetate buffer at pH 7.4; when the hydrogen bond between O.sub.2
and the proximal histidine is broken, the O.sub.2 seems to gain
superoxide-like character. NO then reacts rapidly to form metHb and
nitrate. Efficient metHb formation actually requires an excess of
NO/oxyHb. In contrast, at lower ratios of NO/Hb (<1:20), it
reacts with the small residual fraction (<1%) of deoxyHb, in
turn producing S-nitroso-hemoglobin extremely efficiently. As the
concentration of NO is increased, there is some reaction with
oxyHb, but the products are nitrite and nitrate, not nitrate alone.
The conclusion is that NO can be incubated in reaction mixtures of
oxyHb without inactivating the O.sub.2 binding functionality by
converting it to nitrate.
Nitrosylhemoglobin can be used in an animal or human as a
therapeutic NO donor for the prevention or treatment of diseases or
medical disorders which can be alleviated by delivery of NO or its
biologically active form to tissues affected by the disease or
medical disorder. Like SNO-Hb, nitrosylhemoglobin can be
administered as a blood substitute, because nitrosylhemoglobin can
be converted to SNO-Hb under physiological conditions. NO is
released from the thiol either by deoxygenation or by conversion to
metHb.
An illustration of nitric oxide reactions with hemoglobin in the
respiratory cycle is presented in FIG. 26. The upper panel in FIG.
26 shows alternative reactions proposed for .beta.-chain nitrosyl
hemes in the T structure. (1) Transfer of NO to the .alpha.-chain
heme irons (this is likely to occur mainly in the microcirculation
and venous system; (2) charge-transfer reaction at the heme iron to
produce methemoglobin and nitroxyl anion (this is more likely to
occur in the microcirculation and venous system when NO synthesis
is high); and (3) NO group exchange with .beta.-Cys93 mediated by
the oxygen-driven allosteric transition to the R structure, forming
SNO-Hb(FeII)O.sub.2 (this seems to occur in the lung, but may also
happen in oxygenated arterial blood). See Example 15, for instance.
Squares, circles and diamonds represent hemes in the T structure, R
structure and met state, respectively.
The lower panel of FIG. 26 shows a model of NO binding to hemes and
thiols of Hb in the circulation. Partially nitrosylated venous
blood enters the lungs in the deoxy or T structure (square)
(presumably maintained by .alpha.-chain binding of NO to hemes and
CO.sub.2 to amines). Specifically, NO can be detected in large
veins as .alpha.(5- and 6- coordinate) nitrosyl heme (Westenberger,
U. et al., Free Rad. Res. Commun. 11:167-178 (1990); Hall, D. M. et
al., J. Appl. Physiol. 77:548-553 (1994); Kosaka, H. et al., Am. J.
Physiol. 266:1400-1405 (1994); and Kagan, V. E. et al., Nature
383:30-31 (1996)), some of which is found in the fraction of blood
that is fully deoxygenated. In the lungs, partially nitrosylated
(carbamino) Hb is exposed to more NO and O.sub.2 tensions
(PO.sub.2) that appear to couple the allosteric transition with NO
group exchange from hemes to .beta.-chain thiols. (For example, see
Examples 24 and 25.) Oxygen serves both to position the .beta.
thiol close to the .beta. heme (Riggs, A. and Wolbach, R. A., J.
Gen Physiol. 39:585-605 (1956)) and thermodynamically to drive the
redox mediated formation of SNO. Accordingly, blood entering the
arterial circuit contains SNO-oxy Hb, that is, Hb in the R
structure (circle) with NO attached to .beta.-Cys93 and O.sub.2 to
the hemes. Low-molecular-mass SNOs present in the lung and arterial
blood will further support SNO-Hb formation by transnitrosation of
R-structure molecules (Example 1). Blood moving into resistance
vessels that control blood pressure and blood flow to tissues is
then exposed to low pO.sub.2 which promotes the T structure in
SNO-Hb and effects NO group release. Some NO will exchange with
low-relative-mass thiols to dilate blood vessels and some will be
autocaptured at the hemes (.beta..fwdarw..alpha.; just as some
endothelial-derived NO is inevitably sequestered by the hemes; see
Example 3). NO oxidation of heme irons (metHb formation) will also
enhance the vasodilator function of SNO-Hb. For instance, see
Example 4. As O.sub.2 delivery is a function of blood flow, the
R.fwdarw.T transition in Hb (and perhaps metHb formation) is
designed to maximize oxygen delivery. Hb can further bind NO at the
heme irons as it progresses through the venous system; the more NO
that binds, the greater the propensity to form metHb and hemoglobin
X [Hb(.alpha.FeIIINO)(.beta.FeIII)]. It is not known whether the
endogenous level of .about.0.1% nitrosyl Hb, which should promote
the T structure in Hb, is sufficient to enhance O.sub.2 delivery,
but higher levels found in endotoxic shock (Kosaka, H. et al., Am.
J. Physiol. 266:1400-1405 (1994)) may do so.
Inhaled NO causes selective pulmonary vasodilation without
influencing systemic responses. A previously-formed rationale
behind its use is that scavenging by Hb prevents adverse systemic
effects. It is illustrated in Examples 14-21 that NO can be used to
produce S-nitrosohemoglobin, which is a potent vasodilator and
antiplatelet agent. Inhaled NO can be used to raise levels of
endogenous S-nitrosohemoglobin. Similarly, treatment of red blood
cells (RBCs) with NO can be used to form SNO-RBCs, or "loaded" red
blood cells.
Compared to SNO-deoxyHb, which is a good NO donor, but which would
release its NO very quickly, or SNO-oxyHb, which would release its
NO more slowly, but has a propensity to form metHb over time,
nitrosyl-deoxyhemoglobin stored in a form such that final ratio of
NO: heme is less than about 1:100 or greater than about 0.75, is
stable. Formation of metHb is prevented at these NO: heme ratios.
For this reason nitrosyl-deoxyhemoglobin stored with such NO: heme
ratios in a physiologically compatible buffer can be administered
to an animal or human as an NO donor. Erythrocytes comprising
nitrosylhemoglobin can also be used as NO donors. Erythrocytes
comprising nitrosylhemoglobin can be made in a process comprising
incubating deoxygenated erythrocytes in a solution comprising
NO.
A blood substitute or therapeutic which can be used as an NO donor,
and which is free of the vasoconstrictor effects of underviatized
Hbs, can be made by obtaining a solution of oxyHb (including
solutions stored in the form of oxyHb) and adding NO as dissolved
gas, yielding SNO-oxyHb. Buffer conditions and NO: Hb ratios can be
optimized, as illustrated in Example 21 and FIG. 19, to yield
S-nitrosothiol without significant production of oxidized Hb
(metHb). For example, NO added to oxyhemoglobin in 10 mM phosphate
buffer, pH 7.4, at a ratio of less than 1:30 NO: Hb resulted in
formation of SNO-oxyHb with minimal formation of metHb. This ratio
can be increased by varying the buffer conditions, for example by
the use of 10 mM phosphate, 200 mM borate at pH 7.4. The buffer
anions as well as the buffer concentration should be chosen
carefully. For instance, acetate and chloride have the opposite
effect from borate, increasing the formation of metHb and nitrite
at 200 mM, pH 7.4.
This can be explained by a competition between free NO and oxygen
for a H-bond with the imidazole of the proximal His residue. If low
concentrations of NO are used, in low ionic strength buffer, e.g.,
10 mM phosphate, metHb does not readily form. If the H-bond is
weakened by increasing the ionic strength of the buffer, NO reacts
more readily with oxyHb, yielding more metHb. Buffers with a low
pK.sub.a relative to pH 7.4 tend to stabilize FeIII. Buffers having
a pK.sub.a at least about two pH units higher than the reaction
condition are preferred.
A blood substitute can be made which acts as a donor of NO.sup.-.
NO can be added to a solution of deoxyHb at a ratio of NO: Hb in
the range of 1:100 to 1:2, with a ratio of NO/heme of approximately
1:10 being preferred. If the ratio of NO:heme is increased, to a
NO: Hb of about 2 (at which Hb is still in the T (deoxy) state), in
the absence of an electron acceptor/free radical scavenger, NO is
released from the .beta. heme as NO.sup.-, with oxidation of the
heme iron to form metHb. The product solution can be used as a
blood substitute or a therapeutic NO donor. NO.sup.- can protect
from N-methyl-D-glutamate-mediated brain injury in stroke; this
effect has not been found for NO.
Nitrosylhemoglobin belongs to a broader class of
nitrosyl-heme-containing donors of NO which can be administered to
an animal or human for the delivery of NO to tissues.
Nitrosyl-heme-containing donors of NO include, for example, the
nitrosated ("nitrosated" as defined herein) hemoglobins
nitrosylhemoglobin and SNO-nitrosylhemogobin, nitrosyl-heme, and
substituted forms of hemoglobin in which a different metal, (e.g.,
Co.sup.++, Mg.sup.++, Cu.sup.++) is substituted for the heme iron,
or nitrosyl-porphyrins are substituted for the heme.
Applicants teach physiologically significant results that provide a
rationale for NO donors to be attached to Hb. Such derivatized Hbs
can themselves serve as NO-donating therapeutics and can ameliorate
the side effects of underivatized Hb administered as a blood
substitute, for example. At one time, it had been thought that
there would be no use for these compounds, because it was thought
that NO released by the Hb would immediately be scavenged by the
heme. It had been thought also that the released NO would oxidize
Hb and limit oxygen delivery. The same rationale has previously
limited the administration of NO donors, such as nitroglycerin and
nitroprusside, because they had been thought to cause the formation
of metHb.
Preferably, NO-donors to be covalently attached to hemoglobin are
relatively long-lived and have at least one functional group that
can be used for the chemical attachment to hemoglobin. Examples of
NO-donors include nitroprusside, nitroglycerin, nitrosothiols, and
the diazeniumdiolte class of compounds (also called "NONOates")
having structure 1. ##EQU1##
A variety of these compounds have been synthesized that, in their
anionic form, release NO without activation at physiological pH
(Keefer, L. K. et al., Am. Chem. Soc. Symposium Ser. 553:136-146
(1994); Hanson, S. R. et al., Adv. Pharmacol. 34:383-398 (1995)).
Systemic administration can result in system-wide effects,
according to equation 1. However, attachment to hemoglobin can be
used to produce tissue-selective delivery of NO and oxygen. For
instance, covalently esterifed NO-donors can be activated
predominantly in the liver. Different NO donors can be chosen to be
linked to hemoglobin for different controlled release rates of NO
from Hb.
Compound 4 (below), for example, is a diazeniumdiolate with a
half-life for NO release, at 37.degree. C. and pH 7.4, of
approximately two weeks. It can be converted to its nucleophilic
N-4 mercaptoethyl derivative, compound 5. Hemoglobin can be
activated toward coupling reactions by reacting it with
.gamma.-maleimidobutyric acid N-hydroxysuccinimide ester. Compound
5 can then be covalently attached to the activated hemoglobin
through its maleimide functionality. The adduct, 6, can generate NO
steadily over several days in pH 7.4 phosphate buffer at 37.degree.
C. This property can alleviate side effects of underivatized blood
substitutes, for example. ##STR1##
Nitric oxide synthase (NOS) working in conjunction with Hb can
reload NO onto the hemes, thus a composition comprising NOS and Hb,
or NOS conjugated to Hb can facilitate delivery of NO to the
tissues. NOS of neurons is preferable for this composition because
the neuronal NOS responds to oxygen tension. At low oxygen tension,
the neuronal NOS produces more NO; at high oxygen tension, NOS
produces less NO. This form of NOS will efficiently reload NO onto
the heme when Hb is deoxygenated. NOS-Hb conjugates can be used
when a blood substitute is indicated, and especially when an
ischemic injury or condition is present.
Biologically compatible electron acceptors, are well known in the
art and include, but are not limited to, superoxide dismutase and
the oxidized forms of nicotinamide adenine dinucleotide
(NAD.sup.+), nicotinamide adenine dinucleotide phosphate
(NADP.sup.+), flavin adenine dinucleotide (FAD), flavin
mononucleotide (FMN), ascorbate, dehydroascorbate and nitroxide
spin traps. One or more electron acceptors can be conjugated to Hb
molecules, and can facilitate the conversion of the
nitrosyl-Hb-electron acceptor form to the SNO-Hb-electron acceptor
form by accepting the electron lost by NO in its transfer, in the
form of NO.sup.+ or as RSNO., to a .beta.93Cys thiol group.
Nitroxides are one such class of electron acceptors which also act
as free radical scavengers. Nitroxides are stable free radicals
that have been shown to have antioxidant catalytic activities which
mimic those of superoxide dismutase (SOD), and which when existing
in vivo, can interact with other substances to perform
catalase-mimic activity. Nitroxides have been covalently attached
to hemoglobin. See Hsia, J-C., U.S. Pat. No. 5,591,710, the
contents of which are incorporated by reference in their entirety.
See also Liebmann, J. et al., Life Sci. 54:503-509 (1994),
describing nitroxide-conjugated bovine serum albumin and
differential nitroxide concentrations among the different organs of
mice tested with the conjugate.
Methods for chemically attaching superoxide dismutase (SOD) to Hb
are known in the art. For example, see Quebec, E. A. and T. M.
Chang, Artif. Cells Blood Substit. Immobil. Biotechnol. 23:693-705
(1995) and D'Agnillo, F. and Chang, T. M., Biomater. Artif. Cells
Immobilization Biotechnol. 21:609-621 (1993). SOD attached to
nitrosylhemoglobin can drive the reaction in which NO is
transferred from the heme to thiol, by serving as an electron
acceptor.
Like NO, CO is known to have vasodilator effects. (See Zakhary, R.
et al., Proc. Natl. Acad. Sci USA 93:795-798 (1996).) A solution of
deoxyhemoglobin can be derivatized with CO by exposing it to
purified CO gas in solution, until the desired extent of CO-bound
Hb is reached. CO-derivatized Hb can be administered as a blood
substitute or co-administered with other heme-based blood
substitutes to alleviate the effects (e.g., hypertension,
intestinal pain and immobility) of underivatized hemoglobin.
Hemoglobins can be derivatized to the extent necessary to overcome
constrictor effects, for example to a ratio of CO/Hb in the range
of approximately 0.1% to 10%.
Because the .alpha. subunits lack thiol groups to serve as NO.sup.+
acceptors from the heme, a blood substitute comprising .alpha.
chains, for example in the form of dimers or tetramers, can be made
which has different properties from a blood substitute comprising
.beta. chains alone, or comprising a combination of .alpha. and
.beta. chains. A blood substitute comprising .alpha. chains of
hemoglobin can be administered to an animal or to a human patient
to alleviate a condition characterized by the effects caused by NO,
for example, in hypotensive shock.
.beta. chains, unlike .alpha. chains, serve as active donors of NO
to the tissues, rather than traps for NO. A blood substitute
comprising .beta. chains, for example in the form of .beta. dimers
or tetramers, can be made. Such a blood substitute can be
administered to a mammal to treat diseases or medical disorders
wherein it is desired to deliver oxygen as well as NO or its
biological equivalent to tissues affected by the disease, for
example, in angina and other ischemic conditions.
Methods are known by which hemoglobin can be separated into its
.alpha. and .beta. subunits and reconstituted. Separated,
heme-free, alpha- and beta-globins have been prepared from the
heme-containing alpha and beta subunits of hemoglobin. (Yip, Y. K.
et al., J. Biol. Chem. 247:7237-7244 (1972)). Native human
hemoglobin has been fully reconstituted from separated heme-free
alpha and beta globin and from hemin. Preferably, heme is first
added to the alpha-globin subunit. The heme-bound alpha globin is
them complexed to the heme-free beta subunit. Finally, heme is
added to the half-filled globin dimer, and tetrameric hemoglobin is
obtained (Yip, Y. K. et al., Proc. Natl. Acad. Sci. USA 74:64-68
(1997)).
The human alpha and beta globin genes reside on chromosomes 16 and
11, respectively. Both genes have been cloned and sequenced,
(Liebhaber, et al., Proc. Natl. Acad. Sci. USA 77:7054-7058 (1980)
(alpha-globin genomic DNA); Marotta, et al., J. Biol. Chem.
252:5040-5053 (1977) (beta globin cDNA); Lawn, et al., Cell 21:647
(1980) (beta globin genomic DNA)).
Recombinant methods are available for the production of separate
.alpha. and .beta. subunits of hemoglobin. For instance, Nagai and
Thorgerson, (Nature 309:810-812 (1984)) expressed in E. coli a
hybrid protein consisting of the 31 amino terminal residues of the
lambda cII protein, an IIe-Glu-Gly-Arg linker, and the complete
human beta globin chain. They cleaved the hybrid immediately after
the linker with blood coagulation factor Xa, thus liberating the
beta-globin chain. Later, (Nagai, K. et al., Proc. Natl. Acad. Sci.
USA 82:7252-7255 (1985)) took the recombinant DNA-derived human
beta globin, naturally derived human alpha globin, and a source of
heme and succeeded in producing active human hemoglobin.
An efficient bacterial expression system for human alpha globin was
reported. (GB 8711614, filed May 16, 1987; see also WO 88/09179).
This led to the production of wholly synthetic human hemoglobin by
separate expression of the insoluble globin subunits in separate
bacterial cell lines, and in situ refolding of the chains in the
presence of oxidized heme cofactor to obtain tetameric hemoglobin.
A synthetic human hemoglobin has been produced in yeast cells (EP
700997A1, filing date Oct. 5, 1990).
The properties of hemoglobin have been altered by specifically
chemically crosslinking the alpha chains between the Lys99 of alpha
1 and the Lys99 of alpha 2. (Walder, U.S. Pat. Nos. 4,600,531 and
4,598,064; Snyder, et al., Proc. Natl. Acad. Sci USA 84:84
7280-7284 (1987); Chaterjee, et al., J. Biol. Chem. 261:9927-9937
(1986)). This chemical crosslinking was accomplished by reacting
bis (3,5-dibromosalicyl) fumarate with deoxyhemoglobin A in the
presence of inositol hexaphosphate. The beta chains have also been
chemically crosslinked. (Kavanaugh, M.P. et al., Biochemistry
27:1804-1808 (1988)). Such linking methods or other suitable
methods can be adapted to methods of producing .alpha. or .beta.
dimers or other multimers, or for the crosslinking of other
polypeptides to the .alpha. and .beta. chains. (For further methods
to derivatize proteins and to conjugate proteins, see Hermansoh, G.
T., Bioconjugate Techniques, Academic Press, 1996.)
The term hemoglobin or Hb as used herein includes variant forms
such as natural or artificial mutant forms differing by the
addition, deletion and/or substitution of one or more contiguous or
non-contiguous amino acid residues, or modified polypeptides in
which one or more residues is modified, and mutants comprising one
or more modified amino acid residues. Hb also includes chemically
modified forms as well as genetically altered forms, such as fusion
proteins, and truncated forms. It also includes Hbs of all animal
species and variant forms thereof. The biological and/or chemical
properties of these variant Hbs can be different from those of
hemoglobins which are found naturally occurring in animals.
It will be appreciated that NO exists in biological systems not
only as nitric oxide gas, but also in various redox forms and as
biologically active adducts of nitric oxide such as
S-nitrosothiols, which can include S-nitrosoproteins,
S-nitroso-amino acids and other S-nitrosothiols (Stamler, J. S.
Cell 78:931-936 (1994)).
A blood substitute can be a biologically compatible liquid which
performs one or more functions of naturally occurring blood found
in a mammal, such as oxygen carrying and/or delivery, NO carrying
and/or delivery, and the scavenging of free radicals. A blood
substitute can also comprise one or more components of such a
liquid which, when infused into a mammal, perform one or more
functions of naturally occurring blood. Examples of blood
substitutes include compositions comprising one or more forms of
hemoglobin. Such compositions can also include other biologically
active components, such as a low molecular weight thiol,
nitrosothiol or NO donating agents, to allow transnitrosation. Low
molecular weight thiols (i.e., relative to proteins and other
biological macromolecules) can include glutathione, cysteine,
N-acetylcysteine, S-nitrosocysteinylglycine, S-nitrosocysteine, and
S-nitrosohomocysteine.
The compounds and therapeutic compositions of this invention to be
used in medical treatment are intended to be used in
therapeutically effective amounts, in suitable compositions, which
can be determined by one of skill in the art. Modes of
administration are those known in the art which are most suitable
to the affected site or system of the medical disorder. Intravenous
infusion is a preferred mode of administration of various forms of
hemoglobin to be used as a blood substitute. Suitable compositions
can include buffers, carriers, stabilizers or inert ingredients
known to those of skill in the art, along with biologically active
components(s).
The term "therapeutically effective amount," for the purposes of
the invention, refers to the amount of modified Hb and/or
nitrosating agent which is effective to achieve its intended
purpose. While individual needs vary, determination of optimal
ranges for effective amounts of each compound to be administered is
within the skill of one in the art. Research animals such as dogs,
baboons or rats can be used to determine dosages. Generally,
dosages required to provide effective amounts of the composition or
preparation, and which can be adjusted by one of ordinary skill in
the art, will vary, depending on the age, health, physical
condition, sex, weight, extent of disease of the recipient,
frequency of treatment and the nature and scope of the desired
effect. Dosages for a particular patient can be determined by one
of ordinary skill in the art using conventional considerations,
(e.g. by means of an appropriate, conventional pharmacological
protocol). For example, dose response experiments for determining
an appropriate dose of a heme-based blood substitute can be
performed to determine dosages necessary to produce a physiological
concentration of approximately 1 nM to 100 .mu.M heme. Suitable
pharmaceutical carriers or vehicles can be combined with active
ingredients employed in a therapeutic composition, if
necessary.
The present invention is further and more specifically illustrated
in the following examples, which are not intended to be limiting in
any way.
Exemplification
Materials and Methods for Assays
Determination of R--S--NO Concentration (Standard Saville
Method)
The concentration of R--S--NO groups in a sample is based on the
method reported in Saville, Analyst 83:670-672 (1958). The
quantification of the NO group, displaced from the thiol by
mercuric ion, forms the basis of this highly sensitive method. The
detection limit is in the range of 0.1-0.5 .mu.M.
2RSNO+Hg.sup.2+.fwdarw.Hg(RS).sub.2 +2NO.sup.- (5)
As shown (equations 5-7), the reaction proceeds in two steps.
First, NO.sup.+ is displaced from the RSNO by mercuric ion and
reacts, under acidic conditions, with sulfanilamide (Ar--NH.sub.2).
In a second step, the diazonium salt (which is formed in amounts
equivalent to the thionitrite) is then coupled with the aromatic
amine, N-(1-naphthyl)-ethylenediamine (Ar'), to form an intensely
colored azo dye which can be measured at 540 nm
(.epsilon..about.50,000 M.sup.-1 cm.sup.-1). The same assay
performed with the mercuric salt omitted allows for the
simultaneous detection of nitrite. In principle, the second part of
the Saville procedure is analogous to the classical Griess reaction
for the detection of nitrite.
The procedure is as follows: Solution A: sulfanilamide 1% dissolved
in 0.5 M HCl. Solution B: same solution as used in A to which 0.2%
HgCl.sub.2 Solution C: 0.02% solution of
N-(1-naphthyl)-ethylenediamine dihydrochloride dissolved in 0.5 M
HCl.
A given volume (50 .mu.l-1 ml) of the sample to be assayed is added
to an equivalent volume of solution A and solution B. The two
samples are set aside for 5 minutes to allow formation of the
diazonium salt, after which an equivalent volume of solution C is
added to each mixture. Color formation, indicative of the azo dye
product, is usually complete by 5 minutes. The sample absorbance is
then read spectrophotometrically at 540 nm. The RSNO is quantified
as the difference in absorbance between solution B and A. (i.e.
B--A). In the event that the background nitrite concentration is
high (i.e. increased background in A), the accuracy of the
measurement can be increased by the addition of an equivalent
volume of 0.5% ammonium sulfamate in acid (45 mM) 5 minutes prior
to the addition of sulfanilamide. The nitrous acid in solution
reacts immediately with excess ammonium sulfamate to form nitrogen
gas and sulfate.
Concentrations of thiol greater than 500 .mu.M in samples may
interfere with the assay if nitrite is also present at micromolar
concentration. Because nitrite will nitrosate indiscriminately
under the acidic conditions employed, thiols will effectively
compete for reaction with sulfanilamide (present at 50 mM in this
assay) as their concentration approaches the millimolar range. This
will lead to artifactual detection of RSNO. The problem can be
avoided by (1) keeping the ratio of thiol to sulfanilamide
<0.01, (2) first alkylating thiols in the solution, or (3)
adding free thiols to standards to correct for the potential
artifact.
Assay for S-Nitrosohemoglobin and Nitrosyl(FeII)-Hemoglobin
A highly sensitive photolysis-chemiluminescence methodology was
employed, which had been used for measuring RSNOs (S-nitrosothiols)
in biological systems (Gaston, B., et al., Proc. Natl. Acad. Sci.
USA 90:10957-10961 (1993); Stamler, J. S., et al., Proc. Natl.
Acad. Sci USA 89:7674-7677(1992)). The method involves photolytic
liberation of NO from the thiol, which is then detected in a
chemiluminesence spectrometer by reaction with ozone. The same
principle of operation can be used to cleave (and measure) NO from
nitrosyl-metal compounds (Antonini, E. and Brunori, M. In
Hemoglobin and Myoglobin in Their Reactions with Ligands, American
Elsevier Publishing Co., Inc., New York, pp. 29-31 (1971)). With
adjustment of flow rates in the photolysis cell, complete
photolysis of the NO ligand of Hb(FeII)NO is achieved. Standard
curves derived from synthetic preparations of SNO-Hb, Hb(FeII)NO,
and S-nitrosoglutathione were linear (R>0.99), virtually
superimposable, and revealing of sensitivity limits of
approximately 1 nM. Two analytical criteria were then found to
reliably distinguish SNO-Hb from Hb(FeII)NO: 1) signals from SNO-Hb
were eliminated by pretreatment of samples with 10-fold excess
HgCl.sub.2, while Hb(FeII)NO was resistant to mercury challenge;
and 2) treatment of SNO-Hb with HgCl.sub.2 produced nitrite (by
standard Griess reactions) in quantitative yields, whereas similar
treatment of Hb(FeII)NO did not. UV/VIS spectroscopy confirmed that
NO remained attached to heme in the presence of excess
HgCl.sub.2.
We linked a photolysis cell directly to the reaction chamber and
detector portion (bypassing the pyrolyzer) of a chemiluminescence
apparatus (model 543 thermal energy analyzer, Thermedix, Woburn,
Mass.). A sample (5 to 100 .mu.l) is either introduced directly or
introduced as a chromatographic effluent from an attached
high-performance liquid or gas chromatography system into the
photolysis cell (Nitrolite, Thermedix, Woburn, Mass.). This cell
consists of a borosilicate glass coil (3 m.times.0.64 cm
o.d..times.1 mm i.d., turned to a diameter of 6 cm and a width of
12 cm). The sample is introduced with a purge stream of helium (5
liters/min) and then irradiated with a 200-W mercury-vapor lamp
(vertically mounted in the center of the photolysis coil on Teflon
towers). The effluent from the photolysis coil is directed to a
series of cold traps, where liquid and gaseous fractions less
volatile than nitric oxide (such as nitrite and nitrate) are
removed. Nitric oxide is then carried by the helium stream into the
chemiluminescence spectrometer, in which free nitric oxide is
detected by reaction with ozone. Signals are recorded on a digital
integrator (model 3393A, Hewlett-Packard). Flow rates and
illumination levels in the photolysis cell were designed to result
in complete photolysis of the S--N bond of S-nitrosothiols, as
confirmed by analysis of effluent from the cell according to the
method of Saville (Saville, B., Analyst 83:670-672 (1958)).
To determine what fraction of the total nitric oxide detected in
samples was derived from S-nitrosothiols, several control
measurements were performed. Mercuric ion was used to displace
nitric oxide selectively from the S-nitrosothiols (Saville, B.,
Analyst 83:670-672 (1958)). Comparison of measured nitric oxide
concentrations from samples alternatively pretreated or not
pretreated with HgCl.sub.2 ensured that nitric oxide obtained by
photolysis was derived specifically from S-nitrosothiols.
Similarly, as an added measure of confirmation, we distinguished
between S-nitrosothiols and free nitric oxide by comparing nitric
oxide concentrations in samples alternatively exposed or not
exposed to photolyzing illumination.
Methods for Spectrophotometric Experiments and Nitrosylhemoglobin
Formation, Examples 14-20
Purified human HbA.sub.0 was obtained from Apex Biosciences
(Antonini, E. and Brunori, M. In Hemoglobin and Myoglobin in Their
Reactions with Ligands, American Elsevier Publishing Co., Inc., New
York (1971)). The spectrophotometer used was a Perkin Elmer UV/vis
Spectrometer Lambda 2S. All measurements were made at 23.degree. C.
in a sealed quartz cuvette to which all additions were made.
Deoxygenation was achieved by argon passage through a Hb solution
within a sealed quartz cuvette. The degree of deoxygenation can be
measured by UV/vis spectrum. Nitrosylation of hemes is achieved by
addition of purified NO gas to deoxyHb and the products quantitated
by the extinction coefficient per Antonini and Brunori, supra.
EXAMPLE 1
Interactions of NO and RSNO with Hb
It was observed that naturally occurring N-oxides, such as NO and
RSNOs (Gaston, B., et al., Proc. Natl. Acad. Sci. USA
90:10957-10961 (1993); Scharfstein, J. S., et al., J. Clin.
Invest., 94:1432-1439 (1994); Clancy, R. M., et al., Proc. Natl.
Acad. Sci. USA 91:3680-3684 (1994)), differed markedly in their
reactions with Hb. NO bound very rapidly to deoxyHb (Hb[FeII]),
forming relatively stable Hb[FeII]NO complexes (FIG. 1A), and
converted oxyHb (Hb[FeII]O.sub.2) to methemoglobin (Hb[FeIII]) and
nitrate (FIG. 1B), confirming previous reports (Olson, J. S.,
Methods in Enzymol. 76:631-651 (1981); Toothill, C., Brit. J.
Anaesthy. 39:405-412 (1967)). In contrast, RSNOs were found to
participate in transnitrosation reactions with sulfhydryl groups of
Hb, forming S-nitrosohemoglobin (SNO-Hb), and did not react with
the heme centers of either deoxyHb or Hb(FeII)O.sub.2 (FIGS. 1C and
1D).
A. Interaction of NO With DeoxyHb
Conversion of deoxyHb (Hb[FeII]) to Hb(FeII)NO is observed upon
incubation of Hb(FeII) with increasing concentrations of nitric
oxide. See FIG. 1A. a. Deoxy Hb. b, c, d. Reaction mixtures of NO
and Hb(FeII) in ratios of 1:1, 2:1 and 10:1, respectively. The
reaction product Hb(FeII)NO formed essentially instantaneously on
addition of NO (i.e. within instrument dead time).
B. Interaction of NO With OxyHb
Conversion of oxyHb (Hb[Fe[II]O.sub.2) to metHb (HbFe[III]) is
observed upon incubation of oxyHb with increasing concentrations of
NO. See FIG. 1B. a. oxy Hb. b, c, d. Reaction mixtures containing
NO and oxyHb in ratios of 1:1, 2:1 and 10:1, respectively.
Methemoglobin formation occurred instantaneously on addition of NO
(i.e. within instrument dead time).
C. Interaction of S-Nitrosothiols With DeoxyHb
Conversion of Hb(FeII) to SNO-Hb(FeII) is observed upon incubation
of either GSNO (shown) or S-nitrosocysteine (CYSNO) with deoxy Hb.
There is little (if any) interaction of RSNO with the heme
functionalities of Hb. See FIG. 1C. a. deoxyHb. b, c, d. Reaction
mixtures of GSNO and Hb(FeII) in ratios of 1:1, 2:1 and 10:1,
respectively. Spectra were taken after 60 min of incubation in b,
c, and 15 min in d. Further analysis of reaction products revealed
the formation of moderate amounts of SNO-Hb in all cases. Yields of
SNO-Hb (S-NO/Hb) in b, c, and d at 60 min were 2.5%, 5% and 18.5%,
respectively. (See FIG. 1D and FIG. 2A.)
D. Interaction of S-Nitrosothiols With OxyHb
Conversion of Hb(FeII)O.sub.2 to SNO-Hb(FeII)O.sub.2 is observed
upon incubation of either GSNO (shown) or CYSNO with oxyHb. There
is little (if any) reaction of GSNO (or CYSNO) at the heme centers
of Hb(FeII)O.sub.2. Specifically, the capacity for O.sub.2 binding
to heme is unaffected by RSNOs. See FIG. 1D. a. oxyHb. b, c, d.
Reaction mixtures of GSNO and oxyHb in ratios of 1:1, 2:1, and
10:1, respectively. Spectra were taken after 60 min of incubation
in the spectrophotometer. Further analysis of reaction products
revealed the formation of SNO-Hb in all cases. Yields of SNO-Hb in
spectra b, c and d were 5%, 10% and 50% (S-NO/Hb), respectively. In
5 other determinations, the yield of S-NO/Hb was 0.37.+-.0.06 using
GSNO (pH 7.4, 10-fold excess over Hb) and .about.2 SNO/tetramer
(1.97.+-.0.06) using CYSNO (vida infra). These last data are in
agreement with reports that human HbA contains 2 titratable SH
groups.
Methods
Human HbA.sub.0 was purified from red cells as previously described
(Kilbourn, R. G., et al., Biochem. Biophys. Res. Comm., 199:155-162
(1994)). Nitric oxide solutions were rigorously degassed and
purified according to standard procedure (Beckman, J. S., et al.,
Methods in Nitric Oxide Research, Feelisch and Stamler, eds., Wiley
Chichester, U.K. (1996)) and saturated solutions were transferred
in air tight syringes. Deoxygenation of Hb was achieved by addition
of excess dithionite (NO studies) or by reduction of
Hb(FeII)O.sub.2 through evacuation in Thunberg tubes (RSNO studies;
as RSNOs react with dithionite). RSNOs were synthesized as
previously described (Gaston, B., et al., (1993); Arnelle, D. R.
and Stamler, J. S., Arch. Biochem. Biophys. 318:270-285 (1995))
Incubations with HbA.sub.0 were made in phosphate buffer, pH 7.4,
0.5 mM EDTA. Quantifications of SNO-Hb were made according to the
method of Saville (Gaston, B., et al., (1993); Stamler, J. S., et
al., Proc. Natl Acad. Sci. USA, 90:444-448 (1992)) after
purification of protein with Sephadex G-25 columns. The Saville
method, which assays free NO.sub.x in solution, involves a
diazotization reaction with sulfanilamide and subsequent coupling
with the chromophore N-(naphthyl)ethylenediamine. No low molecular
weight S-NO complexes survived this purification and all activity
was protein precipitable. The reactions and spectra were carried
out using a Perkin Elmer UV/Vis Spectrometer, Lambda 2S.
EXAMPLE 2
Allosteric Function of O.sub.2 in Regulation of Hb
S-Nitrosylation
Oxygenation of Hb is associated with conformational changes that
increase the reactivity of cys.beta.93 to alkylating reagents
(Garel, C., et al., J. Biochem., 123:513-519 (1982); Jocelyn, P.
C., Biochemistry of the SH Group, Academic Press, London, p. 243
(1972); Craescu, C. T., et al., J. Biol. Chem., 261:14710-14716
(1986)). The physiological importance of this effect has not been
explained previously. It was observed here that rates of
S-nitrosation of Hb were markedly dependent on conformational
state. In the oxy conformation (R state), S-nitrosation was more
rapid than in the deoxy conformation (T state) (FIG. 2A). The rate
of S-nitrosation was accelerated in both conformations by alkaline
conditions (i.e., rate at pH 9.2>pH 7.4), which tends to expose
the cys.beta.93 that is otherwise screened from reaction by the
C-terminal histidine 146.beta.. The salt bridge (asp .beta.94 --his
.beta.146) tying down the histidine residue is loosened at high pH.
These data suggest that the increase in thiol reactivity associated
with the R state derives, at least in part, from improved NO access
rather than a conformation-induced change in pK.
A. Oxygenation Accelerates S-Nitrosylation of Hb
Rates of Hb S-nitrosation by S-nitrosocysteine (CYSNO) are faster
in the oxy conformation (Hb[FeII]O.sub.2) than in the deoxy state
(Hb[FeII]).
Methods
Incubations were performed using 10-fold excess CYSNO over protein
(50 .mu.M) in aerated 2% borate, 0.5 mM EDTA (oxyHb), or in a
tonometer after rapid O.sub.2 evacuation (deoxyHb). At times shown
in FIG. 2A, samples were rapidly desalted across G-25 columns
(preequilibrated with phosphate buffered saline, 0.5 mM EDTA, pH
7.4) to remove CYSNO, and analyzed for SNO-Hb by the method of
Saville (Stamler, J. S., et al., Proc. Natl. Acad. Sci. USA,
89:444-448 (1992)).
B. Deoxygenation Accelerates Denitrosylation of Hb
Rates of RSNO decomposition (and transfer) are much faster in the
deoxy conformation [SNO-Hb(FeII)] than in the oxy state
[SNO-Hb(FeII)O.sub.2 ]. The decomposition of SNO-Hb(FeII) is
further accelerated by the presence of excess glutathione. Within
the dead time of measurements according to this method (.about.15
seconds), a major fraction of SNO-Hb(FeII) was converted to
GSNO.
Methods
Hbs in PBS (0.5 mM EDTA, pH 7.4) were incubated in air (oxy) or in
a tonometer previously evacuated of O.sub.2 (deoxy)
SNO-Hb(FeII)O.sub.2 decomposition was determined by the method of
Saville (Saville, B., Analyst 83:670-672 (1958)). Spontaneous
decomposition of SNO-Hb(FeII) was followed spectrophotometrically
by formation of Hb(FeII)NO. Transnitrosation reactions with
glutathione were performed by addition of 100-fold excess
glutathione over protein (50 .mu.M), immediate processing of the
reaction mixture under anaerobic conditions followed by rapid TCA
precipitation, and analysis of RSNO in the supernatant. Rates of NO
group transfer were too rapid to measure accurately by the standard
methods used in this study.
EXAMPLE 3
NO-Related Interactions With Cysteine Residues of Hb in
Physiological Systems
Given that Hb is largely contained within red blood cells,
potential mechanisms by which S-nitrosation of intracellular
protein might occur were explored. Incubation of oxygenated rat red
blood cells with S-nitrosocysteine resulted in very rapid formation
of intracellular SNO-Hb(FeII)O.sub.2 (FIG. 3A). Rapid oxidation of
Hb was not observed under these conditions. Intraerythrocytic
SNO-Hb also formed when red blood cells were treated with
S-nitrosohomocysteine or S-nitrosocysteinylglycine, but not with
S-nitrosoglutathione (GSNO). Thus, erythrocyte access of RSNOs is
thiol group specific. Exposure of oxygenated red blood cells to NO
resulted primarily in metHb formation.
Endothelium-Derived Relaxing Factor (EDRF) and Hemoglobin
Hb-mediated inhibition of endothelium-dependent relaxations is
commonly used as a marker of NO responses. Inasmuch as reactions
with either metal or thiol centers of Hb should lead to attenuated
NO/EDRF (endothelium-derived relaxing factor) responses,
experiments were performed to elucidate the molecular basis of
inhibition. Hb preparations in which .beta.93 thiol groups had been
blocked with N-ethylmaleimide (NEM) or the hemes blocked by cyanmet
(FeIIICN)-derivitization were studied in an aortic ring bioassay,
and their activities were compared with that of native Hb. Both
cyanmet-Hb and NEM-Hb caused increases in vessel tone and
attenuated acetylcholine (EDRF)-mediated relaxations (FIG. 3B).
However, native Hb was significantly more effective than either of
the modified Hb preparations (FIG. 3B). Taken in aggregate, these
studies suggest that both the thiol and metal groups of Hb
contribute to its NO-related activity. To verify formation of an
S-nitrosothiol in Hb, a bioassay was used in which 2 cm segments of
thoracic aorta were interposed in Tygon tubing, through which 3 cc
of Krebs solution containing Hb (4 .mu.M) and ACh (2 .mu.M) were
circulated by roller pump (1.5 cc/min.times.5 min). Analysis of the
effluent (Gaston, B., et al., (1993)) revealed the formation of
SNO-Hb (20.+-.4 nM) in 5 of 5 experiments.
A. S-Nitrosation of Intraerythrocytic Hb
Incubation of rat erythrocytes with S-nitrosocysteine (equimolar to
heme (5 mM); phosphate buffer pH 7.4, 25.degree. C.) leads to rapid
formation of intracellular SNO-Hb(FeII)O.sub.2. MetHb does not form
rapidly. Separation of intracellular RSNOs across G-25 columns
reveals that only a small percentage exists as low molecular weight
S-nitrosothiol (e.g. GSNO) at most time points. By 60 min, 3 of the
4 available SH groups of Hb were S-nitrosated (note that rat Hb
contains 4 reactive SH groups). See FIG. 3A. Inset shows spectra of
SNO-Hb isolated from rat erythrocytes and related analyses.
Spectrum A is that of SNO-Hb isolated from erythrocytes following
G-25 chromatography. Treatment of A with dithionite results in
reduction of the S-NO moiety, liberating free NO which is
autocaptured by deoxy Hb, forming Hb(FeII)NO (note that dithionite
simultaneously deoxygenates Hb) (spectrum C). This spectrum (C)
reveals a stoichiometry of .about.3 S-NO's per tetramer. The
spectrum of Hb(FeII)NO containing 4 NO's per tetramer is shown for
comparison (inset, spectrum B).
Methods
At shown intervals, red blood cells were pelleted rapidly by
centrifugation, washed three times, lysed in deionized water at
4.degree. C., and the cytosolic fraction subjected to rapid
desalting across G-25 columns. Intracellular SNO-Hb was measured by
the method of Saville (Gaston, B., et al., (1992); Stamler, J. S.,
et al., Proc. Natl. Acad. Sci. USA, 89:444-448 (1992)), and
confirmed spectroscopically (inset of FIG. 3A) as described
above.
B. Molecular Basis of EDRF/Hb Interaction
The effects of native Hb on EDRF responses were compared with Hb
preparations in which the thiol or heme centers had been blocked by
alkylation or cyanmet derivitization, respectively. All
preparations of Hb elicited contractions; however, those of native
Hb (in which both SH and metal centers are free for interaction)
were most pronounced. See FIG. 3B. Likewise, acetylcholine (ACh)
mediated relaxations were most effectively inhibited by native Hb.
Relaxations were inhibited to lesser degrees by cyanmet Hb
(CN-Hb)(in which hemes were blocked from reaction) and NEM-Hb (in
which thiol groups were alkylated by N-ethylmaleimide). See Table
1. These data illustrate that both heme and .beta.93SH groups of Hb
contribute to reversal of EDRF responses. Direct measurement of
SNO-Hb, formed from EDRF under similar conditions, is described in
Example 8.
Methods
Descending rabbit thoracic aorta were cut into 3 mm rings and
mounted on stirrups attached to force transducers (model FT03,
Grass Instruments, Quincy, Mass.) for measurement of isometric
tone. The details of this bioassay system have been previously
described (Stamler, J. S., et al., Proc. Natl. Acad. Sci. USA,
89:444-448 (1992)). Cyanmet Hb was prepared from human HbA
according to published protocols (Kilbourn, R. G. et al. Biochem.
Biophys. Res. Comm., 199:155-162, (1994)). Alkylation of HbA with
N-ethylmaleimide was followed by desalting across G-25 Sephadex to
remove excess NEM. Removal of unmodified Hbcys.beta.93 was achieved
by passage through Hg-containing affinity columns. The alkylation
of free SH groups was verified using 5,5'-dithio-bis[2-nitrobenzoic
acid].
TABLE 1 % INCREASE IN ADDITIONS TENSION % ACh RELAXATION Hb (1
.mu.M) 40.8 .+-. 2.3 (n = 7) 31.9 .+-. 6.9 (n = 7) NEM-Hb (1 .mu.M)
29.4 .+-. 1.3** (n = 7) 60.5 .+-. 3.9* (n = 7) CN-Hb (1 .mu.M) 12.9
.+-. 3.0** (n = 6) 80.7 .+-. 1.0** .dagger. (n = 4) ACh (1 .mu.M)
98.3 .+-. 0.6 (n = 10) *, P < 0.01; **, P < 0.001, Compared
to Hb; .dagger., P < 0.001, Compared to ACh
EXAMPLE 4
Transduction of SNO-Hb Vasoactivity
Arterial red blood cells contain two physiologically important
forms of hemoglobin: Hb(FeII)O.sub.2 and Hb(FeIII) (Antonini, E.
and Brunori, M. In Hemoglobin and Myoglobin in Their Reactions with
Ligands, American Elsevier Publishing Co., Inc., New York, pp.
29-31 (1971)). Arterial-venous differences in the S-nitrosothiol
content of intraerythrocytic Hb suggest that the NO group is
released during red cell transit. Such findings raise the
possibility of functional consequences, perhaps influenced by the
redox state of heme and its occupation by ligand.
SNO-Hb(FeII)O.sub.2 was found to possess modest NO-like activity
when tested in a vascular ring bioassay. Specifically, the
contraction elicited by SNO-Hb(FeII)O.sub.2 was less than that of
native Hb(FeII)O.sub.2, indicating that S-nitrosation partially
reverses the contractile effects of Hb (FIG. 4A). By comparison,
SNO-Hb(FeIII) was found to be a vasodilator (FIG. 4A). Notably,
free NO was devoid of relaxant activity in the presence of
Hb(FeII)O.sub.2 or Hb(FeIII).
Red blood cells contain millimolar concentrations of glutathione.
As equilibria among RSNOs are rapidly established through
RSNO/thiol exchange (Arnelle, D. R. and Stamler, J. S., Arch.
Biochem. Biophy., 318:279-285 (1995)), the vasoactivity of SNO-Hb
was reassessed in the presence of glutathione. FIG. 4B illustrates
that glutathione potentiated the vasodilator activity of both
SNO-Hb(FeII)O.sub.2 and SNO-Hb(FeIII). GSNO formation under these
conditions (confirmed chemically and in bioassay experiments)
appeared to fully account for this effect. Further kinetic analyses
revealed that transnitrosation involving glutathione was more
strongly favored in the equilibrium with SNO-Hb(FeIII) than
SNO-Hb(FeII)O.sub.2 (FIG. 4C). Given the findings of steady-state
levels of SNO-Hb in red blood cells (Table 2 and FIG. 3A), these
results suggest that 1) the equilibrium between naturally occurring
RSNOs and Hb(cys.beta.93) lies toward SNO-Hb under physiological
conditions; 2) that transnitrosation reactions involving SNO-Hb and
GSH are likely to occur within red blood cells (in these studies,
low molecular weight RSNOs have been found in erythrocytes loaded
with SNO-Hb); and 3) that oxidation of the metal center of Hb shift
the equilibrium toward GSNO, thereby potentially influencing
bioactivity.
Additional mechanisms of NO group release from SNO-Hb were sought.
Arterial-venous differences in levels of SNO-Hb raised the
possibility that S-NO bond stability may be regulated by the
changes in Hb conformation accompanying deoxygenation. To test this
possibility, the rates of NO group release from SNO-Hb(FeII)O.sub.2
and SNO-Hb(FeIII) were compared. Deoxygenation was found to enhance
the rate of SNO-Hb decomposition (FIG. 2B). These rates were
accelerated greatly by glutathione in a reaction yielding GSNO
(FIG. 2B). The results illustrate that O.sub.2 -metal interactions
influence S-NO affinity, and suggest a new allosteric function for
Hb.
For SNO-Hb to be of physiological importance it must transduce its
NO-related activity across the erythrocyte membrane. This
possibility was explored by incubating erythrocytes containing
SNO-Hb in physiologic buffer, and measuring the accumulation of
extracellular RSNOs over time. FIG. 4D illustrates that red blood
cells export low molecular weight (trichloroacetic acid soluble)
S-nitrosothiols under these conditions. Importantly, the degree of
hemolysis in these experiments was trivial (<0.5%), and
correction for lysis did not significantly impact on rates of RSNO
release. These results establish that an equilibrium exists between
low molecular weight and protein RSNOs within the red cell, and
that intracellular location is unlikely to be a limiting factor in
the transduction of such NO-related activity to the vessel
wall.
A. Concentration-Effect Esponses of Different SNO-Hb
Preparations
Contractile effects of Hb(FeII)O.sub.2 (.tangle-solidup.) are shown
to be partially reversed by S-nitrosation (SNO-Hb[FeII]O.sub.2
(.box-solid.); P=0.02 by ANOVA vs Hb(FeII)O.sub.2) (See FIG. 4A.).
Oxidation of the metal center of SNO-Hb
(SNO-Hb[FeIII](.circle-solid.)) converts the protein into a
vasodilator (P<0.0001 by ANOVA vs. SNO-Hb[FeII]O.sub.2), with
potency comparable to that of other S-nitrosoproteins (Stamler, J.
S., et al., Proc. Natl. Acad. Sci. USA, 89:444-448 (1992)). The
contractile properties of Hb(FeIII) are shown for comparison
(.quadrature.); n=6-17 for each data point.
Methods
Details of the vessel ring bioassay have been published (Stamler,
J. S., et al., Proc. Natl. Acad. Sci. USA 89:444-448 (1992)).
SNO-Hb(FeII)O.sub.2 preparations were synthesized with 10-fold
excess S-nitrosocysteine (CYSNO) over Hb(FeII)O.sub.2 protein (2%
borate, 0.5 mM EDTA, .about.15 min incubation), after which
desalting was performed across Sephadex G-25 columns. CYSNO was
synthesized in 0.5 N HCl, 0.5 mM EDTA and then neutralized (1:1) in
1 M phosphate buffer containing 0.5 mM EDTA. SNO-Hb(FeIII)
preparations followed a similar protocol, but used Hb(FeIII) as
starting material. The latter was synthesized by treatment of
Hb(FeII)O.sub.2 with excess ferricyanide, followed by desalting
across G-25 columns. SNO-Hb concentrations were verified
spectroscopically and the S-nitrosothiol content was determined by
the method of Saville (Stamler, J. S., et al., Proc. Natl. Acad.
Sci USA 89:444-448 (1992)). The S-NO/tetramer stoichiometry for
both SNO-Hb preparations was .about.2. Oxidation of the heme was
undetectable by uv-spectrophotometric methods.
B. Potentiation of SNO-Hb Effects by Glutathione
Addition of glutathione (100 .mu.M) to bioassay chambers
potentiates the dose-response to both SNO-Hb(FeII)O.sub.2
(.box-solid.) and SNO-Hb(FeIII) (.circle-solid.) (See FIG. 4B.
n=6-12; p<0.0001 for both by ANOVA, compared with the respective
tracings in FIG. 4A. Glutathione had a transient affect on baseline
tone in some experiments, and did not significantly influence the
response to Hb(FeII)O.sub.2 (.tangle-solidup.).
C. Transnitrosation Between SNO-Hb and Glutathione
Rates of NO group transfer from SNO-Hb (100 .mu.M) to glutathione
(10 mM) are displayed for SNO-Hb(FeII)O.sub.2 (oxy) and
SNO-Hb(FeIII) (met) (n=5). Data are presented as the amount of GSNO
formed relative to the starting SNO-Hb concentration. The transfer
is more rapid for SNO-Hb(FeIII) than SNO-Hb(FeII)O.sub.2
(p<0.002 by ANOVA), suggesting that the GSNO/SNO-Hb equilibrium
is shifted toward GSNO by formation of metHb.
Methods
Thiol/SNO-Hb exchange, forming GSNO, was verified chemically
(Stamler, J. S., et al., Proc. Natl Acad. Sci. USA, 89:444-448
(1992)) following trichloroacetic acid precipitation (n=5). These
results were verified in separate experiments by measuring the
residual SNO-Hb concentration, following separation of reaction
mixtures across G-25 columns.
D. Export of S-Nitrosothiols by Red Blood Cells
Human red blood cells containing SNO-Hb are shown to export low
molecular weight RSNOs over time. Hemolysis, which ranged from
0-<0.5% over one hour and did not correlate with rates of RSNO
release, could account for only a trivial fraction of the measured
extracellular RSNO.
Methods
Packed human red blood cells were obtained by centrifugation,
washed, and resuspended in phosphate buffered saline containing 5
mM SNOCYS (0.5 mM EDTA, pH 7.4) for one hour. This results in a red
cell preparation containing SNO-Hb (FeIIO.sub.2 /FeIII mixture)
with a stoichiometry of 0.5 S-NO/tetramer. The red blood cells were
then washed repeatedly to remove residual CYSNO (verified), and
incubated in Krebs' solution (1:4). The accumulation of
extracellular RSNO was measured over time by the method of Saville
(Saville, B., Analyst, 83:670-672 (1958)). Hemolysis was determined
by spectral analysis of red blood cell supernatants following
centrifugation.
EXAMPLE 5
SNO-Hb Bioactivity In Vivo
Systemic administration of cell-free Hb results in hypertensive
responses which have been attributed to NO scavenging by the heme
(Vogel, W. M., et al., Am. J. Physiol. 251:H413-H420 (1986); Olsen,
S. B., et al., Circulation 93:329-332 (1996)). To determine if
SNO-Hb is free of this adverse affect, and to explore if in vitro
mechanisms of NO release extend to the in vivo circumstance, we
compared responses to Hb and SNO-Hb infused as a bolus into the
femoral vein of anesthetized rats. As illustrated in FIG. 5,
Hb(FeII)O.sub.2 (200 nmol/kg) caused an increase in mean arterial
pressure of 20.+-.3 mm Hg (n=4; P<0.05). In contrast,
SNO-Hb(FeII)O.sub.2 did not exhibit hypertensive effects and
SNO-Hb(FeIII) elicited hypotensive responses (FIG. 5). Thus, the
profiles of these compounds in vivo closely resemble those seen in
vitro (FIG. 4A). Moreover, to demonstrate that the physiological
responses of red cells are comparable to those of cell-free Hb
preparations, erythrocytes containing SNO-Hb were injected into the
femoral vein of rats pretreated with L-NMMA (50 mg/kg) to deplete
endogenous RSNOs. At levels of SNO-Hb comparable to those found in
the normal rat (0.1-0.5 .mu.M), SNO-Hb containing red blood cells
elicited hypotensive responses (8.+-.1 mm Hg; mean.+-.SEM; n=9),
whereas native (SNO-Hb depleted) red blood cells did not (P=0.001).
These changes in mean blood pressure of .about.10% are on the order
of those that differentiate normotension from hypertension in man,
and in the therapeutic range of some antihypertensive regimens. The
effects of both Hb and SNO-Hb--whether cell-free or contained
within red cells--were transient, suggesting that S-nitrosylation
of Hb and metabolism of SNO-Hb is occurring in vivo, with
consequent restoration of blood pressure. The bioactivity of SNO-Hb
in blood, where S-NO/heme stoichiometries approach 1:50,000, is a
dramatic illustration of the resistance of this NO-related activity
to Hb(Fe) inactivation.
In Vivo Effects of Cell-Free Hb and SNO-Hbs
Administration of 2-200 nmol/kg Hb(FeII)O.sub.2 (as a bolus) into
the femoral vein of a Sprague-Dawley rat is shown to increase mean
arterial pressure in a dose-dependent manner. At 200 nmol/kg, mean
arterial pressure increased by 25 mm Hg (20.+-.3 mm Hg; n=4;
P<0.05). Elevations in blood pressure reversed within 10-15
minutes. SNO-Hb(FeII)O.sub.2 infusions (over the same dose range)
are shown to ameliorate Hb(FeII)O.sub.2 -induced hypertension
without causing overt changes in blood pressure. A similar response
was seen at higher doses. By comparison, SNO-Hb(FeIII) infusions
caused a significant fall in mean arterial pressure (pre 108.+-.4
mm Hg; post 74.+-.6 mm Hg, n=5; P<0.05) at the highest dose (200
nmol/kg). Hypotensive responses tended to be transient with blood
pressure normalizing over 10 minutes. A fall in blood pressure was
also seen with injection of erythrocytes containing SNO-Hb.
Methods
Rats were anesthetized by intraperitoneal injection of
pentobarbital and the femoral arteries and veins accessed by local
cut down. The artery was then cannulated and the blood pressure
monitored continuously using a Viggo Spectramed pressure transducer
attached to a Gould recorder. An IBM PC (DATA Q Codas) was used for
data acquisition.
EXAMPLE 6
Loading of Red Blood Cells With S-Nitrosothiols
Incubation of rat erythrocytes with S-nitrosocysteine (equimolar to
heme (5 mM); phosphate buffer pH 7.4, 25.degree. C.) leads to rapid
formation of intracellular S-nitrosothiols. MetHb does not form
rapidly. Separation of cell content across G-25 columns establishes
the formation of intraerythrocytic low molecular weight
S-nitrosothiol, e.g. S-nitrosoglutathione, (GSNO). By 2 minutes,
one can achieve as much as millimolar GSNO.
Method for Assay of RSNO
S-nitrosocysteine (5 mM) treated red blood cells are pelleted
rapidly by centrifugation, washed three times, lysed in deionized
water at 4.degree. C., and the cytosolic fraction subjected to
rapid desalting across G-25 columns. Intracellular RSNO is measured
by the method of Saville and can be confirmed
spectroscopically.
Effects on Blood Pressure From Loaded Red Blood Cells
Red blood cells treated with S-nitroscysteine (to produce SNO-RBCs)
and introduced into the femoral vein of a Sprague-Dawley rat
decreased mean arterial pressure in a dose-dependent manner. For
red blood cells in which SNO-Hb was assayed at 0.3 .mu.M (the
endogenous in vivo SNO-Hb concentration), arterial pressure
decreased by 8.+-.1 mm Hg (mean.+-.SEM for 9 experiments;
p<0.001 compared to untreated red blood cell controls). For red
blood cells in which SNO-Hb was assayed at 0.5 .mu.M, arterial
pressure decreased by 10 mm Hg. For red blood cells in which SNO-Hb
was assayed at 0.1 .mu.M (a sub-endogenous SNO-Hb concentration),
arterial pressure decreased by 6 mm Hg. The administration of
untreated red blood cells caused no effect or a slight increase in
arterial blood pressure. Administration of L-monomethyl-L-arginine
(L-NMMA; 50 mg/kg) caused an increase in blood pressure of about 20
mm Hg. Changes in blood pressure from a bolus administration of
loaded red blood cells lasted 15-20 minutes.
Further Methods
Rats were anesthetized by intraperitoneal injection of
pentobarbital and the femoral arteries and veins accessed by local
cut down. The artery was then cannulated and the blood pressure
monitored continuously using a Viggo Spectramed pressure transducer
attached to a Gould recorder. An IBM PC (DATA Q Codas) was used for
data acquisition.
EXAMPLE 7
Effects of SNO-Hb on Coronary Vasodilation, Coronary Flow and Blood
Pressure
SNO-Hb was synthesized as described in Example 4A. Completion of
the reaction was determined as described in Example 4A. Twenty-four
healthy mongrel dogs (25-30 kg) were anesthetized with intravenous
thiamylal sodium (60-80 mg/kg) and subjected to left thoracotomy in
the fourth intercostal space. The left circumflex coronary artery
distal to the left atrial appendage was minimally dissected. A pair
of 7-MHz piezoelectric crystals (1.5.times.2.5 mm, 15-20 mg) was
attached to a Dacron backing and sutured to the adventitia on
opposite surfaces of the dissected vessel segment with 6-0 prolene.
Oscilloscope monitoring and on-line sonomicrometry (sonomicrometer
120-2, Triton Technology, San Diego, Calif.) were used to ensure
proper crystal position. A pulse Doppler flow probe (10 MHz, cuff
type) was implanted distal to the crystals. An inflatable balloon
occluder was also placed distal to the flow probe. All branches of
the circumflex artery between the crystals and the occluder were
ligated. Heparin sodium-filled polyvinyl catheters were inserted
into the left ventricular cavity via the apex, into the left atrium
via the atrial appendage, and into the ascending aorta via the left
internal thoracic artery. The catheters, tubing, and wires were
tunnelled to a subcutaneous pouch at the base of the neck.
After a 10 to 15 day recovery period, the catheters and wires were
exteriorized under general anesthesia, and 2-3 days later, each dog
was given a bolus injection of SNO-Hb (0.4 mg) to evaluate vascular
response. Two dogs that demonstrated <5% dilation of epicardial
coronary vessels were excluded from subsequent studies, and two
were excluded because of other technical reasons.
Dogs were trained and studied while loosely restrained and lying
awake in the lateral recumbent position. The laboratory was kept
dimly illuminated and quiet. Aortic pressure, left ventricular
end-diastolic pressure dP/dt external coronary diameter and
coronary flow were monitored continuously. In 10 dogs, 0.1 ml of
SNO-Hb solution, 50 nM/kg, was injected via the left atrial
catheter. To verify potential effects of solvent on vasculature,
0.1 ml injections of 30% ethanol in distilled water were given as
vehicle control. Between injections, phasic coronary blood flow and
coronary artery diameter were allowed to return to preinjection
levels (minimum 15 minutes). Allowing a 15 minute period between
injections resulted in no modification of repeated does injections.
To assess the direct and potential flow mediated indirect
vasodilation effects of SNO-Hb on the conductance vessels, the dose
was repeated in 6 of 10 dogs with partial inflation of the
adjustable occluder to maintain coronary blood flow at or slightly
below preinjection levels. The response to acetylcholine chloride
(Sigma Chemical) was assessed in another group of 10 dogs following
a similar protocol to that used for SNO-Hb.
Epicardial coronary diameter, coronary blood flow, heart rate, and
aortic and left ventricular end-diagnostic pressures were compared
before and after each SNO-Hb injection. The maximum changes in
coronary dimension and blood flow were expressed as a function of
increasing doses of SNO-Hb. The response of coronary dimension to
increasing doses followed a characteristic sigmoid dose-response
curve that could be described by the following equation
##EQU2##
where K.sub.D is the drug-receptor complex dissociation constant
and is the dose at which 50% of the maximum response (EC.sub.50) is
achieved. In each animal, a nonlinear least-squares regression
(r.sup.2 >0.90) was performed on the dose-response data. The
regression was constrained to the above equation. From the
regression, values for maximum response and K.sub.D were obtained
for each individual animal. The mean of these values was then
calculated to obtain an average K.sub.D and maximum response for
the study group. These values were used to generate a mean curve,
which was plotted with the mean dose-response values. (See FIGS.
6A-6F.)
EXAMPLE 8
Endogenous Levels of S-Nitrosohemoglobin and
Nitrosyl(FeII)-Hemoglobin in Blood
To determine if SNO-Hb is naturally occurring in the blood, and if
so, its relationship to the O.sub.2 transport capacity and
nitrosylated-heme content of red cells, an analytical approach was
developed to assay the S-nitrosothiol and nitrosyl-heme content of
erythrocytes (Table 2). Arterial blood was obtained from the left
ventricle of anesthetized rats by direct puncture and venous blood
was obtained from the jugular vein and inferior vena cava. Hb was
then purified from red cells and assayed for RSNO and (FeII)NO
content. Arterial blood contained significant levels of SNO-Hb,
whereas levels were virtually undetectable in venous blood (Table
2). Measurements made 45 minutes after infusion of the NO synthase
inhibitor N.sup..omega. -monomethyl-L-arginine (L-NMMA) (50 mg/kg),
showed a depletion of SNO-Hb as well as total Hb-NO (82 and
50.+-.18%, respectively; n=3-5; p<0.05). These data establish
the endogenous origin of SNO-Hb, although some environmental
contribution is not excluded. The arterial-venous distribution seen
for SNO-Hb was reversed in the case of Hb(FeII)NO, which was
detected in higher concentrations in partially deoxygenated
(venous) erythrocytes (Table 2). Accordingly, the proportion of
nitrosylated protein thiol and heme appears to depend on the
oxygenation state of the blood. Consistent with these findings,
Wennmalm and coworkers have shown that Hb(FeII)NO forms mainly in
venous (partially deoxygenated) blood (Wennmalm, A., et al., Br. J.
Pharmacol. 106(3):507-508 (1992)). However, levels of Hb(FeII)NO in
vivo are typically too low to be detected (by EPR) and SNO-Hb is
EPR-silent (i.e., it is not paramagnetic). Thus,
photolysis-chemiluminesence represents an important technological
advance, as it is the first methodology capable of making
quantitative and functional assessments of NO binding to Hb under
normal physiological conditions.
TABLE 2 Endogenous Levels of S-Nitrosohemoglobin and Nitrosyl
(FeII) -Hemoglobin in Blood Hb (FeII) NO Site SNO-Hb (nM) (nm)
Arterial 311 .+-. 55* 536 .+-. 99 .dagger. Venous 32 .+-. 14 894
.+-. 126 *P < 0.05 vs venous; .dagger. P < 0.05 for paired
samples vs venous
Methods
Blood was obtained from the left ventricle (arterial) and jugular
vein (venous) of anesthetized Sprague-Dawley rats. Comparable
venous values were obtained in blood from the inferior vena cava.
Red blood cells were isolated by centrifugation at 800 g, washed
three times in phosphate buffered saline at 4.degree. C., lysed by
the addition of 4-fold excess volume of deionized water containing
0.5 mM EDTA, and desalted rapidly across G-25 columns according to
the method of Penefsky at 4.degree. C. In 24 rats, Hb samples were
divided in two aliquots which were then treated or not treated with
10-fold excess HgCl.sub.2 over protein concentration as measured by
the method of Bradford. Determinations of SNO-Hb and Hb(FeII)NO
were made by photolysis-chemiluminescence. In 12 additional rats,
further verification of the presence of SNO-Hb was made by assaying
for nitrite after HgCl.sub.2 treatment. Specifically, samples (with
and without HgCl.sub.2) were separated across Amicon-3 (Centricon
filters, m.w. cut off 3,000) at 4.degree. C. for 1 h, and the low
molecular weight fractions collected in airtight syringes
containing 1 .mu.M glutathione in 0.5 N HCl. Under these
conditions, any nitrite present was converted to
S-nitrosoglutathione, which was then measured by
photolysis-chemiluminescence (detection limit .about.1 nM). SNO-Hb
was present in all arterial samples, and levels determined by this
method (286.+-.33 nM) were virtually identical to and not
statistically different from those shown in Table 2. In venous
blood, SNO-Hb was undetectable (0.00.+-.25 nM); levels were not
statistically different from those given above.
Method for Assay of S-Nitrosohemoglobin
A highly sensitive photolysis-chemiluminescence methodology was
employed. A somewhat similar assay has been used for measuring
RSNOs (S-nitrosothiols) in biological systems (Gaston, B., et al.,
Proc. Natl. Acad. Sci. USA 90:10957-10961 (1993); Stamler, J. S.,
et al., Proc. Natl. Acad. Sci. USA 89:7675-7677 (1992)). The method
involves photolytic liberation of NO from the thiol, which is then
detected in a chemiluminesence spectrometer by reaction with ozone.
The same principle of operation can be used to cleave (and measure)
NO from nitrosyl-metal compounds (Antonini, E. and Brunori, M. In
Hemoglobin and Myoglobin in Their Reactions with Ligands, American
Elsevier Publishing Co., Inc., New York, pp. 29-31 (1971)). With
adjustment of flow rates in the photolysis cell, complete
photolysis of the NO ligand of Hb(FeII)NO could be achieved.
Standard curves derived from synthetic preparations of SNO-Hb,
Hb(FeII)NO, and S-nitrosoglutathione were linear (R>0.99),
virtually superimposable, and revealing of sensitivity limits of
approximately 1 nM. Two analytical criteria were then found to
reliably distinguish SNO-Hb from Hb(FeII)NO: 1) signals from SNO-Hb
were eliminated by pretreatment of samples with 10-fold excess
HgCl.sub.2, while Hb(FeII)NO was resistant to mercury challenge;
and 2) treatment of SNO-Hb with HgCl.sub.2 produced nitrite (by
standard Griess reactions) in quantitative yields, whereas similar
treatment of Hb(FeII)NO did not. UV/VIS spectroscopy confirmed that
NO remained attached to heme in the presence of excess
HgCl.sub.2.
EXAMPLE 9
Inhibition of Platelet Aggregation by S-Nitrosohemoglobins
Methods to prepare human HbA.sub.0 were as described in Example 1
"Methods" section. Methods to make SNO-Hb(FeII)O.sub.2 were as
described for Example 2A. Methods to make SNO-Hb(FeIII) were as in
Example 1 (see parts B, C, and "Methods" in Example 1).
Quantifications of SNO-hemoglobins were made as in Example 1
according to the method of Saville (Saville, B., Analyst 83:670-672
(1958)) and by the assay as described in Example 8, "Method for
assay of S-nitrosohemoglobin."
Venous blood, anticoagulated with 3.4 nM sodium citrate, was
obtained from volunteers who had not consumed acetylsalicylic acid
or any other platelet-active agent for at least 10 days.
Platelet-rich plasma was prepared by centrifugation at 150.times.g
for 10 minutes at 25.degree. C. and was used within 2 hours of
collection. Platelet counts were determined with a Coulter counter
(model ZM) to be 1.5 to 3.times.10.sup.8 /ml.
Aggregation of platelet-rich plasma was monitored by a standard
nephelometric technique in which results have been shown to
correlate with bleeding times. Aliquots (0.3 ml) of platelets were
incubated at 37.degree. C. and stirred at 1000 rpm in a PAP-4
aggregometer (Biodata, Hatsboro, Pa.). Hemoglobins were
preincubated with platelets for 10 min and aggregations were
induced with 5 .mu.M ADP. Aggregations were quantified by measuring
the maximal rate and extent of change of light transmittance and
are expressed as a normalized value relative to control
aggregations performed in the absence of hemoglobin.
The results of the aggregation assays are shown in FIGS. 7A, 7B and
7C. Standard deviations are shown as vertical bars.
SNO-Hb[Fe(II)]O.sub.2 causes some inhibition of platelet
aggregation at the higher concentrations tested. SNO-Hb[Fe(III)]
also inhibits platelet aggregation when present at concentrations
of 1 .mu.M and above, but to a much greater extent than
SNO-Hb[Fe(II)]O.sub.2.
EXAMPLE 10
Effect of SNO-Hbs on cGMP
Platelet rich plasma (PRP) was incubated with either hemoglobin,
SNO-oxy Hb, or SNO-metHb for 5 min, after which the assay was
terminated by the addition of 0.5 ml of ice cold trichloroacetic
acid to 10%. Ether extractions of the supernatant were performed to
remove trichloroacetic acid, and acetylation of samples with acetic
anhydride was used to increase the sensitivity of the assay.
Measurements of cyclic GMP were performed by radioimmunoassay
(Stamler, J. et al., Circ. Res. 65:789-795 (1989)).
Results are shown in FIG. 8. For all concentrations of Hb tested
(1, 10 and 100 .mu.M), the concentration of cGMP measured for
SNO-Hb(FeIII) was less than that of native Hb.
EXAMPLE 11
Polynitrosation of Hb A. HbA.sub.0 (oxy) was incubated with
S-nitrosoglutathione at a ratio of 6.25
S-nitrosoglutathione/HbA.sub.0 for 240 minutes at pH 7.4 at
25.degree. C. and desalted over Sephadex G-25 columns. Spectra were
run in the presence (spectrum B, FIG. 9A) and absence (spectrum A,
FIG. 9A) of dithionite. The shift in the spectrum is indicative of
2 SNO groups/tetramer. B. HbA.sub.0 was incubated with 100-fold
excess S-nitrosoglutathione over protein for 240 minutes at pH 9.2,
followed by desalting over a G-25 column. A portion was then
treated with dithionite. The spectra in FIG. 9B indicate that Hb
has been nitrosated at multiple sites. C. HbA.sub.0 was treated
with 100-fold excess S-nitroscysteine over tetramer at pH 7.4,
25.degree. C. for 5-20 min. After various times of treatment, the
protein was desalted over a G-25 column and treated with
dithionite. The spectra show progressive polynitrosation of Hb with
time (spectra A to F in FIG. 9C). After 5 minutes of treatment with
100-fold excess S-nitrosocysteine, 0.09 NO groups had added per
tetramer (spectrum A of FIG. 9C); after 20 minutes, at least 4 NO
groups had added (spectrum F). At intermediate time points, 0.4 NO
groups (spectrum B), 1.58 NOs (spectrum C), 2.75 NOs (spectrum D)
or 2.82 NOs had added per tetramer (spectrum E). D. Rat Hb was
treated with 100x S-nitrosoglutathione excess over tetramer for 3
hours at pH 7.4. The protein was then desalted by passage through a
G-25 column. A portion of the desalted protein was treated with
dithionite (spectrum B in FIG. 9D; the protein of spectrum A was
left untreated by dithionite). Spectrum B in FIG. 9D is
illustrative of a ratio of 6 RNOs/Hb. E. A time course experiment
tracking the extent of nitrosation of HbA.sub.0 with time was
performed (FIG. 9E). Treatment of HbA.sub.0 was with 10.times.
excess S-nitrosocysteine at pH 7.4, 25.degree. C. or with
100.times. excess S-nitroscysteine under the same conditions.
Analysis for SNO and NO was performed by the method of Saville and
by UV spectroscopy as in Jia, L. et al., Nature 380:221-226 (1996).
Under these conditions the heme is ultimately oxidized; the rate is
time dependent.
Treatment with 10.times. excess S-nitrosocysteine nitrosylates only
the thiol groups of the two reactive cysteine residues of
HbA.sub.0. Inositol hexaphosphate is known to shift the allosteric
equilibrium towards the T-structure (ordinarily, the deoxy form).
Treatment with 100.times. excess nitrosates additional groups;
i.e., the product has more than 2 NO groups/tetramer.
EXAMPLE 12
Effect of SNO-Hb(FeII)O.sub.2 on Blood Flow
SNO-Hb(FeII)O.sub.2, having a SNO/Hb ratio of 2, was prepared (from
HbA.sub.0) by reaction with S-nitrosothiol. Rats breathing 21%
O.sub.2 were injected (time 0) with Hbs prepared from HbA.sub.0 as
indicated in FIG. 10 (open circles, SNO-Hb (100 nmol/kg); filled
circles, SNO-Hb (1000 nmol/kg); filled squares, unmodified Hb (1000
nmol/kg)). Three rats were used per experiment. Blood flow was
measured in brain using the H.sub.2 clearance method;
microelectrodes were placed in the brain stereotactically.
Concomitant PO.sub.2 measurements revealed tissue PO.sub.2 =20.
Thus, SNO-Hb improves blood flow to the brain under normal
physiological conditions, whereas native Hb decreases blood flow.
NO group release is promoted by local tissue hypoxia.
EXAMPLE 13
Effects of SNO-Hb(FeII)O.sub.2, SNO-Hb(FeIII) and (NO).sub.x
-Hb(FeIII) on Tension of Rabbit Aorta
Hemoglobin was treated with either 1:1, 10:1 or 100:1
S-nitrosocysteine to Hb tetramer for 1 hour, processed as in
Example 4. The products of the reactions done with 1:1 and 10:1
excess were assayed by the Saville assay and by standard
spectrophotometric methods. The product of the reaction done at the
1:1 ratio is SNO-Hb(Fe)O.sub.2 ; SNO-Hb(FeIII) is found following
reaction with 1:10 excess CYSNO/tetramer.
The aortic ring bioassay was performed as described in Example 4.
The product of the reaction in which a ratio of 100:1 CYSNO/Hb
tetramer was used, contains 2 SNOs as well as NO attached to the
heme. The potency of the 100:1 CYSNO/Hb product is much greater
than that of SNO-Hb(FeIII) and is indicative of polynitrosation
(see FIG. 11).
EXAMPLE 14
Effect of Oxygenation on Partially Nitrosylated Hemoglobin
The effect of oxygenation on partially nitrosylated Hb was examined
by following spectral changes in the Soret region upon the addition
of air to partially nitrosylated Hb. Hemoglobin A (17 .mu.M) was
deoxygenated by bubbling argon through a 1 ml solution in 100 mM
phosphate (pH 7.4), for 45 minutes. Nitric oxide was added by
injection of 0.5 .mu.l of a 2 mM solution, stored under nitrogen.
The final heme:NO ratio was 68:1. The solution was slowly aerated
by sequential 50 .mu.l injections of room air. FIG. 12 shows that
the initial additions of air failed to produce a true isosbestic
point, indicating changes in the concentrations of at least three
absorbent species. Later additions of air did produce a true
isosbestic point, indicative of the conversion of deoxyhemoglobin
to oxyhemoglobin, with the loss of nitrosyl heme. The results show
that nitrosylated Hb is not a stable end product.
EXAMPLE 15
Conversion of Nitrosylhemoglobin to SNO-Hemoglobin
The hypothesis that the nitric oxide is transferred from the heme
iron to a thiol residue, forming nitrosothiol upon oxygenation, was
tested. Hemoglobin A (400 .mu.M) was deoxygenated by bubbling argon
through a 1 ml solution in 100 mM phosphate (pH 7.4), for 45
minutes. Nitric oxide was added by injection of an appropriate
volume of a 2 mM solution, stored under nitrogen, to achieve
different NO/Hb ratios. The solutions were then exposed to air by
vigorous vortexing in an open container. Samples were then analyzed
by Saville assay and by chemiluminescence after UV photolysis. Data
are shown as mean.+-.standard error (n>3). FIG. 13 shows that
S-nitrosothiol is formed in this manner, and that the efficiency of
this reaction is greatest at high ratios of heme to nitric oxide.
Amounts are highest at very high NO/Hb ratios, i.e., >2:1. This
result implies that nitrosyl Hb entering the lung is converted into
SNO-Hb, as under physiological conditions the ratio of heme to NO
is high.
EXAMPLE 16
Effects Dependent Upon Heme:NO Ratio
It was proposed that the binding of nitric oxide to the heme of the
.beta. chain was inherently unstable, and that the reason for lower
yields of SNO-Hb at higher concentrations of nitric oxide, was a
loss of bound nitric oxide as a result of this instability.
Hemoglobin A (17.5 .mu.M) was deoxygenated by bubbling argon
through a 1 ml solution in 100 mM phosphate (pH 7.4), for 45
minutes. Nitric oxide was added by sequential injections of an
appropriate volume of a 2 mM solution, stored under nitrogen. FIG.
14A: Difference spectra of the nitric oxide hemoglobin mixture and
the starting deoxyhemoglobin spectrum are shown. FIG. 14B: The peak
wavelength of the difference spectra plotted against the
concentration of nitric oxide added to the solution. These data
show that addition of small amounts of nitric oxide (heme:NO ratios
of approximately 70:1) produce predominantly nitrosylhemoglobin and
some oxidized hemoglobin. However, nitric oxide additions of the
order of 10 .mu.M result in the formation of oxidized hemoglobin.
Heme:NO ratios at this point are approximately 7:1. As the
concentration of nitric oxide is increased by further additions of
nitric oxide, the predominant species formed becomes
nitrosylhemoglobin (heme:NO ratio 1:1). The results in FIGS. 14A
and 14B show that under anaerobic conditions, the addition of
increasing quantities of nitric oxide to Hb results first in the
production of nitrosylhemoglobin and then oxidized Hb (metHb). At
very high levels of nitric oxide, nitrosyl-hemoglobin is once again
seen as the nitric oxide first reduces metHb to deoxyHb (producing
nitrite), then binds NO. This drives the conformational change of
T-structure Hb to R-structure, stabilizing the .beta. heme-nitric
oxide bond. The appearance of oxidized Hb at heme to nitric oxide
ratios of approximately 10:1 indicates the decay of the heme/NO
bond to produce oxidized Hb and nitric oxide anion (nitroxyl). The
presence of nitric oxide anion was confirmed by detection of
N.sub.2 O in the gas phase by gas chromatography mass spectrometry
and by the production of NH.sub.2 OH.
EXAMPLE 17
Effects Upon Oxygenation of Nitrosyl-DeoxyHb
Hemoglobin A (20.0 .mu.M) was deoxygenated by bubbling argon
through a 1 ml solution in 100 mM phosphate (pH 7.4), for 45
minutes. In both FIG. 15A and FIG. 15B, the lowest to the highest
spectra indicate the sequential additions of air. These are
difference spectra in which the pure deoxyHb spectrum occurs at
zero absorbance. The peak at 419 nm is from nitrosylhemoglobin;
oxidized hemoglobin absorbs at 405 nm.
In the experiments shown in FIG. 15A, hemoglobin was gradually
oxygenated by sequential additions of 10 .mu.l of room air by
Hamilton syringe. Spectra are shown as difference spectra from the
initial deoxyhemoglobin spectrum. In the experiments shown in FIG.
15B, nitric oxide (1 .mu.M) was added by injection of 0.5 .mu.l of
a 2 mM solution, stored under nitrogen. Final heme:No ratio was
80:1. The solution was gradually oxygenated by sequential additions
of 10 .mu.l of room air. Spectra are shown as difference spectra
from the initial deoxyhemoglobin spectrum. These data show the
initial formation of a nitrosylhemoglobin peak, along with some
formation of oxidized hemoglobin, which disappears after the
addition of approximately 30 .mu.l of air. The results indicate
that a small quantity of nitrosyl Hb is formed upon addition of low
ratios of nitric oxide to deoxy Hb, and that this nitrosyl Hb is
lost upon oxygenation.
EXAMPLE 18
Role of .beta.93Cys in Destabilizing Nitrosyl-Heme
Recombinant hemoglobins were obtained from Clara Fonticelli at the
University of Maryland School of Medicine. .beta.93Ala represents a
single amino acid substitution within human hemoglobin A, whilst
.beta.93Cys represents a wild type control. Recombinant hemoglobin
(5 .mu.M containing either a wild type cysteine (.beta.93Cys) or a
mutant alanine (.beta.93Ala) at position .beta.93 was deoxygenated
as in FIGS. 15A and 15B. Nitric oxide (1 .mu.M) was added by
injection of 0.5 .mu.l of a 2 mM solution, stored under nitrogen.
The final heme:NO ratio was 20:1. The solution was gradually
oxygenated by sequential additions of 10 .mu.l of room air. The
absorption at 418 nm of difference spectra versus initial
deoxyhemoglobin spectra is shown in FIG. 16. These data indicate
that within the mutant, a nitrosyl adduct was formed that was not
lost upon addition of room air. However, the nitrosyl adduct formed
within the wild type was lost after addition of greater than 10
.mu.l of room air. This shows that NO is not lost from this
nitrosyl (FeII) heme in a mutant Hb that does not possess a thiol
residue at position .beta.93. Therefore, this thiol, which is in
close proximity to the heme within the R-structure, is critical for
destabilizing the heme nitric oxide bond.
EXAMPLE 19
SNO-Hb From Nitrosyl-Hb Driven by O.sub.2
Hemoglobin A (400 .mu.M) was prepared in a 1 ml solution, in 100 mM
phosphate (pH 7.4). Nitric oxide was added by injection of an
appropriate volume of a 2 mM solution, stored under nitrogen. The
solutions were vortexed vigorously in an open container. Samples
were then analyzed by Saville assay and by chemiluminescence after
UV photolysis. The results in FIG. 17 show that S-nitrosothiol Hb
can be formed from oxyHb, but that the efficiency of this formation
is critically dependent upon the ratio of heme to nitric oxide.
EXAMPLE 20
Formation of Oxidized Hb Dependent on Protein Concentration
Hemoglobin A was diluted to the concentrations indicated by the
different symbols in FIG. 18A and FIG. 18B, in 50 ml of 100 mM
phosphate buffer (pH 7.4). Nitric oxide was added by sequential
injections of an appropriate volume of a 2 mM solution, stored
under nitrogen. After each injection, the absorbance at 415 and 405
nm was measured. The ratio of these two absorbances was used to
calculate the percentage content of oxidized hemoglobin (FIG. 18A),
and the absolute yield of oxidized hemoglobin (FIG. 18B).
.diamond-solid. represents 1.26 .mu.M hemoglobin, .box-solid.
represents 5.6 .mu.M hemoglobin, .tangle-solidup. represents 7.0
.mu.M hemoglobin, X represents 10.3 .mu.M hemoglobin, {character
pullout}represents 13.3 .mu.M hemoglobin, and .circle-solid.
represents 18.3 .mu.M hemoglobin. These data show that only a small
proportion of the nitric oxide added results in the formation of
oxidized hemoglobin (<10%). Furthermore, this tendency to form
oxidized hemoglobin is reduced at higher protein
concentrations.
EXAMPLE 21
Effect of Ionic Strength and NO: Hb Ratio on Extent of MetHb
Formation
We proposed that the degree of hydrogen bonding between bound
oxygen and the distal histidine was critical in determining the
degree of oxidation of hemoglobin by nitric oxide. Therefore, we
examined the degree of oxidation of hemoglobin by nitric oxide in a
variety of buffers. 5 ml of phosphate buffer containing 300 .mu.M
hemoglobin A (.about.95% oxyHb) was placed in a 15 ml vial. Nitric
oxide was added from a stock solution, 2 mM, stored under nitrogen.
Immediately after nitric oxide addition, the absorbance at 630 nm
was measured, and the concentration of oxidized (metHb) was
plotted, using 4.4 as the extinction coefficient for metHb at 630
nm. Experiments were performed in 1 M, 100 mM, and 10 mM sodium
phosphate buffer (pH 7.4). The data in FIG. 19 show higher oxidized
hemoglobin formation in 1 M phosphate, which is indicative of a
higher effective substrate concentration, as would be predicted by
phosphate destabilization of the hydrogen bond between iron bound
oxygen and the distal histidine. At the lowest concentrations of
nitric oxide added, S-nitrosothiol was formed under all conditions
(approximately 5 .mu.M). Additions of nitric oxide at
concentrations of 30 .mu.M or greater resulted in the additional
formation of nitrite. The presence of 200 mM borate within the
buffer reduced oxidized hemoglobin and nitrite formation, whilst
the presence of either 200 mM or chloride increased the formation
of oxidized hemoglobin and nitrite. Addition of nitric oxide to
hemoglobin in 10 mM phosphate buffer at a ratio of less than 1:30
(NO: Hemoglobin A) resulted in the formation of S-nitrosothiol
without production of oxidized hemoglobin. S nitrosothiol formation
was optimized by adding the nitric oxide to hemoglobin in 10 mM
phosphate, 200 mM borate, pH 7.4. Therefore, the balance between
oxidation and nitrosothiol formation is dependent upon the ratio of
nitric oxide to hemoglobin and the buffer environment.
EXAMPLE 22
Oxygen-Dependent Vasoactivity of S-Nitrosohemoglobin Contraction of
Blood Vessels in R-Structure and Dilation in T-Structure
The details of this bioassay system have been published (Osborne,
J. A., et al., J. Clin. Invest. 83:465-473 (1989)). In brief, New
Zealand White female rabbits weighing 3-4 kg were anesthetized with
sodium pentobarbital (30 mg/kg). Descending thoracic aorta were
isolated, the vessels were cleaned of adherent tissue, and the
endothelium was removed by gentle rubbing with a cotton-tipped
applicator inserted into the lumen. The vessels were cut into 5-mm
rings and mounted on stirrups connected to transducers (model TO3C,
Grass Instruments, Quincy, Mass.) by which changes in isometric
tension were recorded. Vessel rings were suspended in 7 ml of
oxygenated Kreb's buffer (pH 7.5) at 37.degree. C. and sustained
contractions were induced with 1 .mu.M norepinephrine.
Best attempts were made to achieve equivalent baseline tone across
the range of oxygen concentrations; i.e., hypoxic vessels were
contracted with excess phenylephrine. Oxygen tension was measured
continuously using O.sub.2 microelectrodes (Model 733 Mini; Diamond
General Co., MI) (Young, W., Stroke, 11:552-564 (1980); Heiss, W.
D. and Traupett, H., Stroke, 12:161-167 (1981); Dewhirst, M. W. et
al., Cancer Res., 54:3333-3336 (1994); Kerger, H. et al., Am. J.
Physiol., 268:H802-H810 (1995)). Less than 1% O.sub.2 corresponds
to 6-7 torr. Hypoxic vessels were contracted with excess
phenylephrine to maintain tone. SNO-Hb[FeII]O.sub.2 (SNO-oxyHb)
preparations were synthesized and quantified as in Example 27; GSNO
was prepared and assayed as described in Stamler, J. S. and
Feelisch, M., "Preparation and Detection of S-Nitrosothiols," pp.
521-539 in Methods In Nitric Oxide Research (M. Feelisch and J. S.
Stamler, eds.), John Wiley & Sons Ltd., 1996.
Hemoglobin is mainly in the R (oxy)-structure in both 95% O.sub.2
or 21% O.sub.2 (room air) (M. F. Perutz, pp. 127-178 in Molecular
Basis of Blood Diseases, G. Stammatayanopoulos, Ed. (W. B. Saunders
Co., Philadelphia, 1987); Voet, D. and Voet, J. G. (John Wiley
& Sons Inc., New York, 1995) pp. 215-235). Hb and SNO-Hb both
contract blood vessels over this range of O.sub.2 concentrations.
That is, their hemes sequester NO from the endothelium. The
functional effects of these hemoproteins in bioassays are not
readily distinguished (FIG. 20A). Concentration-effect responses of
SNO-Hb are virtually identical to those of native Hb in 95% O.sub.2
--i.e., in R-structure (curves are not different by ANOVA; n=12 for
each data point). Comparable contractile effects were seen with up
to 50 .mu.M SNO-oxyHb/oxyHb--i.e., at doses where the responses had
plateaued. Similar concentration-effect responses were observed in
21% O.sub.2 under which condition Hb/SNO-Hb is .about.99%
saturated.
On the other hand, hypoxia (<1% O.sub.2 [.about.6 mm Hg]
simulating tissue PO.sub.2) which promotes the T-structure (M. F.
Perutz, pp. 127-178 in Molecular Basis of Blood Diseases, G.
Stammatayanopoulos, Ed. (W. B. Saunders Co., Philadelphia, 1987);
Voet, D. and Voet, J. G. (John Wiley & Sons Inc., New York,
1995) pp. 215-235), differentiates Hb and SNO-Hb activities: Hb
strongly contracts blood vessels in T structure whereas SNO-Hb does
not (FIG. 20B). Concentration-effect responses of SNO-Hb and Hb are
significantly different <1% O.sub.2 (.about.6 torr), i.e. in
T-structure. Native deoxyHb is a powerful contractile agent whereas
SNO-deoxyHb has a modest effect on baseline tone. (In most
experiments SNO-Hb caused a small degree of contraction at lower
doses and initiated relaxations at the highest dose; in some
experiments (see FIG. 21C) it caused dose-dependent relaxations.)
n=13 for each data point; *P<0.05; ***P<0.001 by ANOVA.
SNO-Hb relaxations are enhanced by glutathione through formation of
S-nitrosoglutathione (GSNO) (FIG. 20C). The potentiation of SNO-Hb
vasorelaxation by glutathione is inversely related to the PO.sub.2
(FIG. 20C) because NO group transfer from SNO-Hb is promoted in the
T-structure. Specifically, transnitrosation of glutathione by
SNO-Hb--forming the vasodilator GSNO--is accelerated in T-structure
(<1% O.sub.2). Addition of 10 .mu.M glutathione to bioassay
chambers potentiates the vasorelaxant response of SNO-Hb. The
potentiation is greatest under hypoxic conditions; i.e., the curve
for <1% O.sub.2 shows a statistically significant difference
from both the 95% and 21% O.sub.2 curves (P<0.001), which are
not different from one another by ANOVA (n=6 for all data points).
High concentrations of glutathione (100 .mu.M-1 mM) further
potentiate SNO-Hb relaxations, such that the response is virtually
identical to that seen in the presence of GSNO in FIG. 20D.
Glutathione at 10 .mu.M has no effect on native Hb
contractions.
In contrast, the vasorelaxant effects of S-nitrosoglutathione are
largely independent of PO.sub.2 (FIG. 20D) and unmodified by
superoxide dismutase. (Data not shown.) Concentration-effect
responses of S-nitrosoglutathione (GSNO) are largely independent of
PO.sub.2 in the physiological range (n=6 at each data point).
Results are consistent with known resistance of GSNO to O.sub.2
/O.sub.2.sup.- inactivation (Gaston, B. et al., Proc. Natl. Acad.
Sci. USA, 90:10957-10961 (1993)). Thus, in T-structure, relaxation
by SNO overwhelms the contraction caused by NO scavenging at the
heme, whereas the opposite is true in R-structure.
EXAMPLE 23
Bioactivity of Intraerythrocytic S-Nitrosohemoglobin (SNO-RBCs)
Contractile effects of red blood cells are reversed by
intracellular SNO-Hb in low but not high PO.sub.2 --i.e., under
conditions that promote the T-structure. Low and high dose effects
of SNO-RBCs are shown in FIGS. 21A and 21B, respectively.
Preparation of vessel rings and methods of bioassay are described
in Example 22. SNO-oxyHb was synthesized and quantified as
described in Example 27. Red blood cells containing SNO-Hb
(SNO-RBCs) were synthesized by treatment with tenfold excess
S-nitrosocysteine over hemoglobin for 5-10 min. Under this
condition, red blood cells are bright red and contain SNO-oxyHb;
metHb was not detectable in these experiments.
Red blood cells containing SNO-Hb (SNO-RBCs) function in vessel
ring bioassays like cell-free SNO-Hb. In particular, low
concentrations of SNO-RBCs (.about.0.1 .mu.M SNO-Hb) elicited
modest contractile effects in 95% O.sub.2, but not under hypoxia
(FIG. 21A). In 95% O.sub.2, both SNO-RBCs (.about.0.1 .mu.M
SNO-Hb[FeII]O.sub.2) and native RBCs produced modest contractile
effects that were not readily distinguished. The contractions by
RBCs tended to be greater under hypoxic conditions (<1%
O.sub.2), whereas those of SNO-RBCs were reversed (slight relaxant
effects were seen). These O.sub.2 -dependent responses of SNO-RBCs
closely resemble those of cell-free preparations. Hemolysis was
minor and could not account for the observed effects.
At higher concentrations, SNO-RBCs produced small transient
relaxations in 95% O.sub.2 and larger sustained relaxations under
hypoxia (FIG. 21B), much like cell-free SNO-Hb in the presence of
glutathione. For example, SNO-RBCs (.about.1 .mu.M
SNO-Hb[FeII]O.sub.2) caused 32.5.+-.1.2% relaxation that lasted
14.5.+-.0.7 min. in 95% O.sub.2 versus 61.+-.10% relaxation that
lasted 23.+-. min. in <1% O.sub.2 (n=3-4; P<0.05) In
contrast, red blood cells containing no SNO-Hb produced small
contractions (less than those of cell-free Hb) that are potentiated
by hypoxia (13.+-.2.0% in 95% O.sub.2 vs. 25.+-.5% in <1%
O.sub.2 ; P<0.05). Hemolysis in these experiments was minor and
could not account for the extent of relaxation by SNO-RBCs.
In 95% O.sub.2, SNO-RBCs (.about.1.mu.M SNO-Hb[FeII]O.sub.2)
produced relaxations of aortic rings, whereas native RBCs produced
slight contractions. Both effects were more prominent at low
PO.sub.2. That is, relaxations and contractions of
intraerythrocytic SNO-Hb and Hb, respectively, were greater and
longer-lived in <1% O.sub.2 than in 95% O.sub.2. The O.sub.2
-dependent responses of SNO-RBCs mimicked those of cell-free SNO-Hb
in the presence of glutathione. Hemolysis in these experiments was
minor and could not account for the extent of relaxation by
SNO-RBCs.
The normal response of systemic arteries to hypoxia is dilation,
and to high PO.sub.2, contraction. The responses of vessel rings to
changes in PO.sub.2 in the presence of SNO-Hb and Hb were tested
(FIG. 21C). Vessel rings were contracted with phenylephrine under
hypoxic conditions (6-7 torr) and then exposed to either 1 .mu.M Hb
or SNO-Hb. Hb produced progressive increases in vessel tone, while
SNO-Hb caused relaxations. Introduction of 95% O.sub.2 led to rapid
contractions in both cases. Thus, structural changes in SNO-Hb
effected by PO.sub.2 are rapidly translated into contractions or
relaxations, whereas Hb contracts vessels in both R- and
T-structures. Thus, Hb opposes the physiological response and
SNO-Hb promotes it (FIG. 21C). Direct effects of O.sub.2 on smooth
muscle operate in concert with SNO-Hb to regulate vessel tone.
EXAMPLE 24
Influence of O.sub.2 Tension on Endogenous Levels of
S-Nitrosohemoglobin (SNO/Hb) and Nitrosyl Hemoglobin (FeNO/Hb)
Allosteric control of SNO-Hb by O.sub.2 was assessed in vivo by
perturbation of the periarteriolar oxygen gradient. In animals
breathing room air (21% O.sub.2) a the precapillary resistance
vessels (100-10 .mu.m) are exposed to PO.sub.2 s as low as 10-20
torr (Duling, B. and Berne, R. M. Circulation Research, 27:669
(1970); Popel, A. S., et al., (erratum Am. J. Physiol. 26(3) pt. 2)
Am. J. Physiol. 256, H921 (1989); Swain, D. P. and Pittman, R. N.,
Am. J. Physiol. 256, H247-H255 (1989); Buerk, D. et al., Microvasc.
Res., 45:134-148 (1993)) (confirmed here) which promotes the
T-structure in Hb. Raising the inspired oxygen concentration to
100% translates to periarteriolar PO.sub.2 S only as high as 40 mm
Hg (Duling, B. and Berne, R. M. Circulation Research, 27:669
(1970); Popel, A. S., et al., (erratum Am. J. Physiol. 26(3) pt.
2). Am. J. Physiol. 256, H921 (1989); Swain, D. P. and Pittman, R.
N. Am. J. Physiol. 256, H247-H255 (1989); Buerk, D. et al.,
Microvasc. Res., 45:134-148 (1993)); i.e., breathing 100% O.sub.2
may not fully maintain the R-structure in Hb in the
microcirculation. Elimination of the periarteriolar O.sub.2
gradient (artery-arteriole and arterial-venous difference in
PO.sub.2) is accomplished in hyperbaric chambers by applying 3
atmospheres of absolute pressure (ATA) while breathing 100% O.sub.2
(Tibbles, P. M. and Edelsberg, J. S., N.E.J.M., 334:1642-1648
(1996)).
Adult male Sprague-Dawley rats (290-350 g) were anesthetized with
sodium pentobarbital (50 mg/kg IP), intubated and ventilated with a
small animal respirator (Edco Scientific Inc., Chapel Hill, N.C.)
at a rate and tidal volume to maintain normal values of PaCO.sub.2
(35-45 mm Hg; PaCO.sub.2 =systemic arterial blood carbon dioxide
tension) The femoral vein and artery were cannulated for infusion
of drugs and for continuous monitoring of systemic blood pressure,
respectively. Aliquots of arterial blood (200 .mu.l) were drawn
periodically to measure blood gas tensions and pH (Instrumentation
Laboratory Co., model 1304 blood gas/pH analyzer). The blood was
replaced intravenously with three volumes of normal saline. The
inspired O.sub.2 concentration was varied using premixed gases
balanced with nitrogen. The tissue PO.sub.2 was measured
continuously with polarographic platinum microelectrodes (50 .mu.m
O.D. coated with hydrophobic gas permeable Nafion) implanted
stereotaxically in both the right and left hippocampus (AP-3.4 mm,
ML+2.2 mm), caudate putamen nucleus and substantia nigra (see
coordinates below) (Young, W., Stroke, 11:552-564 (1980); Heiss, W.
D. and Traupett, H., Stroke, 12:161-167 (1981); Dewhirst, M. W. et
al., Cancer Res., 54:3333-3336 (1994); Kerger, H. et al., Am. J.
Physiol., 268:H802-H810 (1995)). The PO.sub.2 electrodes were
polarized to -0.65 V against a distant Ag/AgCl reference located on
the tail and the current flow was measured using a low-impedance
nA-meter. Regional arterial PO.sub.2 was adjusted by changing the
inspired O.sub.2 concentration and atmospheric pressure.
Polarographic hydrogen (H.sub.2)-sensitive microelectrodes were
implanted stereotaxically in the substantia nigra (AP -5.3 mm, ML
-2.4 mm to the bregma, depth 3.2 mm), caudate putamen nucleus (CPN)
(AP +0.8 mm, ML -2.5 mm, depth 5.2 mm) and parietal cortex, for
measurement of regional blood flow (Young, W., Stroke, 11:552-564
(1980); Heiss, W. D. and Traupett, H., Stroke, 12:161-167 (1981)).
The microelectrodes were made from platinum wire and insulated with
epoxy, with the exception of the tip (1 mm) which was coated with
Nafion. For placement, the electrodes were mounted on a
micromanipulator and the rat's head was immobilized in a Kopf
stereotaxic frame. H.sub.2 -sensitive electrodes were polarized to
+400 mV against a distant reference electrode on the tail, and the
polarographic current was measured using a low-impedance nA meter
during and after the inhalation of hydrogen gas (2.5%) for 1 min.
Both the hydrogen clearance curves and voltage for oxygen
measurements were made using PC WINDAQ (software, DI-200 AC, Dataq
Instruments, Inc., Akron, Ohio). Cerebral blood flow was calculated
using the initial slope method (Young, W., Stroke, 11:552-564
(1980); Heiss, W. D. and Traupett, H., Stroke, 12:161-167 (1981)).
Regional blood flow responses were monitored for 30 min. prior to
and 30 min. following drug administration; hemoglobins were given
at time 0.
Blood was drawn from indwelling catheters in the carotid artery
(arterial blood that perfuses the brain) and superior vena
cava/right atrium (venous return to the heart) of 5 rats exposed
first to room air (21% O.sub.2) and then 100% O.sub.2 +3 ATA in a
hyperbaric chamber. Levels of SNO-Hb and nitrosyl Hb (Hb[Fe]NO)
were determined from these samples as a measure of SNO-Hb and
nitrosyl Hb (Hb[Fe]NO; FeNO/Hb in FIG. 22) in blood that perfuses
the brain.
Blood samples were transported on ice for immediate processing and
analysis. After centrifugation at 800 g for 10 min, the packed red
blood cells were isolated, washed with a 2-fold volume excess of
PBS, pH 7.4, resuspended, and the PBS removed after a further
centrifugation. Hemolysis was then accomplished by incubation for
10 min with 4-fold excess deionized water containing 0.5 mM EDTA,
followed by purification of hemoglobin by rapidly desalting over a
G-25 Sephadex chromatography spin column (10 to 30-fold volume
excess) in PBS at room temperature. Total Hb concentration was
determined by the visible spectrophotometric method. Hb species
present were converted to Hb(FeII)NO (by addition of dithionite in
the presence of excess S-nitrosocysteine), which was then measured
using the millimolar extinction coefficient of 135.4 at 418 nm.
Each sample of hemoglobin was diluted to 200 .mu.M and paired
aliquots were treated with an equal volume of either distilled
water or 7.5-fold molar excess HgCl.sub.2 (which selectively
cleaves thiol-bound NO). Higher concentrations of HgCl.sub.2 cause
Hb to precipitate (A. F. Riggs, R. A. Wolbach, J. Gen. Physiol.
39:585, 1956). The HgCl.sub.2 concentration can be reduced to
4-fold excess over protein with quite comparable results. A 6-fold
excess HgCl.sub.2 concentration and an incubation time of 1-10
minutes has been used also (determined empirically for each
reaction mixture). Organic mercurials have been used instead of
hemoglobin. They do not cause precipitation, even at high
concentrations, but they react more slowly. In all cases, the
mercurial selectively cleaves the NO group from thiols and
preserves binding at the heme. NO was measured by the
photolysis-chemiluminescence method, in which NO is photolytically
liberated from thiols (SNO-Hb) or hemes (Hb[FeII]NO) and the
chemiluminescent product of its reaction with ozone is measured.
Standard curves were generated using S-nitrosoglutathione. See
"Assay for S-Nitrosohemoglobin and Nitrosyl(FeII)-Hemoglobin" in
Materials and Methods for Assays section at beginning of
Exemplification and Methods section for Example 8.
The mean O.sub.2 saturation of venous blood (room air) was 69%; of
arterial blood (room air) was 93%; of venous blood (100%+3 ATA) was
also 93% and of arterial blood (100%+3 ATA) was 100% (FIG. 22).
Numerous statistical comparisons were highly significant. For
example, SNO-Hb venous 100% O.sub.2 +3 ATA vs. SNO-Hb venous 21%
O.sub.21, P=0. 004; and nitrosyl Hb venous 21% O.sub.2 vs. arterial
21% O.sub.2 P=0.008. On the other hand, SNO-Hb and nitrosyl Hb were
not different in artery 21% O.sub.2 compared with venous 100%+3 ATA
(which have identical O.sub.2 saturations), nor did the differences
reach significance between venous and arterial 100% O.sub.2 +3 ATA.
n=5 for all measurements.
In 21% O.sub.2, venous blood contained mostly nitrosyl Hb, whereas
arterial blood contained significant amounts of SNO-Hb (FIG. 22).
On the other hand, SNO-Hb predominated in both arterial and venous
blood in 100% O.sub.2 +3 ATA (FIG. 22). In hyperbaric conditions,
the tissues are oxygenated primarily by O.sub.2 dissolved in
plasma. Physiologically circumventing the unloading of O.sub.2 by
Hb alters the endogenous SNO/nitrosyl Hb balance. The data show
that SNO-Hb appears to form endogenously in R-structure whereas SNO
is released in the T-structure (compare venous 21% O.sub.2
(T-state) with arterial 100% O.sub.2 +3 ATA (R-state)).
This structure-function relationship in vivo is consistent with
both the in vitro pharmacology and the molecular model suggesting
that 1) O.sub.2 is an allosteric effector of Hb S-nitrosylation; 2)
binding of NO to hemes of Hb is favored in the T-structure; (some
of the NO released during arterial-venous (A-V) transit appears to
be autocaptured at the hemes) and 3) maintaining endogenous SNO-Hb
in the R-structure by eliminating the A-V O.sub.2 gradient
preserves levels of SNO (compare venous 100% O.sub.2 +3 ATA with
arterial 21% O.sub.2). Thus, it can be predicted that SNO-Hb should
improve cerebral blood flow in 21% O.sub.2 under which condition
SNO is readily released during A-V transit, but not under the
hyperoxic conditions that maintain the R-structure in artery and
vein.
EXAMPLE 25
O.sub.2 -Dependent Effects of SNO-Hb and Hb on Local Cerebral Blood
Flow
The cerebrovascular effects of SNO-Hb were measured in adult male
Sprague-Dawley rats using O.sub.2 and H.sub.2 (blood
flow)-sensitive microelectrodes that were placed stereotaxically in
several regions of the brain as for Example 24.
SNO-Hb increases blood flow under tissue hypoxia, whereas it
decreases blood flow under hyperoxia. In contrast, Hb decreases
blood flow irrespective of the PO.sub.2. Comparative effects of
SNO-Hb (.circle-solid.) and Hb (.box-solid.) (1 .mu.mol/kg infused
over 3 minutes) on local blood flow in substantia nigra (SN),
caudate putamen nucleus, and parietal cortex are shown for three
different conditions. In 21% O.sub.2, SNO-Hb improved blood flow in
all three regions of the brain tested, whereas native Hb decreased
local blood flow, paradoxically attenuating O.sub.2 delivery to
hypoxic tissues (FIGS. 23A, 23B and 23C; all curves are highly
statistically significantly different from one another and from
baseline by ANOVA). In rats breathing 100% O.sub.21 where the
periarteriolar O.sub.2 gradient has been essentially eliminated,
the increase in flow to SNO-Hb was significantly attenuated (i.e.,
only the SN increase reached statistical significance), but the
Hb-mediated decrease in flow was preserved (FIGS. 23D, 23E and 23F;
all curves remain different from one another by ANOVA to
P<0.05). In 100% O.sub.2 +3 ATA, both SNO-Hb and Hb tended to
decrease cerebral flow to similar extents (FIGS. 23G, 23H and 23I;
curves are not different by ANOVA). S-nitrosoglutathione (GSNO)
increased brain perfusion in 100% O.sub.2 and 100% O.sub.2 +3 ATA,
reversing protective vasoconstriction. Baseline blood flow was
decreased by .about.10% under 100% O.sub.2 +3 ATA as compared to
100% O.sub.2. n=7 for all data points. Values of
tissue/microvascular PO.sub.2 ranged from 19-37 mm Hg in 21%
O.sub.2 ; from 68-138 mm Hg in 100% O.sub.2 ; and from 365-538 mm
Hg in 100%+3 ATA (Duke University Medical Center Hyperbaric
Chambers).
The effects of Hb and SNO-Hb on local blood flow in the parietal
cortex can be seen by comparing the results shown in FIGS. 23C, 23I
and 23F. In 21% O.sub.2 (tissue/microvascular PO.sub.2 19 to 37 mm
Hg), Hb reduced blood flow whereas SNO-Hb augmented blood flow
(FIG. 23C). The increase in blood flow in response to SNO-Hb was
significantly attenuated in 100% O.sub.2 (FIG. 23F; tissue PO.sub.2
68 to 138 mm Hg) and was converted to decreases in flow in 100%
O.sub.2 plus 3 ATA (FIG. 23I; tissue PO.sub.2 365 to 538 mm Hg). In
contrast, responses to Hb or GSNO were not oxygen-dependent: Hb
decreased blood flow while GSNO increased it, irrespective of
PO.sub.2. Thus, SNO-Hb uniquely regulates blood flow in response to
the physiological oxygen gradient in resistance arterioles.
SNO-Hb acts like native Hb (net NO scavenger) when it is in the R
(oxy)-structure and like GSNO (net NO donor) in the T
(deoxy)-structure. The results are consistent with the conclusion
that SNO-Hb is a nitrosothiol whose vasoactivity is allosterically
controlled by PO.sub.2.
EXAMPLE 26
Hemodynamics of Cell Free and Intraerythrocytic SNO-Hb, Hb and GSNO
at Different O.sub.2 Concentrations
Rats were anesthetized by intraperitoneal injection of
pentobarbital, and the femoral arteries and veins accessed by local
cutdown. The artery was then cannulated and the blood pressure
monitored continuously using a P23 XL pressure transducer (Viggo
Spectramed, Oxnard, Calif.) attached to a Gould recorder. The
femoral vein was used for infusion of drugs and red blood cells
containing SNO-Hb (1 ml over 1 min.) and an IBM PC (WINDAQ 200,
Dataq Instruments, Inc.; Akron, Ohio) was used for data
acquisition.
Drugs were infused through the femoral vein at 1 .mu.mol/kg infused
over 1 minute after blood pressure had stabilized (approximately 30
min). Measurements shown (FIG. 24A) were taken at 10 min.
post-infusion of drug. Similar responses were seen at 3 and 20 min.
SNO-Hb produced significantly less of an increase in blood pressure
than Hb (P<0.05), whereas GSNO decreased blood pressure.
P<0.05 vs. SNO-Hb; *P<0.05, **P<0.01) vs. baseline blood
pressure. n=5-6 for each drug.
Infusions of SNO-RBCs also lowered blood pressure consistent with a
GSNO-like effect (FIG. 24B). SNO-RBCs produced dose-dependent
hypotensive effects (similar to those of cell-free SNO-Hb)
(P<0.001 at all points vs. baseline). The hypotensive effects of
SNO-RBCs were potentiated by pre-administration of the NO synthase
inhibitor N.sup.G -monomethyl-L-arginine (L-NMMA; 50 mg/kg). n=8
for each data point. Curves different by ANOVA (P<0.01),
*P<0.05 vs. L-NMMA. The amount of hemolysis in these experiments
was trivial. Infusion of the hemolysate had no effect on blood
pressure.
NO synthase inhibition increases tissue O.sub.2 consumption by
relieving the inhibition of mitochondrial respiration produced by
NO in the tissues (King, C. E. et al., J. Appl. Physiol.,
76(3):1166-1171 (1994); Shen, W. et al., Circulation, 92:3505-3512
(1995); Kobzik, L. et al., Biochem. Biophys. Res. Comm.,
211(2):375-381 (1995)). This should, in turn, increase the
periarteriolar O.sub.2 gradient which might explain some of the
potentiation. However, other factors, such as a change in tone or
distribution of blood flow imposed by L-NMMA, may well contribute.
The effects of SNO-Hb on blood pressure are consistent with SNO
being released in resistance arterioles to compensate for NO
scavenging at the heme iron.
EXAMPLE 27
Synthesis of S-Nitrosohemoglobins Materials and Methods
L-cysteine hydrochloride, glutathione, sulfanilamide, and
N-(1-naphthyl)ethylenediamine (NED) were purchased from Sigma
Chemical Co. (St. Louis, Mo.). Sodium nitrate and potassium
ferricyanide were purchased from Aldrich Chemical Co. (Fairlawn,
N.J.). G-25 Sephadex (fine) was purchased from Pharmacia Biotech
(Uppsala, Sweden). Purified (.about.99.00%) human HbA.sub.0 was
prepared as previously described, and was stored at -80.degree. C.
(R. G. Kilbourn, G. Joly, B. Cashon, J. DeAngelo, J. Bonaventura.
Biochem. Biophys. Res. Comm. 199:156, 1994). The final buffer was
lactated Ringer's solution, pH 7.4. Nitric oxide solutions were
prepared in tonometers by bubbling high-purity, KOH-scrubbed NO gas
through rigorously degassed solutions of phosphate-buffered saline
(PBS) or deionized water. EDTA is ethylenediaminetetraacetic
acid.
Synthesis of S-Nitroso-Oxyhemoglobin (SNO-Hb[Fe(II)]O.sub.2)
The principal technical challenges in synthesis of
S-nitrosooxyhemoglobin (SNO-Hb[FeII]O.sub.2) are to selectively
nitrosylate specific thiols and to avoid oxidation of the heme. The
method of preparation of SNO-Hb(FeII)O.sub.2 is thus quite
different from that previously described for the synthesis of other
S-nitrosoproteins (J. S. Stamler, D. I. Simon, J. A. Osborne, M. E.
Mullins, O. Jaraki, T. Michel, D. J. Singel, J. Loscalzo. Proc.
Natl. Acad. Sci. 89:444, 1992). The rate of S-nitrosylation is
accelerated, while the rate of oxidation of the hemes is slowed, in
alkaline buffer.
Hemoglobin (Hb)A.sub.0 was purified from human red blood cells as
previously described (Kilbourn, R. G. et al., Biochem. Biophys.
Res. Commun., 199:155-162 (1994)). HbA.sub.0 (0.5-1.0 mM) was
dialyzed against 2% aerated borate, 0.5 mM EDTA (pH 9.2) at
4.degree. C. for 12-16 hours. The oxyHb concentration was
determined based on the optical absorbance at 577 nm (i.e., using
the millimolar extinction coefficient 14.6). An excess of
nitrosylating agent is used for effective synthesis, but steps must
be taken to ensure the selective modification of Cys.beta.93 (over
other thiols and hemes in Hb).
Hb was reacted with 10-fold molar excess S-nitrosocysteine (CysNO)
which was synthesized immediately before use in high concentration
by a modification of standard procedure (see, for example, Stamler,
J. S. and Feelisch, M., "Preparation and Detection of
S-Nitrosothiols," pp. 521-539 in Methods In Nitric Oxide Research,
M. Feelisch and J. S. Stamler, eds., John Wiley & Sons Ltd.,
1996) as follows. L-cysteine hydrochloride (1.1 M) dissolved in 0.5
N HCl/0.5 mM EDTA was reacted with an equal volume of 1 M
NaNO.sub.2 (sodium nitrite) dissolved in water, to form CysNO (the
ratio of cysteine to nitrite influences the SNO-Hb product and
activity profile), and is neutralized prior to addition to the
hemoglobin solution by dilution in 100-200 mM PBS (pH 7.4 to 8.0,
with 0.5 mM EDTA). The concentration of CysNO was then adjusted by
dilution in PBS, pH 8.0, to yield a working CysNO solution (pH
6-7). oxyhemoglobin (>100 .mu.M in borate, pH 9.1-9.2) was
S-nitrosylated by incubation with a 10-fold molar excess of CysNO
over Hb (ratio influences product critically). Periods of
incubation are determined by the desired synthetic preparation;
i.e., a desired ratio of SNO/tetramer, desired met- to oxy- to
nitrosyl-Hb ratios, polynitrosated or non-polynitrosated. For
example, 10 min. is a preferred time for SNO-oxyHb with 2 SNO per
tetramer. The reaction was stopped by rapid transfer of the
reaction mixture to a column of fine Sephadex G-25 (bed volume
should be 10 to 30-fold excess over that of the reaction mixture)
preequilibrated with 100 mM PBS pH 7.4, 0.5 mM EDTA. Typically, a
150 .mu.l sample of the mixture was added to a 4.5 ml column
measuring 12 mm (inner diameter). The column was then centrifuged
at 800-1200 g for 60 seconds and the effluent containing purified
SNO-oxyHb collected in a 1.5 ml airtight plastic vial that was
subsequently kept on ice and protected from light.
Total Hb concentration was determined by the sequential addition of
dithionite and excess CysNO, which results in the conversion of the
Hb species present to Hb(FeII)NO. The millimolar extinction
coefficient of Hb(FeII)NO is 135.4 (based on heme) at 418 nm.
SNO-Hb(FeII)O.sub.2 is quite stable, although it is best that
samples be made fresh daily.
The S-nitrosothiol content of SNO-Hb was determined by a
modification of the method of Saville. By this method, Hg.sup.++
(derived from HgCl.sub.2 or from organic mercurials such as
4-(chloromercuri)benzenesulfonate; 5 to 10-fold excess over
Hb)-displaced NO equivalent was assayed by the diazotization of
sulfanilamide and the subsequent coupling with the chromophore
N-(1-napthyl)ethylenediamine (NED). From measurements of the
optical density at 540 nm, SNO concentrations were determined
against those of the standard, S-nitrosoglutathione (GSNO). These
assays were carried out in a 96-well microplate reader (Molecular
Devices Corp. Sunnyvale, Calif.). Each sample well contained 5 Al
SNO-Hb/95 .mu.l 0.5 N HCl/100 .mu.l sulfanilamide/100 .mu.l NED.
Triton X-100 (0.03-0.1%) has been used if necessary to prevent
precipitation of hemoglobin.
Incubations of CysNO with HbA resulted in different synthetic
products (and different activities) over time. For example, with 10
min incubations, the Hb preparation contained 1.857.+-.0.058 SNO
groups per tetramer, and is approximately 12-15% metHb and 1-3%
nitrosyl(FeII)-hemoglobin. Capillary electrophoretic analysis
revealed a mixture of three protein peaks. MetHb was then reduced
(lowered from 13% to 2% with 100-fold excess NaCNBH.sub.3 dissolved
in PBS, pH 8.0) under anaerobic conditions (achieved by purging
with argon gas for a minimum of 10 minutes). Lower concentrations
of NaCNBH.sub.3 or treatment of the samples under aerobic
conditions were not effective in lowering the metHb concentration,
and alternative measures to reduce the heme resulted in SNO
reduction. The resulting mixture was rapidly added to a column of
fine Sephadex G-25 (20 to 30-fold volume excess) preequilibrated
with 100 mM PBS pH 7.4, 0.5 mM EDTA. The final S-nitrosothiol/Hb
tetramer ratios were not significantly different from those
measured in samples degassed and treated with PBS only: losses in
SNO/Hb ratio relative to the starting ratios were consistent with
the expected time-dependent decay of SNO-deoxyHb and could be
reduced to an insignificant loss by taking preparation time into
consideration. NaCNBH.sub.3 treatment of a sample with a mean
SNO/Hb ratio of .about.1 decreased the metHb content from 5.6% to
0.63%.
Variations in Methods of Synthesis
Nitrosyl(FeII)-hemoglobin and metHb contamination of SNO-oxyHb
preparations or compositions of less than about 2% are acceptable,
inasmuch as they do not seem to alter bioactivity of SNO-oxyHb, and
enable O.sub.2 binding measurements, that is, P.sub.50
determinations. Bioactivity can be modified and varied by
controlling the proportion of SNO-metHb (high and low spin) and
nitrosyl(FeII)-hemoglobin in the composition. The spin state of
metHb can be controlled by the heme ligand: cyan-metHb is low spin
and aquo-met Hb (H.sub.2 O bound as ligand) is high spin. The
desired proportion of nitrosylhemoglobin can be controlled (see,
inter alia, Example 16). High yield SNO-metHb,
SNO-nitrosyl(FeII)-hemoglobin can be formed by using the
heme-liganded protein as starting material. Carbomonoxy Hb can be
made by gassing with CO under anaerobic conditions. HbCO can then
be used as starting material to make SNO-carboxyl-Hb. Various
combinations of Hb[FeNO] [FeCO] can also be used as starting
material.
Synthesis of S-Nitroso-Deoxyhemoglobin (SNO-Hb[Fe(II)])
Isolated SNO-deoxyHb is synthesized in an anaerobic environment
(glove box) using the general approach described for SNO-oxyHb. The
hemoglobin solution and other materials for synthesis are allowed
to equilibrate overnight in the glove box. The UV-visible spectrum
of the Hb solution should be that of pure deoxyHb (Soret region
peak at 430 nm) before synthesis is initiated. Purified SNO-deoxyHb
samples are transferred to tonometers or sealed cuvettes before
removal from the glove box. Isolated SNO-deoxyHb is highly unstable
and must be used immediately.
Synthesis of S-Nitroso-Methemoglobin (Abbreviated as SNO-MetHb or
SNO-Hb[Fe(III)])
Isolated methemoglobin was produced by reacting oxyhemoglobin
(0.5-1.0 mM, pH 7.4 in 150 mM phosphate buffer solution, with 0.5
mM EDTA) with a 10-fold molar excess (over hemoglobin tetramer) of
either NaNO.sub.2 or potassium ferricyanide K.sub.3 Fe(CN).sub.6 at
room temperature for 10 min. The reaction mixture was desalted
across a column of fine G-25 Sephadex (10-fold volume excess,
preequilibrated with PBS, pH 7.4, with 0.5 mM EDTA) by
centrifugation. The completeness of conversion to methemoglobin was
then confirmed spectrophotometrically. (V. G. Kharitonov, J.
Bonaventura, V. S. Sharma. Interactions of nitric oxide with heme
proteins using UV-vis spectroscopy. In: Methods in Nitric Oxide
Research, M. Feelisch and J. S. Stamler, eds., 1996. John Wiley and
Sons Ltd., Chichester, England.) Methemoglobin was S-nitrosylated
by incubation with CysNO, with the duration determined by the
desired extent of S-nitrosylation, as described in the synthesis of
SNO-Hb(FeII)O.sub.2. The reaction was stopped by rapid transfer of
the reaction mixture to a G-25 Sephadex chromatography column (10
to 30-fold volume excess over reaction mixture), followed by rapid
centrifugation and collection of the effluent in a plastic vial.
Purified SNO-metHb is inherently unstable and should be
resynthesized at frequent intervals.
Measurement of NO/SNO Content of Partially Nitrosylated DeoxyHb and
S-Nitroso-Oxyhemoglobin in Reaction Mixtures Under Physiological
Conditions
Isolated SNO-Hb(FeII)O.sub.2 and Hb(FeII)NO were prepared at
relatively physiological ratios of NO to Hb. SNO-Hb(FeII)O.sub.2
was synthesized as described above and diluted to 1 .mu.M in 100
.mu.M Hb(FeII)O.sub.2 /PBS pH 7.4/0.5 mM EDTA, resulting in a 1:100
ratio of SNO-Hb(FeII)O.sub.2 to Hb(FeII)O.sub.2. Hb(FeII)NO (1
.mu.M NO: 100 .mu.M deoxy Hb) was prepared by the addition of
saturated NO solution to 100 .mu.M (final) deoxyHb/PBS pH 7.4/0.5
mM EDTA under anaerobic conditions. NO was measured by the
photolysis-chemiluminescence method in the absence (NO bound) or
presence (NO post Hg) of HgCl.sub.2 (final 600 .mu.M). Data (n=5)
in FIG. 25 represent mean .+-.SEM.
Equivalents
Those skilled in the art will know, or be able to ascertain using
no more than routine experimentation, many equivalents to the
specific embodiments of the invention described herein. These and
all other equivalents are intended to be encompassed by the
following claims.
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