U.S. patent application number 10/465475 was filed with the patent office on 2003-11-13 for monooxygenase assays.
Invention is credited to Chenna, Ahmed, Gibbons, Ian, Salimi-Moosavi, Hossein, Singh, Sharat.
Application Number | 20030211565 10/465475 |
Document ID | / |
Family ID | 22712020 |
Filed Date | 2003-11-13 |
United States Patent
Application |
20030211565 |
Kind Code |
A1 |
Singh, Sharat ; et
al. |
November 13, 2003 |
Monooxygenase assays
Abstract
Cytochrome P-450 assay methods and kits for the methods are
provided employing a cytochrome P-450 enzyme, substrates
characterized by having an oxidizable methylene group oxidized to
an aldehyde and a fluorescent hydrazine. A fluorescent hydrazine is
added to the reaction mixture and the resulting hydrazone analyzed
by capillary electrophoresis. The method finds use in evaluating
compounds for enzyme modulating activity.
Inventors: |
Singh, Sharat; (San Jose,
CA) ; Chenna, Ahmed; (Sunnyvale, CA) ;
Salimi-Moosavi, Hossein; (Sunnyvale, CA) ; Gibbons,
Ian; (Portola Valley, CA) |
Correspondence
Address: |
ACLARA BIOSCIENCES, INC.
1288 PEAR AVENUE
MOUNTAIN VIEW
CA
94043
US
|
Family ID: |
22712020 |
Appl. No.: |
10/465475 |
Filed: |
June 19, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10465475 |
Jun 19, 2003 |
|
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09820289 |
Mar 28, 2001 |
|
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60193034 |
Mar 29, 2000 |
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Current U.S.
Class: |
435/25 |
Current CPC
Class: |
G01N 2333/795 20130101;
Y10S 435/968 20130101; G01N 2333/90245 20130101; G01N 2333/80
20130101; C12Q 1/26 20130101 |
Class at
Publication: |
435/25 |
International
Class: |
C12Q 001/26 |
Claims
What is claimed is:
1. A kit comprising a cytochrome P-450 enzyme, at least one P-450
enzyme substrate comprising an ether group bonded to a methylene,
which is oxidized to an aldehyde by said P-450 enzyme, and a
fluorescent hydrazine.
2. A kit according to claim 1, wherein said P-450 enzyme is the 3A4
isoform and said fluorescent hydrazine is fluorescein
hydrazine.
3. A kit according to claim 1, wherein said P-450 enzyme substrate
is at least one of 7-benzyloxyresorufin or
7-benzyloxyquinoline.
4. A kit according to claim 1 wherein said fluorescein hydrazine is
N-(.alpha.-hydrazylacetyl) 5-aminofluorescein.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of application Ser. No.
09/820,289, filed Mar. 28, 2003, which claimed priority to
provisional application serial No. 60/193,034, filed Mar. 29, 2000,
which disclosures are incorporated herein by reference
FIELD OF THE INVENTION
[0002] The field of this invention is monooxygenase assays.
BACKGROUND OF THE INVENTION
[0003] Monooxygenase include the numerous isoforms of the
cytochrome P-450 enzymes. Because of the importance of these
enzymes, particularly their activity in the liver, there is
substantial interest in being able to assay for their activity and
identify compounds that can modulate that activity. These enzymes
serve to clear the blood of foreign factors. Unfortunately, in many
cases these foreign factors are drugs, whose half-life is
substantially diminished by virtue of being processed by the P-450
enzymes into inactive products. Also, reduced or modified activity
of the P-450 enzymes may lead to poisoning or sensitivity to
various agents, which in the normal person would be rapidly
detoxified. The enzyme preparations are very expensive and each
enzyme has multiple binding sites. Thus, multiple assays have to be
performed to screen the enzyme(s).
[0004] With the advent of nanotechnology, there is an increased
ability to perform numerous chemical and physical operations with
very small volumes. This opportunity comes with the requirement
that determinations have enhanced sensitivity to detect the fewer
molecules that are present to provide the detectable signal. Part
of the increased sensitivity may come from more sensitive
detectors, but these are usually more expensive and are not readily
available in most laboratories. The other opportunity is to provide
assays that are more efficient in providing for detectable
products, uses compounds that are readily accepted by the enzymes
as substrates, and provide products with a strong signal, for
fluorescent compounds, a high emission efficiency.
[0005] There is, therefore, substantial interest in providing P-450
assays that are rapid, accurate and can be performed in small
volumes with low levels of enzyme and expensive reagents.
BRIEF DESCRIPTION OF THE PRIOR ART
[0006] U.S. Pat. No. 5,179,013 and references cited therein
describe assaying for novel cytochrome P-450 enyzmes. Assays for
P-450 enzymes are also described in Schwaneberg, et al., Anal
Biochem 1999, 1:269:359-66; Tremblay, et al., Anal Biochem 1999,
276:215-26; Jansen, et al., J Chromatogr B Biomed Appl 1996,
684:133-45 and Eguchi et al., Xenobiotica 1996, 26:755-63. Other
references that may be of interest include Hartmann and Frotscher,
Arch Pharm 1999, 332:358-62; Ubeaud, et al., Eur J. Pharm Sci 1999,
8:255-60; Ertl, et al., Toxicol Appl Pharmcol 1999, 157:157-65; and
Sanderson, et al., Toxicol Appl Pharmacol 1996, 137:316-25.
SUMMARY OF THE INVENTION
[0007] Methods and compositions are provided for determinations of
monooxygenase enzymes, particularly P-450 enzymes, using a
substrate having an ether group comprising an oxidizable
a-hydrogen, resulting in an aldehyde product. The aldehyde is
reacted in situ with a fluorescent hydrazine and the resulting
reaction mixture separated by capillary electrophoresis. The
hydrazone product indicates the reaction occurrence and the area
under the peak may be used for quantitation. Kits can be provided
for performing the assay.
BRIEF DESCRIPTION OF THE FIGIIRES
[0008] FIG. 1 shows an electropherogram of the reaction of
benzaldehyde with fluorescein using capillary electrophoresis
technique (PAGE);
[0009] FIG. 2 shows a calibration curve for benzaldehyde and
fluorescein hydrazine to form the hydrazone;
[0010] FIG. 3 is a graph of a kinetic study of the reaction between
fluorescein-hydrazine and benzaldehyde;
[0011] FIG. 4 is a graph of the effect of concentration of
7-benzyloxyresorufin on enzymatic activity;
[0012] FIG. 5 is the effect of the enzyme concentration on
enzymatic activity;
[0013] FIGS. 6A and 6B are two calibration curves over different
concentration ranges for 7-benzyloxyresorufin;
[0014] FIGS. 7 and 8 are graphs of the effect of concentration of
7-benzyloxyquinoline and enzyme, respectively, on enzymatic
activity;
[0015] FIGS. 9 and 10 are graphs of the effect of a non-ionic
detergent on enzymatic activity with 7-benzyloxyquinoline as the
substrate, with varying concentration of substrate enzyme;
[0016] FIG. 11 is a graph of comparison calibration curves in the
presence of a non-ionic detergent;
[0017] FIG. 12 is a calibration curve for 7-hydroxyquinoline;
[0018] FIG. 13 is an electropherogram of the product of
benzaldehyde and fluorescein hydrazine;
[0019] FIG. 14 is an electropherogram of the oxidative product of
CYP3A4 and 7-benzyloxyquinoline, followed by reaction with
fluorescein hydrazine; and
[0020] FIGS. 15-17 are electropherograms of control reactions,
where enzyme, cofactor or substrate, respectively, are excluded
from the reaction; and
[0021] FIG. 18 is a graph of a comparison between capillary
electrophoresis (A) and micro titer plate results (B).
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
[0022] Methods and compositions are provided for sensitive
detection of monooxygenase activity, particularly the cytochrome
P-450 enzyme isoforms, such as 3A4, 2E1, 1A2, 2A6, etc. The method
employs an in vitro enzyme preparation, coenzyme, and at least one
substrate. The substrates are characterized by having a turnover
number under the conditions of the assay of 10.sup.-5, preferably
at least about 10.sup.-3, min.sup.-1, and comprising an ether,
where one group is an aromatic compound, usually a heterocyclic
compound, and the other group has an oxidizable .alpha.-hydrogen to
produce an aldehyde (that is, the .alpha.-carbon atoms has two
hydrogens). Where a mixture of substrates is included in the assay
determination, each of the substrates will produce a product that
will have a different mobility in the form of a fluorescent
hydrazone. Prior to, during or after sufficient time for formation
of the aldehyde product, a fluorescent hydrazine is added and the
components of the reaction mixture separated under mildly acidic
conditions by capillary electrophoresis.
[0023] A large number of monooxygenase substrates are known, which
fulfill the desired requirements. The ethers are usually phenolic
ethers, where the aromatic group may be carbocyclic or
heterocyclic. Generally, the aromatic group will be at least about
4 carbon atoms, usually at least about 5 carbon atoms (6 annular
members per ring) and not more than about 30 carbon atoms, usually
not more than about 20 carbon atoms, and having from 0 to 8,
usually 0 to 6 heteroatoms, which are for the most part O, N, S.
Illustrative aromatic groups include phenyl, benzyl, naphthyl,
acenaphthyl, coumarin, resorufin, quinoline, warfarin,
methoxymephenytoin, mephenytoin, paclitaxel, debrisoquine,
bufuralol, midazolam, in effect, any group which permits enzyme
catalytic oxidation to result in a carbonyl. See, for example,
Gentest Corp. 1999-2000 catalog (info@gentest.com for further
information.) The other group of the ether will have a methylene
bonded to the oxygen of the ether, where the remaining moiety may
be hydrogen, aliphatic, alicyclic, aromatic or heterocyclic. When
other than hydrogen, the remaining moiety will be at least one
carbon atom, usually at least about 2 carbon atoms and not more
than about 16 carbon atoms, usually not more than about 12 carbon
atoms. There may be from 0 to 4, usually 0 to 3 heteroatoms, such
as O, S and N. The aldehyde reacts rapidly with a mono-substituted
hydrazine with a change in the mobility of the hydrazine in
capillary electrophoresis.
[0024] Specific substrates include 7-benzyloxyquinoline,
7-methoxyquinoline, AHMC, 7-benzyloxy-4-(trifluoromethyl)coumarin,
7-methoxy-4-(trifluoromethyl)coumarin, etc.
[0025] Various groups bonded to the methyleneoxy include methyl,
substituted methyls, where the group bound to the ether oxygen is
hydroxyethyl, ethyl ester of carboxymethyl, methoxypropyl,
cyclohexylmethyl, benzyl and substituted benzyls, e.g.
p-nitrobenzyl, p-cyanobenzyl, m-tolyl, o-anisole, etc.
[0026] The mono-substituted hydrazine will be substituted with a
convenient fluorescer, where the hydrazone product will have a
different mobility from the parent hydrazine. By employing a mildly
acidic pH, the hydrazine will be protonated, while the hydrazone
will be only partially, if at all, protonated. The difference in
molecular weight and charge of the product in comparison to the
hydrazine reactant provides for a significant change in mobility.
Various fluorescers may be used, since the formation of the
hydrazone is a secondary reaction and does not involve the P-450
enzyme. Selection of the fluorescer will be based on convenience,
rate of reaction, water solubility, excitation and emission
wavelengths and quantum yield (emission efficiency). Conventional
fluorescers may be used, such as fluorescein, rhodamine, BODIPY,
Texas Red, dansyl, Cascade blue, NBD, Cy-5, squarates, Lucifer
yellow, Rhodol green, pyrene, acridine orange, etc.
[0027] In carrying out the assay, the enzyme concentration is
conveniently in the range of about 1 nM to 500 nM, more usually in
the range of about 25 to 250 nM and may vary outside the indicated
range, depending upon the isoform of the cytochrome P-450. One may
use an individual substrate or a mixture of substrates in order to
determine the substrate profile of the enzyme. Each of the
substrates would produce an aldehyde product that would have a
different mobility when derivatized as a hydrazone. One could use
homologous series, differential substitution, differences in charge
and/or mass, or the like to obtain the different mobilities. A
homologous series may include aliphatic molecules, aliphatic
substituted aromatics or the like. Alternatively, one may have
substituted benzenes, where one would have different substituents,
such as halogen, oxy, amino, cyano, nitro, carboxy, e.g. esters,
etc. Depending on the concentration of enzyme, nature of the
substrate(s) and solubility in water, the turnover rate for each
substrate, the rate of reaction of each of the products with the
substituted hydrazine and its quantum yield, and the total number
of substrates. The concentration range of each substrate will be
about 50 to 5000 .mu.M, more usually 100 to 2000 .mu.M. Generally,
the concentration of the substrate will be in the range of about
0.25 to 0.75 of K.sub.m.
[0028] Coenzyme, if any, will be present in excess, so as not be
rate limiting. Generally, with the concentrations of enzyme
indicated above, the concentration of coenzyme will be at least
about 0.1 mM, usually at least about 1 mM and not more than about
25 mM. The coenzyme solution should be prepared freshly for each
series of determinations.
[0029] Various buffers may be used that do not interfere with the
enzyme activity. These buffers include PBS, Tris, MOPS, HEPES,
phosphate, etc. The pH will vary depending upon the particular
monooxygenase being assayed, generally being in the range of about
7-7.5, where the pH is selected to provide for at least about
maximum enzyme activity. The concentration of buffer will be
sufficient to prevent a significant change in pH during the course
of the reaction, generally being in the range of about 0.1 to 50
mM, more usually 0.5 to 20 mM.
[0030] The reaction time will usually be at least about 5 min, more
usually at least about 30 min and not more than about 180 min,
preferably not more than about 120 min, depending upon the
temperature, concentrations of enzyme and substrate, etc. By using
a specific time period for the reaction or taking aliquots at 2
different times, the rate of reaction can be determined for
comparison with other determinations. The temperature will
generally be in the range of about 20 to 50.degree. C., more
usually in the range of about 25 to 40.degree. C.
[0031] In many instances, it may be advantageous to add a small
amount of a non-ionic detergent. Generally the detergent will be
present in from about 0.01 to 0.1 vol. %. Illustrative non-ionic
detergents include the polyoxyalkylene diols, e.g. Pluronics,
Tweens, Triton X-100, etc.
[0032] After sufficient time for a detectable amount of product,
the reaction is quenched. Various quenching agents may be used,
both physical and chemical. Conveniently, a small amount of a
water-soluble inhibitor may be added, such as acetonitrile, DMSO,
SDS, methanol, DMF, etc. The amount of inhibitor will vary with the
nature of the inhibitor and may be determined empirically. A
sufficient amount of the fluorescent hydrazine derivative is then
added, usually at least stoichiometric, generally at least about
2-fold excess of the anticipated maximum amount of product, and may
be 5-fold or more. Too much of the hydrazine should not be added,
which can be determined empirically so as to avoid a broad band
that might interfere in the separation from the hydrazone product.
The hydrazone forming reaction is allowed to proceed for sufficient
time for the reaction to be at least substantially complete and the
reaction mixture is then separated using capillary electrophoresis
under conventional conditions at a pH in the range of about 4-8,
more usually 4-6, employing conventional buffers to achieve the pH,
e.g. acetate, carbonate, EDPA, etc. The hydrazone peak is read and
integrated to determine the activity of the enzyme. The conditions
of the capillary electrophoresis are conventional and may be
optimized for each hydrazone.
[0033] The fluorescent hydrazine derivatives are novel compounds.
The compounds are N-(.alpha.-hydrazinylacyl) 5-aminofluorescein,
where the acyl group is of from 2 to 6, usually 2 to 4, carbon
atoms. The product is made from 5-haloacetamidofluorescein and
hydrazine.
[0034] The following examples are offered by way of illustration
and not by way of limitation.
1TABLE 1 Results from the reaction of F1-NH.sub.2 and benzaldehyde
Benzaldehyde Incub. time Peak Peak P H P A Migration time
Concentration (.mu.M) (min) Dilution Height Area (Nor.) (Nor.)
(min) 133 30 50 6.297 10.212 6.297 10.212 2.153 133 200 50 4.791
11.818 4.072 10.045 2.533 33 30 50 0.886 2.134 0803 1.632 2.377 33
200 50 0.953 3.012 0.753 2.381 2.723 16 30 50 0.474 1.813 0.394
1.508 2.587 16 200 50 0.612 1.809 0.470 1.389 2.803 3 30 50 0.073
0.363 0.057 0.282 2.770 3 200 50 0.112 0.635 0.081 0.462 2.960
F1-NH.sub.2 -- 50 0.064 0.244 0.047 0.174 2.930 3 60 10 1.973 8.597
1.176 5.123 3.613 0.3 60 10 0.346 4.285 0.174 2.156 4.280
F1-NH.sub.2 -- 10 0.316 2.797 0.166 1.476 4.080
[0035] Reaction conditions were in phosphate buffer (3.3 mM and
pH=5.6), fluorescein hydrazine ("F1-NH2") concentration was 1.6 mM,
and 50 times dilution before injection: Separation buffer,
phosphate (10 mM, pH=7.1): 27 cm capillary (I.D. 50 .mu.m, O.D. 360
em); pressure injection and 10 kV for separation. FIG. 1 shows an
electropherogram of the reaction of benzaldehyde with fluorescein
using capillary electrophoresis technique (PAGE).
[0036] A linear calibration curve was achieved for benzaldehyde
(see FIG. 2) in the range of 3-133 .mu.M (for 30 min incubation and
50 times dilution before the injection). Higher sensitivity was
achieved with using lower dilution (see Table 1). The results from
the kinetic study of this reaction are depicted in FIG. 3, which
shows a maximum of reaction product is reached after 30 min and
then levels off.
[0037] 2-Cytochrome P-450 assay using CYP3A4: Micro titer plate. In
order to evaluate the assay, two sets of substrates were chosen:
the first one was 7-benzyloxyresorufin (7-BR) and the second one
was 7-benzyloxyquinoline (7-BQ).
[0038] I: 7-Benzyloxyresoufin (7-BR)
[0039] The effect of enzyme and substrate concentration as well as
incubation time on the enzymatic activity for CYP3A4 was evaluated
using a micro titer plate (reaction volume was 50 .mu.l). The
results from these studies are shown in FIGS. 4-6. FIG. 4 shows the
effect of 7-BR concentration as a function of time (enzyme
concentration was 200 nM). As can be seen, regardless of substrate
concentration, reaction was reached to a maximum after 30-40 min
and then leveled off. In addition, the activity increases
significantly upon an increase in substrate concentration. FIG. 5
shows the effect of enzyme concentration on enzymatic activity for
four different substrate concentrations. As can be seen in this
Figure, the effect of enzyme concentration for this specific
substrate is not that significant, since the reactivity of enzyme
is limited by the solubility of the substrate (7-BR has a very
limited solubility in water). FIGS. 6(A) and 6(B) show the
calibration curve for resorufin (product of enzymatic activity)
over a concentration range of 5-50,000 nM.
[0040] II: 7-Benzyloxyquinoline (7--BQ)
[0041] The effect of enzyme and substrate concentration as well as
incubation time on the enzymatic activity for CYP3A4 and 7-BQ was
evaluated using a micro titer plate (reaction volume was 50 .mu.l).
The results from these studies are shown in FIGS. 7-8.
[0042] FIG. 7 shows the effect of 7-BQ concentration as a function
of time (enzyme concentration was 40 nM). As can be seen,
regardless of substrate concentration, the reaction reached a
maximum after 25-30 min and then leveled off. In addition, the
activity, as in the case of 7-BR, increases significantly upon an
increase in substrate concentration. FIG. 8 shows the effect of
enzyme concentration on enzymatic activity for four different
substrate concentrations. As can be seen in this Figure, an
increase of enzyme concentration will cause an increase in the
enzymatic activity for this specific substrate.
[0043] III: Effect of Pluronic F68
[0044] The effect of the addition of Pluronic F68 on the enzymatic
activity of CYP3A4 using 7-BQ as a substrate on a micro titer plate
was studied. The results from these studies are shown in FIGS.
9-11. FIG. 9 shows the effect of 7-BQ concentration as a function
of time (enzyme concentration was 150 nM). As can be seen,
regardless of substrate concentration, reaction reached to a
maximum after 25 min and then leveled off. In addition, the
activity, like in the previous cases, increases significantly upon
an increase in substrate concentration. FIG. 10 shows the effect of
enzyme concentration on enzymatic activity for three different
substrate concentrations. As can be seen in this Figure, an
increase in enzyme concentration will cause an increase in the
enzymatic activity. FIG. 11 shows a comparison between the effect
of the presence (A) and absence (B) of Pluronic F68 on the
enzymatic activity of CYP3A4. Comparing the slopes of these two
calibration curves shows a 96% increase in the enzymatic activity
upon an addition of 0.026% of Pluronic F68.
[0045] FIG. 12 shows the calibration curve for 7-Hydroxyquinoline
(product of enzymatic activity) over a concentration range of 1-500
.mu.M.
[0046] The following table shows a comparison of the turnover rates
(TO) between resorufin and 7-BQ, which shows a significantly higher
number for 7-BQ as compared to resorufin.
2.45.times.10.sup.-3.+-.9.+-.10.sup.-3 min.sup.-1; 7-BQ 17.+-.11
min.sup.-1.
[0047] CYP450 Assay Using F1-NH.sub.2 as a Universal Substrate and
Capillary Electrophoresis I-Assay protocol.
[0048] 1. Enzymatic reaction. The reaction mixture contains 10
.mu.l of buffer solution for CYP3A4 (provided by PanVera, pH=7.4),
10 .mu.l of enzyme CYP3A4 (PanVera), 1 .mu.l 7-BQ (20 mM), 29 .mu.l
of water, and 10 .mu.l of a fresh solution of NADPH (10 mM). The
reaction mixture was incubated for 120 min at 37.degree. C. after
the addition of NADPH. Then the reaction was stopped by an addition
of 10 .mu.l of acetonitrile followed by a 2 .mu.l addition of
F1-NH.sub.2 (5 mM). Separation conditions were: buffer, 10 mM
Acetate and 5 mM SDS (pH=4.95); 27 cm capillary (I.D. 50 .mu.m,
O.D. 360 .mu.m); Pressure injection for 5 s and 20 kV for
separation.
[0049] 2. Control 1 (no enzyme). The reaction mixture was prepared
containing 10 .mu.l of Buffer (pH=7.4, provided from PanVera for
CYP3A4), 29 .mu.l of water, 1 .mu.l of 7-BQ (20 mM), and 10 .mu.l
NADPH (10 mM), then incubated for 2 h at 37.degree. C., followed by
an addition of 10 .mu.l of acetonitrile to stop the reaction
followed by a 2 .mu.l addition of F.sub.1-NH.sub.2 (5 mM).
[0050] 3. Control 2 (no NADPH). The reaction mixture was prepared
containing 10 .mu.l of Buffer (pH=7.4, provided from PanVera for
CYP3A4),10 .mu.l of CYP3A4 (PanVera), 29 .mu.l of water, and 1
.mu.l of 7-BQ (20 mM), then incubated for 2 h at 37.degree. C.
followed by an addition of 10 .mu.l of acetonitrile to stop the
reaction followed by a 2 .mu.l addition of F.sub.1-NH.sub.2 (5
mM).
[0051] 4. Control 3 (no 7-BQ). The reaction mixture was prepared
containing 10 .mu.l of buffer solution for CYP3A4 (provided by
PanVera, pH=7.4), 10 .mu.l of enzyme CYP3A4 (from PanVera), 20
.mu.l of water, and 10 .mu.l of a fresh solution of NADPH (10 mM),
and was incubated for 120 min at 37.degree. C. after the addition
of NADPH. Then the reaction was stopped by an addition of 10 .mu.of
acetonitrile followed by a 2 .mu.l addition of F.sub.1-NH.sub.2 (5
mM).
[0052] FIG. 13 shows the reaction of benzaldehyde (160 .mu.M) with
fluorescein hydrazine under conditions, which are used for the
Cytochrome P450 assay. The result from the enzymatic reaction of
CYP3A4 with 7-BQ followed by the reaction with fluorescein
hydrazine is shown in FIG. 14. As can be seen, the product
(benzaldehyde) peak can be clearly identified from the other peaks
resulting from either F.sub.1-NH.sub.2 or possibly from other
enzymatic reaction products. No such a peak was seen in the 1 st
control (FIG. 15, no enzyme was added), or 2.sup.nd control (FIG.
16, no NADPH), or 3.sup.rd control (FIG. 17, no 7-BQ).
[0053] IV. Comparison of Results from CF and Micro Titer Plate for
7-BQ
[0054] FIG. 18 shows the comparison between the sensitivity from
the CE and the micro titer plate. As can be seen, a significantly
higher sensitivity was achieved with CE as compared to that from
the micro titer plate (the slope of calibration curve from CE
results was 0.1593 as compared to 0.0052 from the micro titer
plate).
[0055] V. Preparation of N-(.alpha.-hydrazinylacetyl)
5-aminofluorescein
[0056] 5-Iodoacetamidofluorescein (100 mg, 0.194 mmol) was
dissolved in 20 ml of anhydrous ethanol. Hydrazine solution (3.5
ml, 1.0M in THF) was added dropwise at 0.degree. C. while stirring.
The color of the reaction mixture changed to dark red shortly after
the initial addition of the hydrazine solution. After completion of
the addition, the temperature was allowed to increase to room
temperature. After two hours, TLC on silica gel (10% MeOH+90%
CH.sub.2Cl.sub.2) showed a new very polar product was formed and
some unconverted. Another 3.0 ml of the hydrazine solution was
added and the reaction monitored with TLC until starting material
was no longer detected. The product precipitated as a red
precipitate. The reaction mixture was centrifuged for 5 min, the
solvent decanted, and the product dried in vacuo for a few minutes
and stored at -80.degree. C. It was used as is. ES/MS for
C.sub.22H.sub.17N.sub.3O.sub.6 m/z 420.1 (M+H).sup.+ (100%).
[0057] It is evident from the above results that by using
cytochrome P-450 substrates having an ether group, which is
oxidized to an aldehyde, the subject method provides for a simple
rapid and sensitive assay for determining P-450 activity. The rate
of reaction at conventional concentrations is rapid and
quantitative. The enzyme activity is found to be related to the
concentrations of substrate and enzyme and reaches a maximum that
is sustained over an extended period of time. Therefore, one may
follow the rate of reaction in the early stages of the assay or
determine the value, when the rate has leveled off.
[0058] All references and patent applications cited herein are
incorporated herein by reference, as if they had been set forth in
their entirety.
[0059] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be readily apparent to those of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the appended claims.
* * * * *