U.S. patent number RE41,461 [Application Number 12/199,157] was granted by the patent office on 2010-07-27 for purification, composition and specificity of heparinase i, ii, and iii from flavobacterium heparinum.
This patent grant is currently assigned to Massachusetts Institute of Technology, University of Iowa Research Foundation. Invention is credited to Charles L. Cooney, Robert S. Langer, Robert J. Linhardt, Daniel L. Lohse, Ramnath Sasisekharan.
United States Patent |
RE41,461 |
Lohse , et al. |
July 27, 2010 |
**Please see images for:
( Certificate of Correction ) ** |
Purification, composition and specificity of Heparinase I, II, and
III from Flavobacterium heparinum
Abstract
A single, reproducible scheme to simultaneously purify all three
of the heparin lyases from F. heparinum to apparent homogeneity is
disclosed herein. The kinetic properties of the heparin lyases have
been determined as well as the conditions to optimize their
activity and stability. Mono-clonal antibodies to the three
heparinases are also described and are useful for detection,
isolation and characterization of the heparinases.
Inventors: |
Lohse; Daniel L. (Bryan,
TX), Linhardt; Robert J. (Iowa City, IA), Sasisekharan;
Ramnath (Bedford, MA), Cooney; Charles L. (Brookline,
MA), Langer; Robert S. (Newton, MA) |
Assignee: |
Massachusetts Institute of
Technology (Cambridge, MA)
University of Iowa Research Foundation (Iowa City,
IA)
|
Family
ID: |
25529915 |
Appl.
No.: |
12/199,157 |
Filed: |
August 27, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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07983367 |
Nov 30, 1992 |
5389539 |
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Reissue of: |
08378789 |
Jan 26, 1995 |
05569600 |
Oct 29, 1996 |
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Current U.S.
Class: |
435/220; 435/850;
435/822; 435/100; 435/232; 435/183 |
Current CPC
Class: |
C12N
9/88 (20130101); Y10S 435/822 (20130101); Y10S
435/85 (20130101) |
Current International
Class: |
C12N
1/20 (20060101); C12N 9/00 (20060101); C12N
9/52 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0670892 |
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May 1997 |
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EP |
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54107584 |
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Aug 1979 |
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JP |
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3108486 |
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Aug 1991 |
|
JP |
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WO 89/12692 |
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Dec 1989 |
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WO |
|
Other References
Langer, R., et al., "An Enzymatic System for Removing Heparin in
Extracorporeal Therapy", Science 217, 261-263, (Jul. 1982). cited
by examiner .
Linhardt, R. J., et al., "Immuno-Affinity Purification of
Heparinase", Int. J. Biochem., 17(11), 1179-1183 (1985). cited by
examiner .
Linker, A., et al., "Hepainase and Heparitinase from
Flavorbacteria", V. Ginsburg, ed., Methods in Enzymology 28,
902-911 (Academic Press, NY 1972). cited by examiner .
Silva, M.E., et al., "Isolation and Partial Characterization of
Three Induced Enzymes from Flavobacterium heparinum Involved in the
Degradation of Heparin and Heparitin Sulfates", Biochemical and
Biophysical Res. Communications 56(4), 965-972 (1974). cited by
examiner .
Yang, V., et al., "Removal of the Anticoagulant Activities of the
Low Molecular Weights Hepain Fractions and Fragments with
Flavobacterial heparinase", Thrombosis Research 44(5), 599-610.
cited by other .
Stecher, et al., Ed., The Merck Index, Eighth Ed., 879 (1968).
cited by other .
Belyavsky, A., et al, "PCR-base cDNA library construction: general
cDNA libraries at the level of a few cells", 17, 1919-2932 (Apr.
1989). cited by examiner .
Yoshizawa et al., A 79107,584 (Japan) Aug. 23, 1970, CA91:20937d.
cited by examiner .
Berstein, H., "A system for Heparin Removal", Ph.D. Massachusetts
Inst. of Technology (1985). cited by examiner .
Charm, S.E., et al., "Scale-up of Protein Isolation", W.B. Jakobv.
ed., Methods in Enzymology, 22, 476-490 (Academic Press, NY, 1971).
cited by examiner .
Galliher, P.M. et al., "Heparinase Production by Flavobacterium
heparinum", Appl. Environ. Microbiol. 41, 360-365, (1981). cited by
other .
Deut5scher, M. P. (ed.) "Guide to Protein Purification", Methods in
Enzymology 182, 603-613, 738-751 (1988). cited by other .
Berger, S., et al., "Guide to Molecular Cloning Techniques",
Methods in Enzymology 152, 393-399, 415-423, 432-447 (1987). cited
by other .
Cerbelaud, E.C., et al., "Sulfur Regulation of Heparinase and
Sulfatases in Flavobacterium heparinum", Appl. Environ. Microbiol.
51, 640-646 (Mar. 1986). cited by other .
Hovingh P. and Linker, A., "The Enzymatic Degradation of Heparin
and Heparitin Sulfate," J. Biol. Chem. 245, 6170-75 (1970). cited
by other .
Yang, V.C., et al., "Purification and Characterization of
Heparinase from Flavobacterium heparinum," J. Biol. Chem. 260,
1849-1857 (Feb. 1985). cited by other .
Nakmura, T., et al., "Purification and Properties of Bacterioides
Heparinolyticus Heparinase (Heparin Lyase) EC 4.2.2.7", J. Clin.
Microbiol. 26, 1070-71 (1988). cited by other .
Ototani, N., et al., "Purification of Heparinase and Heparitinase
by Affinity Chromatography on Glycosaminoglycan-Bound AH-Sepharose
4B," Carbohydr. Res. 88, 291-303 (1981). cited by other .
Yang, V., et al., "Removal of the Anticoagulant Activities of the
Low Molecular Weights Heparin Fractions and Fragments with
Flavobacterial Heparinase", Thrombosis Research 44(5), pp. 599-610,
(1985). cited by examiner .
Berstein, H. "A System for Heparin Removal," Ph.D. Dissertation,
Massachusetts Institute of Technology (1985). cited by other .
Bohmer et al., J. Biol. Chem., 265:1 3609-13617 (1990). cited by
other .
Bradford, M.M., Anal. Biochem., 72:248-254 (1976). cited by other
.
Casu et al., Adv. Carbohydr. Chem. Biochem., 43:51-134 (1985).
cited by other .
Cerbelaud et al., Appl. Environ. Microbiol., 51:640-646 (1986).
cited by other .
Charm et al., W.B. Jakoby ed. Methods Enzymol., 22:476-490,
Academic Press., NY (1971). cited by other .
Cohen et al., Biopolymers, 30:733-741 (1990). cited by other .
Comfort et al., Biotechnol. Bioengin., 32:554-563 (1988). cited by
other .
Deutsche, M.P. (Ed.), Methods Enzymol., 182:603-613 (1990). cited
by other .
Deutsche, M.P. (Ed.), Methods Enzymol., 182: 738-751 (1990). cited
by other .
Dietrich et al., Biochem. Biophys. Acta, 237:430-441 (1971). cited
by other .
Dietrich et al., Biochem. Biophys. Acta, 343:34-44 (1974). cited by
other .
Dietrich et al., J. Biol. Chem., 248:6408-6415 (1973). cited by
other .
Dietrich, C.P., Biochem. Biophys. Res. Commun., 56:965-972 (1974).
cited by other .
Galliher et al., Appl. Environ. Microbial., 41:360-365 (1981).
cited by other .
Galliher et al., Eur. Appl. Micobiol. Biotechnol., 15:252-257
(1982). cited by other .
Gebelein, C.G. (Ed.), Biometric Polymers, 135-173, New York Plenum
Press (1975). cited by other .
Hovingh et al., J. Biol. Chem., 245:6170-6175 (1970). cited by
other .
Kanwar et al., Proc. Natl. Acad. Sci U S A, 76:4493-7 (1979). cited
by other .
Klein et al., J. Lab. Clin. Med., 102:828-837 (1983). cited by
other .
Langer et al., Sci., 217:261-263 (1982). cited by other .
Langer et al., Trans. Am. Soc. Artif. Intern. Organs, 28:387-390
(1982). cited by other .
Lauer et al., Anal. Chem., 58:166-170 (1986). cited by other .
Lindahl, et al., J. Biol. Chem., 255:5094-5100 (1980). cited by
other .
Lindahl, et al., Trends Biochem. Sci., 11:221-225 (1986). cited by
other .
Lindahl and Kjellen, The Biology of Extracellular Matrix
Proteoglycans, Wight and Mecham Eds., 59-104, Acadmic Press, NY.
cited by other .
Lindhardt et al., Biochem., 29:2611-2617 (1990). cited by other
.
Lindhart et al., Appl. Biochem. Biotech., 12:135-176 (1986). cited
by other .
Lindhart et al., Appl. Biochem. Biotech., 9:41-55 (1984). cited by
other .
Lindhart et al., Biochem. J., 254-781-787 (1988). cited by other
.
Lindhart et al., Biochem., 29:2611-2617 (1990). cited by other
.
Lindhart et al., J. Biol. Chem., 267-2380-2387 (1992). cited by
other .
Lindhart et al., Chem. Ind., 2:45-50 (1991). cited by other .
Lindhart et al., Intl. J. Biochem., 17:1179-1183 (1985). cited by
other .
Lindhart et al., J. Biol. Chem., 257:7310-7313 (1982). cited by
other .
Linker et al., Methods Enzymol., V. Ginsberg, Ed., 28:902-911,
Academic Press, NY (1972). cited by other .
Linker et al., J. Biol. Chem., 240:3724-3728 (1965). cited by other
.
Loganathan et al., Biochem., 29:4362-4368 (1990). cited by other
.
Lohse et al., Dissertation Abstract No. 9235874XP002028829, 87-20,
(1992). cited by other .
Lohse et al., J. Biol. Chem., 267: 24347-24354 (1992). cited by
other .
Lowry et al., J. Biol. Chem., 193:265-275 (1951). cited by other
.
McLean et al., Proceedings of the 8.sup.th International Symposium
on Glycoconjugates, 73-74, Paegar Publishers, NY (1985). cited by
other .
Merchant et al., Biochem. J., 229:369-377 (1985). cited by other
.
Michelacci et al., Biochem. Biophys. Res. Commun., 56:973-980
(1974). cited by other .
Moffat et al., Eur. J. Biochem., 202:531-541 (1991). cited by other
.
Moffatt et al., Eur. J. Biochem., 197:449-459 (1991). cited by
other .
Nader et al., J. Biol. Chem., 265:16807-16813 (1990). cited by
other .
Nakamura et al., J. Clin. Microbiol., 26:1070-1071 (1988). cited by
other .
Ototani et al., Carbohydr. Res., 70:295-306 (1979). cited by other
.
Ototani et al., Carbohydr. Res., 88:291-303 (1981). cited by other
.
Ototani et al., J. Biochem. (Tokyo), 84:1005-1008 (1978). cited by
other .
Ototani et al., Proceedings of the 6.sup.th International Symposium
on Glycojugates, 411-412 , Japan Scientific Press (1981). cited by
other .
Panyim et al., Arch. Biochem. Biophys., 130:337-346 (1969). cited
by other .
Payza et al., Nature, 177:88-89 (1956). cited by other .
Pitney et al., Brit. Med. J., 4:139-141 (1970). cited by other
.
Rice et al., Carbohydr. Res., 190:219-233 (1989). cited by other
.
Sasisekharan, R. Ph.D., "Cloning and Biochemical Characterization
of Heparinese from Flavobacterium heparinum," Harvard University
(1991). cited by other .
Salyers et al., Appl. Environ. Microbiol., 33:319-322 (1977). cited
by other .
Seikayaky America, Inc. Biochem.--Prod. Life Sci. Catalog (1988).
cited by other .
Sharath et al., Immunopharmacol., 9:73-80 (1985). cited by other
.
Silva et al., Biochem. Biophys. Res. Commun., 56:965-974 (1974).
cited by other .
Stecher et al., The Merk Index, Eighth Ed., 879 (1968). cited by
other .
Sticher et al., Glycoconjugates J., 8:45-54 (1991). cited by other
.
Turnbull et al., Biochem. J., 251:597-608 (1988). cited by other
.
Warnick et al., Biochem., 11:568-572 (1972). cited by other .
Yang et al., Appl. Biochem. Biotech., 16:35-50 (1987). cited by
other .
Yang et al., J. Biol. Chem., 260:1849-1857 (1985). cited by other
.
Yang et al., Thrombosis Res., 44:599-610 (1986). cited by other
.
Yoshida et al., Annual Symposium of Glycoconiugates (1989). cited
by other .
Yoshizawa et al., Chem. Abstracts, 91: 302-303 (1979). cited by
other .
Zapater et al., Prep. Biochem., 20:263-296 (1990). cited by other
.
Zimmermann et al., Appl. Biochem. Biotech., 30:137-148 (1991).
cited by other .
Al-Hakim et al., Electrophoresis, 11:23-28 (1990). cited by other
.
Belvavsky et al., Nucleic Acids Research , 17:2919-2932 (1989).
cited by other .
Berger et al., Methods Enzymmol., 152:393-399 (1987). cited by
other .
Berger et al., Methods Enzymmol., 152:415-423 (1987). cited by
other .
Berger et al., Methods Enzymmol., 152:432-447 (1987). cited by
other.
|
Primary Examiner: Naff; David M.
Assistant Examiner: Ware; Deborah K.
Attorney, Agent or Firm: Fish & Richardson P.C.
Parent Case Text
.[.This is.]. .Iadd.This application is a reissue of U.S. Pat. No.
5,569,600, which was filed as U.S. application serial no.
08/378,789 on Jan. 26, 1995, which is .Iaddend.a divisional of
.[.copending.]. .Iadd.U.S. .Iaddend.application .[.Ser. No..].
.Iadd.serial no. .Iaddend.07/983,367, .[.issue as a U.S. Pat.
No..]. .Iadd.filed Nov. 30, 1992, which issued as a U.S. Pat. No.
.Iaddend.5,389,539.[., filed in the U.S. Patent & Trademark
Office on Nov. 30, 1992.]. .
Claims
We claim:
1. A method for cleaving hexosamine-glucuronic acid linkages in
linear .[.polysacharides.]. .Iadd.polysaccharides .Iaddend.of
D-glucosamine linked to hexuronic acid comprising reacting heparin
or heparan sulfate with .[.a.]. purified heparinase selected from
the group consisting of .[.heparinase II present in.].
Flavobacterium heparinum .Iadd.heparinase II .Iaddend.free of lyase
activity other than heparinase II activity, having a molecular
weight of 84,100, cleaving heparin and heparan sulfate and having a
.[.pH optimum.]. .Iadd.pI .Iaddend.of 8.9-9.1 and .[.heparinase III
which is expressed in.]. Flavobacterium heparinum .Iadd.heparinase
III .Iaddend.free of lyase activity other than heparinase III
activity, having a molecular weight of 70,800, cleaving heparan
sulfate, and having a .[.pH optimum.]. .Iadd.pI .Iaddend.of
9.9-10.1.
2. The method of claim 1 wherein the heparin is in extracellular
matrix of cells or tissues.
.Iadd.3. The method of claim 1 wherein the heparinase II has a pH
optimum of 6.9 on heparan sulfate and the heparinase III has a pH
optimum of 7.6 on heparan sulfate..Iaddend.
Description
BACKGROUND OF THE INVENTION
This invention generally relates to the purification and
characterization of heparinase I, II, and III from Flavobacterium
heparinum and antibodies thereto.
Heparin and heparan sulfate represent a class of glycosaminoglycans
characterized by a linear polysaccharide of D-glucosamine
(1.fwdarw.4) linked to hexuronic acid (Linhardt, R. J. (1991) Chem.
Ind. 2, 45-50; Casu, B. (1985) Adv. Carbohydr. Chem. Biochem. 43,
51-134). Heparin and heparan sulfate are complex carbohydrates that
play an important functional role in the extracellular matrix of
mammals. These polysaccharides modulate and regulate tissue level
events that take place either during development under normal
situations or wound healing and tumor metastasis under pathological
conditions.
Much of the current understanding of heparin and heparan sulfate
sequence has relied on studies of their biosynthesis (Linhardt, R.
J., Wang, H. M., Loganathan, D., and Bae, J. H. (1992) Biol. Chem.
267, 2380-2387; Lindahl, U., Feingold, D., and Roden, L. (1986)
Trends Biochem. Sci. 11, 221-225; Jacobson, I., and Lindahl U.
(1980) J. Biol. Chem. 255, 5094-5100; Lindahl, U., and Kjellen, L.
(1987) in The Biology of Extracellular Matrix Proteoglycans (Wight,
T. N., and Mecham R., eds) pp. 59-104, Academic Press, New York).
Recent efforts (Linhardt, R. J., Rice, K. G., Kim, Y. S., Lohse, D.
L., Wang, H. M., and Loganathan, D. (1988) Biochem. J. 254,
781-787; Linhardt, R. J., Turnbull, J. E., Wang, H. M., Loganathan,
D., and Gallagher, J. T. (1990) Biochemistry 29, 2611-2617) have
focused on the application of enzymatic methods to depolymerize
these complex polysaccharides into oligosaccharides that could then
be structurally characterized (Linhardt, et al. (1992) Biol. Chem.
267, 2380-2387; Linhardt, et al., (1988) Biochem. J. 254, 781-787;
Loganathan, D., Want, H. M., Mallis, L. M., and Linhardt, R. J.
(1990) Biochemistry 29, 4362-4368).
Enzymatic methods for heparin and heparan sulfate depolymerization
are very specific and require mild conditions giving
oligosaccharide products that closely resemble the
glycosaminoglycans from which they were derived. Two types of
enzymes that degrade heparin and heparan sulfate glycosaminoglycans
are the polysaccharide lyases from prokaryotic sources that act
through an eliminative mechanism (Linhardt, R. J., Galliher, P. M.,
and Cooney, C. L. (1986) Appl. Biochem. Biotech. 12, 135-176), and
the glucuronidases (hydrolases) from eukaryotic sources that act
through a hydrolytic mechanism.
Prokaryote degradation of heparin and heparan sulfate has primarily
been studied using enzymes derived from Flavobacterium heparinum
(Linker, A., and Hovingh, P. (1965) J. Biol. Chem. 240, 3724-3728;
Linker, A., and Hovingh, P. (1970) J. Biol Chem. 245, 6170-6175);
Dietrich, C. P., Silva, M. E., and Michelacci, Y. M. (1973) J.
Biol. Chem. 248, 6408-6415; Silva, M. E., Dietrich, C. P., and
Nader, H. B. (1976) Biochem. Biophys. Acta 437, 129-141). This
bacterial degradation begins with the action of three (or possibly
more) eliminases. These heparin lyases produce oligosaccharides
with .DELTA..sub.4,5-unsaturated uronic acid residues a their
non-reducing termini. These eliminases probably act in concert to
convert heparin and heparan sulfate to disaccharides.
Heparin lyases are a general class of enzymes that are capable of
specifically cleaving the major glycosidic linkages in heparin and
heparan sulfate. Three heparin lyases have been identified in
Flavobacterium heparinum, a heparin-utilizing organism that also
produces exoglycuronidases, sulfoesterases, and sulfamidases that
further act on the lyase-generated oligosaccharide products (Yang,
V. C., Linhardt, R. J., Berstein, H., Cooney, C. L., and Langer, R.
(1985) J. Biol. Chem. 260, 1849-1857; Galliher, P. M., Linhardt, R.
J., Conway, L. J., Langer, R., and Cooney, C. L. (1982) Eur. J.
Appl. Microbiol. Biotechnol. 15, 252-257). These lyases are
designated as heparin lyase I (heparinase, EC 4.2.2.7), heparin
lyase II (heparinase II, no EC number) and heparin lyase III
(heparitinase EC 4.2.2.8). Although the specificities of these
enzymes are not completely known, studies using partially purified
enzymes with heparin, heparan sulfate, and structurally
characterized heparin oligosaccharides have led to an understanding
of the linkages susceptible to enzymatic cleavage (Lindhart, et
al., (1990), Lohse (1992), Rice, K. G., and Linhardt, R. J. (1989)
Carbohydr. Res. 190, 219-233). The three purified heparin lyases
differ in their capacity to cleave heparin and heparan sulfate;
Heparin lyase I primarily cleaves heparin, heparin lyase III
specifically cleaves heparan sulfate and heparin lyase II acts
equally on both heparin and heparan sulfate (Linhardt, et al.,
1986; Linhardt, et al., 1990).
Several Bacteroides sp. (Saylers, A. A., Vercellotti, J. R., West,
S.E.H:, and Wilkins, T. D. (1977) Appl. Environ. Microbiol. 33,
319-322; Nakamura, T., Shibata, Y., and Fujimura, S. (1988) J.
Clin. Microbiol. 26, 1070-1071) also produce heparinases, however,
these enzymes are not well characterized. A heparinase has also
been purified to apparent homogeneity from an unidentified soil
bacterium (Bohmer, L. H., Pitout, M. J., Steyn, P. L., and Visser,
L. (1990) J. Biol. Chem. 265, 13609-13617). This enzyme differs
from those isolated from Flavobacterium heparinum in its molecular
weight (94,000), pI (9.2), amino acid composition and kinetic
properties (K.sub.m of 3.4 .mu.M and V.sub.max of 36.8 .mu.mol/min,
pH optimum of 7.6).
Three other heparin lyases, partially purified from Flavobacterium
sp. Hp206, have molecular weights of 64,000, 100,000 and 72,000, as
reported by Yoshida, K., Miyazono, H., Tawada, A., Kikuchi, H.,
Morikawa, L., and Tokuyasu, K. (1989) 10th Annual Symposium of
Glycoconjugates, Jerusalem, different from heparin lyases
I-III.
The heparin lyases of F. heparinum are the most widely used and
best studied (Lindhardt, (1986)). Linker and Hovingh (1970) first
separated these lyase activities, fractionating a crude lyase
fraction into a heparinase (heparin lyase I) and a heparitinase
(heparin lyase III). Both activities were purified by 50-100-fold,
but no physical characterization of these enzymes was
performed.
Dietrich and co-workers (Dietrich, et al., 1973); Silva, et al.,
(1976); Silva, M. E., and Dietrich, C. P. (1974) Biochem. Biophys.
Res. Commun. 56, 965-972; Michelacci, Y. M., and Dietrich, C. P.
(1974) Biochem. Biophys. Res. Commun. 56, 973-980) and Ototani and
Yosizawa (Ototani, N., and Yosizawa, Z. (1978) J. Biochem. (tokyo)
84, 1005-1008; Ototani, N., and Yosizawa, Z. (1979) Carbohydr. Res.
70, 295-306; Ototani, N., Kikiuchi, M., and Yosizawa, Z. (1981)
Carbohydr. Res. 88, 291-303; Ototani, N., and Yosizawa, Z. (1981)
Proceedings of the 6th International Symposium on Glycoconjugates,
pp. 411-412, September 20-25, Tokyo, Japan Scientific Press, Tokyo)
isolated three lyases, a heparinase (heparin lyase I) and two
heparitinases, from F. heparinum. The heparinase acted on heparin
to produce mainly trisulfated disaccharides (Dietrich, C. P., and
Nader, H. B. (1974) Biochem. Biophys. Acta 343, 34-44; Dietrich, C.
P., Nader, H. B., Britto, L. R., and Silva, M. E. (1971) Biochem.
Biophys. Acta 237, 430-441); Nader, H. B., Porcionatto, M. A.,
Tersariol, I.L.S., Pinhal, M. S., Oliveira, F. W., Moracs, C. T.,
and Dietrich, C. P. (1990) J. Biol. Chem. 265, 16807-16813)
purified two heparitinases (called heparitinase I and II, possibly
corresponding to heparin lyases II and III, although no physical
properties of these enzymes were presented) and characterized their
substrate specificity toward heparin and heparan sulfate.
Heparitinase I degraded both N-acetylated and N-sulfated heparan
sulfate while heparitinase II degraded primarily N-sulfated heparan
sulfate.
McLean and Co-workers described the specificity of a partially
purified heparinase II (Moffat, C. F., McLean, M. W., Long, W. F.,
and Williamson, F. B. (1991) Eur. J. Biochem. 197, 449-459; McLean,
M. W., Long, W. F., and Williamson, F. B. (1985) in Proceedings of
the 8th International Symposium on Glycoconjugates, pp. 73-74,
September, Houston, Paeger Publishers, New York; McLean, M. W.,
Bruce, J. S., Long, W. F., and Williamson, F. B. (1954) Eur. J.
Biochem. 145, 607-615). Although no evidence of homogencity or any
physical properties for heparinase II were presented, the broad
specificity on various polymeric substrates (Moffat, et al.,
(1991)) identifies the enzyme as heparin lyase II (Lindhardt, et
al., (1990); McLean, et al., (1985).
Linhardt et al. (1984) Appl. Biochem. Biotech. 9, 41-55) reported
the purification of heparinase (heparin lyase I) to a single band
on SDS-PAGE. Affinity purification of heparin lyase I on
heparin-Sepharose failed, apparently due to degradation of the
column matrix. Sufficient quantities of pure heparin lyase I for
detailed characterization studies and amino acid analysis were
first prepared by Yang et al. (1985). Heparin lyase I was used to
prepare polyclonal antibodies in rabbits for affinity purification
of heparin lyase I, but excessively harsh conditions required to
elute the enzyme resulted in substantial loss of activity
(Lindhardt, (1985)). Yang, V. C., Berstein, H., Cooney, C. L., and
Langer, R. (1987) Appl. Biochem. Biotech. 35-50)) also described a
method to prepare heparin lyase I.
Seikagaku Co. has recently orally reported the molecular weights of
their commercial enzymes corresponding to heparin lyase I-III to be
43,000, 84,000, and 70,000, respectively (Yoshida, K. (1991)
International Symposium on Heparin and Related Polysaccharides,
September 1-6, Uppsala, Sweden). These reports are in close
agreement to the molecular weights described herein, but no details
of their purification or characterization methods have been
published.
Heparin lyases have been used to establish the presence of heparin
in mixtures of proteoglycans (Kanwar, Y. S., and Farguhar, M. G.
(1979) Presence of heparan sulfate in the glomerular basement
membrane, Proc. Natl. Acad. Sci., USA 76, 1303-1307), to
depolymerize heparin and heparan sulfate to characterize the
structure of the resulting oligosaccharides (Linhardt, R. J.,
Loganathan, D. Al-Hakim, A., Wang, H.-M., Walenga, J. M.,
Hoppensteadt, D., and Fareed, J. (1990) Oligosaccharide mapping of
low molecular weight heparins: structure and activity differences.
J. Med. Chem. 33, 1639-1645; Linhardt, R. J., Rice, K. G., Kim, Y.
S., Lohse, D. L., Wang, H. M., and Loganathan, D. (1988). Mapping
and quantification of the major oligosaccharide components of
heparin. Biochem. J. 254, 781-787; Merchant, Z. M., Kim, Y. S.,
Rice, K. G., and Linhardt, R. J. (1985). Structure of
heparin-derived tetrasaccharides. Biochem. J. 229, 369-377;
Turnbull, J. E., and Gallagher, J. T. (1988) Oligosaccharide
mapping of heparan sulphate by polyacrylamide-gradient-gel
electrophoresis and electrotransfer to nylon membrane. Biochem J.
251, 597-608), to produce low molecular weight heparin preparations
with anticoagulant and complement inhibitory activities (Linhardt,
R. J., Grant, A., Cooney, C. L., and Langer, R. (1982) Differential
anticoagulant activity of heparin fragments prepared using
microbial heparinase J. Biol. Chem. 257, 7310-7313; Linhardt, R.
J., and Loganathan, D. (1990a). Heparin, heparinoids and heparin
oligosaccharides: structure and biological activity. In C. G.
Gebelein (Ed.) Biomimetic Polymers (pp. 135-173). New York: Plenum
Press; Sharath, M. D., Merchant, Z. M., Kim, Y. S., Rice, K. G.,
Linhardt, R. J., and Weiler, J. M. (1985) Small heparin fragments
regulate the amplification pathway of complement.
Immunopharmacology 9, 73-80) and to remove heparin from the
circulation (Langer, et al., 1982). Heparin depolymerising enzymes
are excellent tools to understand the role of heparin-like
molecules in the extracellular matrix or to be used in different
tissue microenvironments to modulate and alter the extracellular
matrix in a highly specific manner. However, studies utilizing
heparin lyases are hampered by difficulties in purifying the
enzymes from Flavobacterium heparinum, especially with regard to
separation of the three enzymes from each other (Linhardt, et al.;
1985). Specifically, the capacity of heparin lyase II to cleave
both heparin and heparan sulfate makes it difficult to distinguish
from heparin lyase I which cleaves heparin and heparin lyase III
which cleaves heparan sulfate.
Although all three of these heparin/heparan sulfate lyases are
widely used, with the exception of heparin lyase I, there is no
information on the purity or physical and kinetic characteristics
of heparinase II and heparinase III. The absence of pure heparin
lyases, resulting in ambiguities with respect to substrate
specificity. This is due to contamination of other lyases in the
preparation, and a lack of understanding of the optimal catalytic
conditions and substrate specificity has stood in the way of the
use of these enzymes as reagents for the specific depolymerization
of heparin and heparan sulfate into oligosaccharides for structure
and activity studies, and for use in clinical studies.
It is therefore an object of the present invention to provide a
method for purification and characterization of heparinase I,
heparinase II, and heparinase III.
It is a further object of the present invention to provide purified
and characterized heparinase I, heparinase II, and heparinase
III.
It is a still further object of the present invention to provide
the conditions for optimal use and peptide map of the purified
heparinase II and heparinase III.
It is another object of the present invention to provide the amino
acid compositions of the three heparinases.
It is another object of the present invention to provide antibodies
for heparinase I, II, and III which can be used in the purification
and characterization of heparinases.
SUMMARY OF THE INVENTION
A single, reproducible scheme to simultaneously purify all three of
the heparin lyases from F. heparinum to apparent homogeneity and
free of contaminating lyases is disclosed herein. Heparin lyase I
(heparinase, EC 4.2.2.7), heparin lyase II (no EC number), and
heparin lyase III (heparitinase, EC 4.2.2.8) have molecular weights
(by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and
isoelectric points (by isoelectric focusing) of M.sub.r 42,800, pI
9.1-9.2 M.sub.r 70,800, pI 9.9-10.1, respectively. Their amino acid
analyses and peptide maps demonstrate that while these proteins are
different gene products they are closely related. The kinetic
properties of the heparin lyases have been determined as well as
the conditions to optimize their activity and stability.
The purification and characterization of heparinase II from
Flavobacterium heparinum is described. The Michelis-Menton
constants are: Heparin lyase II (with heparin), V(max)=15.04,
K.sub.m=9.23 .mu.M (0,129 mg/ml); Heparin lyase II (with heparan
sulfate), V(max)=46.95, K.sub.m=43.43 .mu.M (0.869 mg/ml). The
approximate pI of the lyase calculated from agarose IEF using a pH
gradient from 9-11 is around 8.9. The optimum temperature for
heparin lyase II (both heparin and heparan sulfate) is 35.degree.
C. The activity is greater at higher temperatures but the stability
is greatly reduced. The optimum pH for activity for the lyase:
(with heparin), pH=7.3 and (with heparan sulfate), pH=6.9.
The purification and characterization of heparinase III (EC
4.2.2.8) from Flavobacterium heparinum is described. The
Michelis-Menton constants are V(max)=277.01, K.sub.m=109.97 .mu.M
(0.780 mg/ml). The approximate pI of the lyase was calculated from
agarose IEF using a pH gradient from 9-11 and was found to be 9.2.
The optimum temperatures for the heparin lyase III activity is
35.degree. C. The activity is higher at higher temperatures for the
enzyme but the stability is greatly reduced. The optimum pHs for
heparin lyase III is pH=7.6. The substrate specificity of
heparinase III is for the hexosamine-glucuronic acid linkages of
the heparan sulfate backbone. The enzyme is a monomeric protein,
very different from heparinase I and II in size and activity. It is
possible to use heparinase III to release heparin-like chains in
the extracellular matrix, for both sequencing and eliciting heparin
based cellular response.
Salt effects were not observed for either heparinase II or
heparinase III. Four different salts were used to confirm that salt
effects and not ion effects were tested.
Methods for the preparation and use of monoclonal antibodies to the
three heparinases are also described. The antibodies are useful for
isolation, detection and characterization of the heparinases,
individually and as a group, and in studies involving substrate
specificity, enzyme inhibition and active site mapping.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph of the HA-HPLC fractionation of heparin lyases.
The protein (A.sub.280) is the solid line. The activity (unit/ml)
toward heparin (solid circles) and activity (unit/ml) toward
heparan sulfate (solid squares) are shown with cross-hatching to
indicate the portion of the peaks that were collected.
FIG. 2 is Mono-S FPLC fractionation of heparin lyases: a, heparin
lyase I, and b, heparin lyase III. The arrow indicates the start of
the salt gradient elution, and the cross-hatching indicates the
portion of the peaks that were collected.
FIG. 3 is a GPC-HPLC fractionation of heparin lyases. a, heparin
lyase I; b, heparin lyase II; c, heparin lyase III; and d,
molecular weight standards (M.sub.r) consisting of thyroglobulin
(bovine, 670,000), gamma globulin (158,000), oval-bumin (44,000),
myoglobin (horse, 17,000), and cyanocobalamin (1350). The
cross-hatching indicates the portion of the peaks that were
collected.
FIG. 4 is an SDS-PAGE in a 12% discontinuous polyacrylamide gel
under reducing conditions. Two .mu.g each of heparin lyase I (lane
a), heparin lyase II (lane b), heparin lyase III (lane c), and
molecular weight standards (lane d). Shown to the right are the
mass of the molecular weight standards in kDa.
FIG. 5. Panel A: Western Blot of SDS-PAGE gel using M2-A9. (a)
heparin lyase I; (b) heparin lyase II; (c) heparin lyase III; (d)
Flavobacterium heparinum cell homogenate. Arrows indicate bands of
interest. This analysis demonstrates the ability of this Mb to
detect the presence of heparin lyases that are either purified or
present in homogenized cellular material.
Panel B: SDS-PAGE analysis of purified heparin lyases. (a) heparin
lyase I; (b) heparin lyase II; (c) heparin lyase III; (d) molecular
weight markers. Arrows indicate bands of interest.
FIG. 6 is a map of the tryptic digest of heparinase II and III.
Panel A is heparinase II and Panel B is heparinase III by
DETAILED DESCRIPTION OF THE INVENTION
I. Purification and Characterization of Heparinase I, II, and
III.
A single, reproducible scheme to simultaneously purify all three of
the heparin lyases from F. heparinum to apparent homogeneity is
described herein.
EXPERIMENTAL PROCEDURES
Materials
Enzyme assays and absorbance measurements were done on a UV 160
spectrophotometer from Shimadzu connected to a Fisher Scientific
Isotamp model 9100 refrigerated circulating water bath.
Fermentations were performed in a two-liter stirred tank fermenter
from Applikon. Centrifugation was done on a Sorval RC-5
refrigerated centrifuge in a GSA rotor from Du Pont. HPLC was
performed using a LDC Milton-Roy Constametric IIIG pump, a Rheodyne
7125 injector, a Jule Linear Gradient Former, and an ISCO model
UA-5 absorbance monitor with a 280-nm filter. The hydroxylapatite
HPLC column 1.times.30 cm connected in series with a 1.times.5 cm
guard column was from Regis, the Mono-S FPLC column was from
Pharmacia LKB Biotechnology Inc., the C.sub.18 column was from
Vydac, and the Bio-Sil gel permeation HPLC column was from Bio-Rad.
The capillary zone electrophoresis system and the silica
capillaries were from Dionex. The Mini-Protein II electrophoresis
chamber, a model 1405 horizontal electrophoresis cell, and a model
1420B power source were from Bio-Rad. The tube gel electrophoresis
equipment was from E-C Apparatus Corp. The precast agarose IEF gels
were from Iso-labs, and the prestained molecular weight markers and
the Rapid Coomassie.TM. stain were from Diversified Biotech. The
Bio-Gel HT hydroxylapatite was from Bio-Rad and the QAE-Sephadex
was from Sigma. Pressure filtration units and 25- and 43-mm PM-10
filters were from Amicon. Heparin (porcine mucosal sodium salt) was
from Celsus, heparan sulfate, dermatan sulfate, and chondroitin
sulfate A, C, D, and E were from Seikagaku. Bovine serum albumin,
lactose, protamine (free base), bromphenol blue, naphthol red,
cyto-chrome c (bovine heart type VA), hyaluronic acid, CAPS,
bis-Tris, HEPES, TES, dithiothreitol, MOPS, mercaptoethanol,
iodoacetamide, and trypsin were for Sigma. The Coomassie reagent
for the protein assay was from Bio-Rad. All water used in reagents
was deionized and distilled in glass.
Assays
The spectrophotometer was adjusted to the optimum temperature of
the particular lyase being assayed. A 700 .mu.l quartz microcuvette
containing 400 .mu.g of substrate in 50 mM sodium phosphate buffer
(containing 100 mM sodium chloride for heparin lyase I) was
thermally equilibrated. A measured quantity of lyase was added,
bringing the final volume to 400 .mu.l and the cuvette was gently
mixed. The microcuvette was then immediately returned to the
spectrophotometer and the change of absorbance at 232 nm was
measured at 10 seconds intervals over 3 min. The activity was
measured from the change of absorbance/unit time using an
extinction coefficient of 3800 M.sup.-1 for products. The specific
activity was then calculated by dividing the micromoles of product
produced per minute by the milligrams of protein in the cuvette.
The molecular weights used for heparin, heparan sulfate, and the
chondroitin sulfates were 14,000, 20,000 and 25,000, respectively,
Rice, K. G., and Linhardt, R. J. (1989) Carbohydr. Res. 190,
219-233. Protein concentration was measured by the Bradford assay,
Bradford, M. M. (1976) Anal. Biochem. 72, 248-254, based on a
bovine serum albumin standard curve.
Fermentation and Enzyme Recovery
F. heparinum (Payza, A. N., and Korn, E. D. (1956) Nature 177,
88-89) (ATCC 13125) was stored at -70.degree. C. in a defined
medium containing dimethyl sulfoxide (Me.sub.2SO) (Zimmermann, J.
J., Oddie, K., Langer, R., and Cooney, C. L. (1991) Appl. Biochem.
Biotech. 30, 137-148). The organism was grown in a two liter
stirred tank fermenter on heparin as the sole carbon source in
defined medium by the method of Galliher, P. M., Cooney, C. L.,
Langer, R. S., and Linhardt, R. J. (1981) Appl. Environ. Microbiol.
41, 360-365). From 5 liters of fermentation broth, an 80 g wet cell
pellet was obtained by centrifugation for 15 min at 12,000 .times.g
at 4.degree. C. This pellet was suspended in 500 ml of 10 mM sodium
phosphate buffer at pH 7.0 and 4.degree. C. Cell suspension (20 ml
at a time) was placed into a 50-ml stainless steel cup and
sonicated with cooling for 10 min at 100 watts using a 40% pulsed
mode. The disrupted cells were centrifuged at 12,500 .times.g for
30 min at 4.degree. C. and the pellet discarded. The 500 ml of
supernatant, obtained by sonification and centrifugation, contained
16.3 mg/ml protein. Protamine free base (2.0 g) was dissolved in 20
ml of 10 mM sodium phosphate buffer, pH 7.0, and added dropwise
with stirring to the 500 ml of supernatant. Centrifugation at
10,000 .times.g, at 4.degree. C. for 20 min, removed the
precipitated DNA and gave 510 ml of supernatant.
Purification of heparin Lyases from F. heparinum
Batch Hydroxylapatite Adsorption and Release
The 510 ml of supernatant containing 15.6 mg/ml protein, used
directly without freezing, was divided equally into four 250 ml
polypropylene centrifuge containers and placed in an ice bath. Dry
hydroxylapatite (HA) (20 g) was added to each container, gently
stirred, lightly compacted with centrifugation at 1000 .times.g for
2 min at 4.degree. C., and the supernatant was decanted away from
the HA matrix. The HA-bound protein was then resuspended in buffers
having increasing concentrations of sodium phosphate and sodium
chloride and recompacted by centrifugation. The supernatants were
again decanted away from the matrix and assayed for enzyme activity
and protein concentration. The buffers used to wash the HA matrix
were prepared by mixing a solution of 10 mM sodium phosphate buffer
at pH 6.8, with a solution of 250 mM sodium phosphate buffer at pH
6.8, containing 500 mM sodium chloride in ratios of 6:0, 5:1, 4:2,
3:3, 2:4, and 0:6 (v/v) at 4.degree. C. The protein supernatant
solutions were placed in dialysis tubing having a molecular weight
cut-off of 14,000 and dialyzed overnight at 4.degree. C. against 50
mM sodium phosphate buffer at pH 7.0.
QAE-Sephadex Chromatography
Lyase activity purified by batch HA was used immediately without
freezing. A quaternary ammonium ethyl (QAE)-Sephadex chromatography
step was performed at 4.degree. C. Three batch HA-purified
fractions (4:2, 3:3, and 2:4), having a total volume of 1.5 liters,
containing more than 89% of the activity toward heparin and 88% of
the activity toward heparan sulfate were consolidated (1.81 mg/ml
protein and 1.72 units/ml toward heparin and 2.16 units/ml toward
heparan sulfate) and applied directly in equal portions to three
columns (2.5.times.20 cm) containing 600 ml of QAE-Sephadex. The
QAE-Sephadex columns had been previously equilibrated with 50 mM
sodium phosphate buffer, pH 7.0, at 4.degree. C. Each column was
then washed with 1-column volume of 50 mM phosphate buffer, pH 7.0,
at 4.degree. C. The fractions containing lyase activity that passed
through the columns without interaction were collected and
combined. The 2.6 liters of eluent was then concentrated to 63 ml
(containing 8.23 mg/ml of protein) by Amicon pressure filtration at
60 psi and 4.degree. C. using a 43 mm PM-10 membrane (10,000
molecular weight cut-off).
Hydroxylapatite HPLC
The 63 ml of QAE-Sephadex-purified and concentrated solution was
divided into twelve 5 ml aliquots and stored at -70.degree. C.
until needed. A 5 ml sample (43 mg of protein) was removed from the
freezer, allowed to thaw at room temperature, and, using a 5 ml
loop, injected onto a HA HPLC column. The HA-HPLC column had been
equilibrated with 50 mM sodium phosphate buffer, pH 7.0. After
loading the sample, the column was washed with 50 mM sodium
phosphate buffer, pH 7.0, at 0.5 ml/min, for 20 min. A 60 ml linear
gradient, from 50 mM sodium phosphate, pH 7.0, to 50 mM sodium
phosphate buffer containing 750 mM sodium chloride, pH 7.0, was
used to elute the column. The elution was monitored continuously at
280 nm. After the gradient was complete, the column was washed with
5.0 ml of 50 mM sodium phosphate containing 1M sodium chloride, pH
7.0, to remove tightly bound proteins, and then re-equilibrated
with the 50 mM sodium phosphate buffer, pH 7.0. This fractionation
step was repeated with the 11 remaining aliquots. The fractions
corresponding to heparin lyase I, heparin lyase II, and heparin
lyase III from each of the 12 fractionations were pooled, dialyzed
against 20 volumes of 50 mM sodium phosphate buffer, pH 7.0, for 12
h at 4.degree. C., and concentrated at 60 psi and 4.degree. C.
using Amicon pressure filtration equipped with PM-10 membranes. The
three lyase preparations were each divided into 1-ml aliquots and
frozen at -70.degree. C.
Mono-S FPLC of heparin Lyases I and III
The concentrated heparin lyase I and heparin lyase III
preparations, isolated from HA-HPLC, were taken from the
-70.degree. C. freezer, thawed at room temperature, and applied to
a Mono-S FPLC HR 5/5 cation-exchange column equilibrated with 50 mM
sodium phosphate buffer, pH 7.0. A portion of each lyase
preparation, 350 .mu.l containing 1.75 mg of protein, was injected
and the column washed at 1 ml/min for 5 min with 50 mM sodium
phosphate buffer, pH 7.0, to elute non-interacting proteins. A
linear gradient from 50 mM sodium phosphate buffer, pH 7.0, to 50
mM sodium phosphate containing 500 mM sodium chloride, pH 7.0, was
used and the elution was monitored at 280 nm. The active heparin
lyase I and heparin lyase III fractions were dialyzed at 4.degree.
C. against 200 mM sodium phosphate buffer, pH 7.0, for 12 h and
concentrated using Amicon Pressure Filtration with a PM-10 membrane
(molecular weight cut-off 10,000).
Gel Permeation HPLC
The heparin lyase I and III preparation obtained from Mono-S FPLC
and the heparin lyase II preparation obtained from HA-HPLC were
applied to a Bio-Sil gel permeation chromatography (GPC) HPLC
column (1.times.25 cm) that had been equilibrated with 200 mM
sodium phosphate buffer, pH 7.0. Each lyase was injected (250 .mu.l
samples containing 800 .mu.g of protein for heparin lyases I and
III; 200 .mu.l samples containing 1.5 mg of protein for heparin
lyase II), eluted at a flow rate of 1 ml/min and absorbance at 280
nm was measured. This separation was repeated 5 times for heparin
lyases I-III. The active fractions were pooled together and assayed
for lyase activity and protein concentration. Each heparin lyase
was dialyzed against 50 mM sodium phosphate buffer, pH 7.0,
concentrated at 60 psi and 4.degree. C. using pressure filtration
with 25 mm PM-10 membranes (molecular weight cut-off 10,000), and
subdivided into 10 .mu.l aliquots and stored at -70.degree. C.
Characterization of the Three heparin Lyases
Assessment of Purity by Electrophoresis
Discontinuous SDS-PAGE was performed on the three heparin lyases
using a modification of a procedure previously described by
Laemmli, U.K. (1970) Nature 227, 680-685 (FIG. 4). The gels were
fixed with 12% (w/v) trichloroacetic acid, rinsed with deionized,
distilled water and stained with a Rapid Coomassie Stain solution,
and desrained.
IEF gel electrophoresis was run on pre-cast agarose gels
(85.times.100 mm). Two electrode wicks were wetted with 1M
phosphoric acid (anolyte) and 1M sodium hydroxide (catholyte).
Electrophoresis was at 5 watts for 5 min, then at 10 watts for 1 h
until the voltage was constant at 1200 V. The gel was immediately
fixed in 15% aqueous trichloroacetic acid, blotted and rinsed with
water, dried overnight, stained by using Coomassie G-250, and
destained.
Continuous acid-urea gel electrophoresis was performed in 10%
polyacrylamide tube gels (Panyim, S., and Chalkley, R. (1969) Arch.
Biochem. Biophys. 130, 337-346). Heparin lyase I-III samples (10
.mu.g) were prepared in acetic acid-urea buffer containing glycerol
and naphthol red as a tracking dye. Electrophoresis was at a
constant current of 2.5 mA/tube gel. The proteins were run toward
the cathode for approximately 2 h, until the 100 .mu.g of
cytochrome c standard (a brown band) was at the bottom of its tube.
Staining and destaining were accomplished as described for
SDS-PAGE.
Capillary zone electrophoresis on the three heparin lyases used a
Dionex Capillary Electrophoresis System on a 375 .mu.m.times.70-cm
capillary by a previously published method for protein analysis
(Lauer, H. H., and McManigill, D. (1986) Anal. Chem. 58, 166-170)
in 20 mM CAPS containing 10 mM potassium chloride, pH 11.0, at 20
kV at room temperature and detection was by absorbance at 280 nm.
Heparin lyase I-III samples (20 nl), each containing 2.74, 2.07,
and 2.45 mg/ml, respectively, were analyzed.
Reversed-phase HPLC
Reversed-phase (RP) HPLC (HP-1090 Hewitt Packard, CA) used a Vydac
C.sub.18 column (Sasisekharan, R. (1991) Ph.D. thesis, Cloning and
Biochemical Characterization of heparinase from Flavobacterium
heparinum, Harvard University). One nmol of each purified enzyme
was injected onto the RP-HPLC column and eluted using a gradient
from 0 to 80% acetonitrile in 0.1 to 1 TFA, H.sub.2O for 120 min.
These elution profiles were monitored at 210 and 280 nm. The enzyme
peaks were isolated for amino acid analysis for composition and
digestion with trypsin for peptide mapping.
Tryptic Peptide Mapping
A nanomole of each RP-HPLC-purified enzyme was denatured in 50
.mu.l of 8M urea containing 400 mM ammonium carbonate and 5 mM
dithiothreitol at 50.degree. C. (Sasisekharan, R. (1991) Ph.D.
thesis). After cooling to room temperature, the proteins were
alkylated with 10 mM iodoacetamide for 15 min in the dark. The
total reaction volume was 200 .mu.l. Trypsin (4%, w/w) was added to
each lyase solution, and the proteins were digested at 37.degree.
C. for 24 h. Proteolysis was terminated by heating at 65.degree. C.
for 2 min. The peptides formed in each digest were completely
soluble and were injected onto RP-HPLC column and were eluted using
a gradient from 0 to 80% acetonitrile in 120 min. The tryptic
peptide maps were monitored at 280 nm.
Amino Acid Compositional and N-terminal Analysis
Amino acid compositional analysis was performed at the Biopolymers
Laboratories at the Massachusetts Institute of Technology on an
Applied Biosystems model 420/130 Derivatizer/Amino Acid Analyzer
using Phenylisothiocyanate pre-column derivatization chemistry.
Gas-phase hydrolysis of samples was performed using a Waters Pico
Tag Hydrolysis Workstation. In pre-column derivatization, free
amino acids are coupled with phenylisothiocyanate to form
phenylthiocarbamyl amino acids that were detected at 254 nm as they
eluated from the reversed-phase column. Hydrolysis used 6N
hydrochloric acid, 0.1% phenol at either 155.degree. C. for 1 h or
100.degree. C. for 22 h. Hydrolysis times of 36 and 48 h were also
examined to ensure that the protein was being fully hydrolyzed with
minimum destruction of amino acid residues N-terminal analysis was
done on 1 nmol of heparin lyase I-III.
Effect of pH on Activity
The activity pH optimum or each of the lyases was obtained by using
succinic acid (4.0-6.5), bis-tris propane (BTP)-HCl (6.5-9.0) and
both Tris-HCl and sodium phosphate (6.0-7.5). Heparin lyase I-III
assay solutions were made by diluting a 10-.mu.l sample of the
purified lyase (2-3 mg/ml protein concentration) with 90 .mu.l of
sodium phosphate buffer at 50 mM, pH 7.0, and placed on ice until
required for assay. The activities of each lyase (I acting on
heparin, II acting on both heparin and heparan sulfate, and III
acting on heparan sulfate) were then determined at different pH
values.
Buffer Selection for Optimum Activity
The buffer giving optimum activity for each heparin lyase was
selected by testing buffers with buffering capacity near the pH
optima calculated in the previous experiments. These buffers were:
Tris-HCl, sodium phosphate, HEPES, MOPS, TES, and BTP-CHl. Each
buffer was prepared at 50 mM, and its pH was adjusted with
hydrochloric acid or sodium hydroxide to 6.9 for heparin lyase II
acting on heparin, 7.15 for heparin lyase I, 7.3 for heparin lyase
II acting on heparan sulfate, and 7.6 for heparin lyase III. The
heparin lyase assay solutions were made by diluting enzyme in 50 mM
sodium phosphate buffer adjusted to the appropriate pH as
previously described. Heparin lyase activity was determined in each
buffer. Activity was assayed both immediately after addition to
each buffer and following incubation for 24 h at 37.degree. C.
Affect of Divalent Metals and Added Salt on Activity
BTP-HCl buffer (50 mM) was prepared containing either 10 mM calcium
chloride, 10 .mu.M or 1 mM copper (II) chloride, 10 .mu.M and 1 mM
mercury (II) chloride, and 1 mM zinc (II) chloride. Each solution
was adjusted to the optimum pH for the lyase being tested, and the
activity of the heparin lyases was measured in the presence and
absence of divalent metals.
The salt concentration for optimum activity was investigated.
Sodium chloride, potassium chloride, and sodium and potassium
acetate were used to differentiate between ionic strength and
specific ion affects. Added salt concentrations varied between 0
and 500 mM and were prepared in 50 mM sodium phosphate buffer after
which the pH was adjusted to each enzyme's optimum and the heparin
lyase activity was measured.
Temperature for Optimum Activity
Temperature for optimum activity was determined for the heparin
lyases at their optimum pH in sodium phosphate buffer (the heparin
lyase I assay buffer contained 100 mM sodium chloride) in 5.degree.
increments at temperatures between 15.degree. and 55.degree. C. The
temperature was adjusted in a temperature-regulated
spectrophotometer and equilibrated for 10 min before the assay was
started.
Temperature Stability Optima
Lyase assay stock solutions were prepared in the appropriate buffer
and placed in water baths at the following temperatures: heparin
lyase I at 30.degree. C., heparin lyase II at 35.degree. C., and
heparin lyase III at both 35.degree. and 40.degree. C. Small
aliquots were taken out at various time intervals (1-22 h) to
measure remaining enzyme activity.
Determination of Kinetic Constants
Michaelis-Menten constants were determined using the optimized
conditions. The final absorbance value for total depolymerization
was divided by 20 to find a value that represented 5% reaction
completion. The purified lyase preparations were diluted so that 5%
of total depolymerization would be reached only at the end of a
3-min assay. The reaction velocities at specific molar
concentrations for each lyase and their substrates were used for
kinetic analysis using EZ-FIT hyperbolic curve-fitting program of
Perella, F. W. (1988) Anal. Biochem. 174, 437-447). Substrate
solutions were prepared from 50 mg/ml heparin and 40 mg/ml for
heparan sulfate stock solutions. These constants were determined at
30.degree. C. in 50 mM sodium phosphate buffer at pH 7.15
containing 100 mM sodium chloride for heparin lyase I and
35.degree. C. for heparin lyase II in 50 mM sodium phosphate buffer
at pH 7.3 for heparin and pH 6.9 for heparan sulfate and at
35.degree. C. in 50 mM sodium phosphate buffer at pH 7.6 for
heparin lyase III.
Activity of the heparin Lyases on Complex Polysaccharides
Each heparin lyase was added to a solution of complex
polysaccharides (1 mg/ml) under optimized assay conditions, and the
reaction was monitored at 232 nm for 30 min. The amount of purified
lyase used was sufficient for complete depolymerization of heparin
or heparan sulfate substrates within 30 min. The initial rate of
depolymerization of each polysaccharide was measured, the reaction
was then continued for 24 h, and the final level of polysaccharide
depolymerization was assessed by measuring the final absorbance at
232 nm and expressed as percent activity.
Stability of the Heparin Lyases
Heparin lyase stabilities toward freeze thawing and lyophilization
were investigated using two expicients, bovine serum albumin (BSA)
at 2 mg/ml and lactose at 0.5 wt %. Each lyase was either dissolved
in 50 mM sodium phosphate buffer, 50 mM sodium phosphate buffer
containing 2 mg/ml BSA, or 50 mM sodium phosphate buffer containing
0.5% lactose at concentrations of 1-3 units/ml. These lyase
solutions were then divided into 3 equal aliquots, and one of each
was subjected to either freeze thawing, lyophilization, or retained
as a control in an ice bath. The activities of heparin lyases I-III
were determined in the presence and absence of excipients after: 1)
brief storage at 4.degree. C.; 2) freezing at -70.degree. C. and
thawing; and 3) -70.degree. C. freezing, lyophilization, and
reconstituting with an equal volume of cold water.
RESULTS
Optimized cell lysis of F. heparinum by sonication was accomplished
in 10 min at 100 watts using a 40% pulse mode without inactivation
of the liberated enzyme. Protamine precipitation increases both the
total and specific activity by 42-fold without decreasing protein
concentration, presumably by removing the polyanionic nucleic acids
that may competitively inhibit the heparin lyases. A batch HA
purification step greatly reduces the protein concentration and
other contaminating activities associated with heparin/heparan
sulfate metabolism, but does not separate the three heparin lyase
activities. QAE-Sephadex is used to remove contaminating acidic
proteins. HA-HPLC resolves the three lyase activities. A linear
sodium chloride gradient is used to elute heparin lyases I-III at
330, 555, and 435 mM sodium chloride, respectively, as shown in
FIG. 1. Chondroitin/dermatan sulfate lyases, also found in this
bacterium, elute from the HA-HPLC column at the end of the
gradient, just behind heparin lyase II. This technique gave good
recovery of total heparin lyase activity while reducing protein
concentration. Heparin lyases I and III were further purified by
cation exchange FPLC, as shown in FIG. 2. Heparin lyase I is
recovered with excellent retention of activity and a large decrease
in protein concentration. The specific activity of heparin lyase
III does not improve using Mono-S FPLC, as it showed a substantial
reduction in total activity. SDS-PAGE analysis, however, revealed
an improvement in the purity of heparin lyase III following this
step. Heparin lyase II was not purified by Mono-S FPLC, since it
does not bind to the column. In the final purification step,
heparin lyases I-III were fractionated using GPC, as shown in FIG.
3.
Following GPC each heparin lyase preparation was shown to be
homogeneous by SDS-PAGE, acid-urea PAGE, IEF, capillary zone
electrophoresis, and reverse phase HPLC. The molecular weights
estimated by SDS-PAGE from heparin lyases I-III were 42,800,
84,100, and 70,800, respectively.
The results obtained using this purification scheme for the three
heparin lyases are summarized in Table I. Heparin lyase I was
purified 3400-fold over the cell homogenate. The scheme provided on
overall yield based on mass of 0.03%, a yield based on total
activity recovery of 10.8%, and had a specific activity of 130
units/mg. Heparin lyase II was purified 5200-fold over the cell
homogenate with an overall yield based on a mass of 0.02%. This
enzyme had a specific activity of 19 units/mg toward heparin with a
1.02% total activity recovery. This enzyme preparation also had a
specific activity of 36.5 units/mg toward heparan sulfate, a 1.54%
total activity recovery. Heparin lyase III was purified 5100-fold
over the cell homogenate, a yield of based on mass of 0.02%, a
yield based on total activity of 2.74%, and had a specific activity
of 63.5 units/mg.
TABLE-US-00001 TABLE I Purification summary of the heparin lyases
Protein Activity % Purification step mg units Unit/mg Activity
Heparin lyase I Cell homogenization 8150 66 8.12 .times. 10.sup.-3
Protam Ppt. 7960 2890 3.63 .times. 10.sup.-1 100 Batch-HA 2720 2580
9.50 .times. 10.sup.-1 89.4 QAE Sepharose 519 2220 4.27 76.8
HA-HPLC 22.6 944 41.8 32.7 Mono-S FPLC 7.36 877 119 30.4 GPC-HPLC
2.40 313 130 10.8 Heparin lyase II acting on heparin Cell
homogenization 8150 66 8.12 .times. 10.sup.-3 Protam Ppt. 7960 2890
3.63 .times. 10.sup.-1 100 Batch-HA 2720 2580 9.50 .times.
10.sup.-1 89.4 QAE Sepharose 519 2220 4.27 76.8 HA-HPLC 19.6 109
5.53 3.8 GPC-HPLC 1.55 29.4 19 1.02 Heparin lyase II acting on
heparan sulfate Cell homogenization 8150 91.5 1.13 .times.
10.sup.-2 Protam Ppt. 7960 3680 4.63 .times. 10.sup.-1 100 Batch-HA
2720 2580 1.19 88.0 QAE Sepharose 519 2220 4.11 57.8 HA-HPLC 19.6
275 14 7.46 GPC-HPLC 1.55 56.5 36.5 1.54 Heparin lyase III Cell
homogenization 8150 91.5 1.13 .times. 10.sup.-2 Protam Ppt. 7960
3860 4.63 .times. 10.sup.-3 100 Batch-HA 2720 3420 1.19 88.0 QAE
Sepharose 519 2130 4.11 57.8 HA-HPLC 23.1 1010 43.6 27.4 Mono-S
FPLC 8.41 348 41.4 9.45 GPC-HPLC 1.59 101 63.5 2.74
Characterization of heparin Lyase Purity and Physical
Properties
The physical, kinetic, and stability characteristics of the three
heparin lyases were investigated. Discontinuous SDS-PAGE (Laemmli,
U.K. (1970)) illustrated the three heparin lyases were apparently
homogeneous. The molecular weights of heparin lyase I, III were
estimated at 42,800, 84,100, and 70,800, respectively. Nonreducing
SDS-PAGE without .beta.-mercaptoethanol revealed the same bandign
pattern, suggesting that no subunits were present. IEF was used to
determine the isoelectric points of the three heparin lyases and to
assess their purity. IEF using a variety of pH gradients (pH 3-10,
7-10, and 8.5-10.5) failed to give accurate pI values for the three
lyases as they each migrated to a position very near the cathode.
An agarose gel with a pH gradient of 9-11 was then used, focusing
the three proteins below the band for cytochrome c standard
(pI=10.25). The pI values measured for heparin lyases I-III were
9.1-9.2, 8.9-9.1, and 9.9-10.1, respectively. Urea-acetic acid PAGE
in tube gels, using the method of Panyim, S., and Chalkley, R.
(1969), confirmed the homogeneity of the three heparin lyases.
Capillary zone electrophoresis electropherograms (Lauer, H. H., and
McManigill, D. (1986)) of each heparin lyase gave a single
symmetrical peak. Heparin lyases I-III had migration times of 12.7,
12.4, and 13.4 min, respectively.
RP-HPLC was used to desalt the three heparin lyases prior to amino
acid compositional analysis and tryptic digestion for peptide
mapping (Sasisekharan, R. (1991) Ph.D. thesis). Interestingly, each
chromatogram shows a very tight doublet of peaks suggesting the
presence of isoforms, possibly due to post-translational
modification. Amino acid analysis of heparin lyase isoforms for I,
II, and III were identical. The isoforms differ slightly in
hydrophilicity, possibly due to some post-translational
modification such as glycosylation or phosphorylation. The major
isoform of heparin lyases I-III had retention times of 38.5, 44.3,
and 42.7 min, respectively, in a RP-HPLC.
The major RP-HPLC peak corresponding to each heparin lyase was
treated exhaustively with trypsin to prepare peptide fragments.
These peptide fragments were again analyzed using RP-HPLC. As shown
in FIG. 6A and 6B, the peptide map of each lyase was distinctly
different although a few common peptide fragments were
observed.
Amino acid analyses of the three heparin lyases are shown in Table
II. The N-terminal amino acid is modified and hence cannot be
detected by amino acid sequencing for all three lyases.
The amino acid composition and peptide mapping demonstrate that
heparin lyases I-III are different gene products and that heparin
lyases I and III are not merely post-translationally processed from
the larger heparin lyase II.
The lyases all contain a high amount of lysine that may contribute
to their high isoelectric points. Computer modeling, using the
amino acid composition of heparin lyase I, gave a calculated
isoelectric point of 9.33 in agreement with the experimental values
obtained by using isoelectric focusing.
TABLE-US-00002 TABLE II Amino Acid Composition for Heparinase I, II
and III Amino Acid Heparinase I Heparinase II Heparinase III ASX 45
91 95 GLX 36 62 67 SER 24 37 38 GLY 30 101 50 HIS 6 14 13 ARG 13 37
35 THR 20 35 25 ALA 26 55 52 PRO 20 40 35 TYR 27 54 37 VAL 18 44 37
MET 2 15 7 ILE 20 31 24 LEU 17 53 42 PHE 17 35 36 LYS 47 47 40
Assuming 727 amino acids for heparinase II (84,000 daltons), and
636 amino acids for heparinase III (70,000 daltons). Cys and Trp
not reported.
Characterization of Optimal Catalytic Activity for the Heparin
Lyases
The optimal reaction conditions for each of the three heparin
lyases was determined in a series of experiments. The first
parameter examined was the pH optimum. A heparin concentration of
2.5 mg/ml for heparin lyases I and II and a heparan sulfate
concentration of 1.0 mg/ml for heparin lyases II and III were
demonstrated to be saturating based on published values (14, 26)
and preliminary experiments. A reaction temperature of 37.degree.
C. was initially chosen as an average of values reported in the
literature (Linhardt, R. J., Turnbull, J. E., Wang, H. M.,
Loganathan, D., and Gallagher, J. T. (1990); Silva, M. E.,
Dietrich, C. P., and Nader, H. B. (1976); Yang, V. C., Linhardt, R.
J., Berstein, H., Cooney, C. L., and Langer, R. (1985)). The
temperature was later modified after the optimum for each lyase was
determined.
The pH optima determined were 7.15 on heparin for lyase I, 7.3 on
heparin and 6.9 on heparan sulfate for lyase II, and 7.6 on heparan
sulfate for lyase III.
The buffer giving optimum activity for each heparin lyase was
selected using six different buffers each adjusted to the optimum
pH for the enzyme and substrate being studied. Heparin lyase I
showed similar initial reaction velocities in Tris-HCl and BTP-HCl,
intermediate activity in sodium phosphate, and reduced activity in
MOPS, TES, and HEPES. After incubation in each buffer at 37.degree.
C. for 24 h, the activity was reduced to 1-20% of its initial
value. Heparin lyase I incubated in MOPS, TES, and HEPES retained
the most activity. Heparin lyase II activity on heparin was
remarkably similar in all six buffers. When acting on heparan
sulfate, however, heparin lyase II also showed a marked reduction
of activity in MOPS, TES, and HEPES. After incubation in each
buffer, MOPS, TES, and HEPES were found to best protect heparin
lyase II activity (30-70% retention of activity) toward both
heparin and heparan sulfate. Heparin lyase III showed only slight
differences in activity in the six buffers studied. MOPS and HEPES
protected heparin lyase III activity (15-30% retention of activity)
following incubation.
The affect of calcium, copper (II), mercury (II), and zinc (II)
ions on heparin lyase initial reaction velocities were investigated
based on prior literature (Silva, M. E., Dietrich, C. P., and
Nader, H. B. (1976); Hovingh, P., and Linker, A. (1970)). BPT-HCl
buffer (50 mM) was chosen because of its compatibility with these
ions.
The ionic strength (0-500 mM) for optimum activity was investigated
for each heparin lyase at its pH optimum in 50 mM sodium phosphate
buffer. Sodium chloride, potassium chloride, sodium acetate and
potassium acetate gave comparable activities at the same ionic
strength. Heparin lyase I showed increased activity in response to
increased salt concentrations, with an optimum activity at 100 mM.
Heparin lyases II and III each show a decrease in activity with
increasing concentration of added salt. At 400 mM of salt, the
activity heparin lyase I-III were almost completely inhibited.
The temperature for optimum activity was determined for the heparin
lyases in 50 mM sodium phosphate buffer at their optimum pH (with
heparin lyase I containing 100 mM sodium chloride) using
temperatures between 15.degree. and 55.degree. C. The temperatures
for maximum activity were 35.degree. C. for heparin lyase I,
40.degree. C. for heparin lyase II acting on both heparin and
heparan sulfate, and 45.degree. C. for heparin lyase III. The
temperature stability optima for the heparin lyases were
established to ensure that thermal inactivation did not influence
experiments aimed at determining the kinetic constants. Heparin
lyases I and III (protein concentration of 650 ng/ml) showed an
exponential decrease in activity. Heparin lyase I lost 80% of its
activity in 5 h at 30.degree. C. Heparin lyase III lost 80% of its
activity in 3.5 h and 0.5 h at 35.degree. C. and 40.degree. C.,
respectively. Heparin lyase II (protein concentration 1-2 .mu.g/ml)
showed a much slower decay in activity, retaining 70% of its
activity on both heparin and heparan sulfate after 25 h at
35.degree. c. All further studies on heparin lyase I-III used 30,
35, and 35.degree. C., respectively, to retain high activity while
maintaining enzyme stability.
The heparin lyases showed less than 0.5% activity toward
chondroitin sulfate C and dermatan sulfate and no activity toward
chondroitin sulfate A, D, and E. No hyaluronidase, glucuronidase
activity and less than 0.5% sulfatase activity was observed.
The specificity of the three heparin lyases was examined using
their polysaccharide substrates. The initial rate and the final
level of heparin and heparan sulfate depolymerization was measured.
Heparin lyase I-III acted at an average of 7, 14, and I sites in
the heparin polymer and 5, 25, and 20 sites in the heparan sulfate
polymer, respectively. Heparin lyase II acted on heparan sulfate at
1.7 times the initial rate observed on heparin. Oligosaccharide
maps, in which the oligosaccharide products were analyzed by strong
anion-exchange HPLC and gradient PAGE (Linhardt, R. J., Turnbull,
J. E., Wang, H. M., Loganathan, D., and Gallagher, J. T. (1990)),
were prepared for each heparin lyase acting on heparin and heparan
sulfate (Lohse, D. L. (1992) Ph.D. thesis, The Heparin lyases of
Flavobacterium heparinum, The University of Iowa). These data are
consistent with the specificity for heparin lyase I-III shown in
FIG. 5.
Determination of the Michaelis-Menten Constants for the Heparin
Lyases
Michaelis-Menten constants were determined using the optimum
reaction conditions in experiments designed to calculate reaction
velocities at each substrate concentration where less than 10% had
been consumed (Table III).
Stability of heparin Lyases
It was necessary to study conditions for the optimal storage of the
heparin lyases as the literature is replete with examples of the
instability of these enzymes. In the absence of excipient, heparin
lyase I stored at 4.degree. C., after a single freeze-thawing and
after freeze-drying, retained 50, 45, and 25% of its activity,
respectively. The addition of 2.0 mg/ml BSA enhanced storage
stability, resulting in greater than 85% retention of activity, as
did the addition of 5% lactose, giving 40-80% retention of
activity. Heparin lyase II retained greater than 75% of its
activity under all storage conditions, and the addition of BSA or
lactose gave little additional stabilization. Heparin lyase III is
very unstable toward freeze-thawing and lyophilization. Heparin
lyase III retains most of its activity during brief storage at
4.degree. C. but lost 70-80% on freeze-thawing or freeze-drying.
The presence of BSA increases the recovered activity by 20-25% but
added lactose destabilizes heparin lyase III.
TABLE-US-00003 TABLE III Kinetic constants of the purified heparin
lyases Heparin lyase Substrate K.sub.m(app).sup.a V.sub.max.sup.a,b
K.sub.cat/K.s- ub.m.sup.c Heparin I 17.8 .+-. 1.50 219 .+-. 3.48
8.82 Heparin II 57.7 .+-. 6.56 16.7 .+-. 0.555 0.405 Heparan
sulfate II 11.2 .+-. 2.18 28.6 .+-. 1.26 3.57 Heparan sulfate III
29.4 .+-. 3.16 141 .+-. 3.88 5.59 .sup.aValues of the apparent
K.sub.m and V.sub.max are derived from initial velocities obtained
at eight or more concentrations (3-500 .mu.M) of either heparin or
heparan sulfate. Protein concentrations for heparin lyases I-III
were 80, 994 and 68 ng/ml, respectively. Standard errors of
apparent K.sub.m and V.sub.max values indicate the precision of
fitting the initial rates and corresponding concentrations of
heparin and heparan sulfate to the Michaelis-Menten equation as
described # under "Materials and Methods". .sup.bV.sub.max is
expressed as .mu.mol/min mg protein. .sup.cK.sub.cat/K.sub.m is
expressed as (s-.mu.M).sup.-1.
The pH optimum calculated for heparin lyase I was 7.15. This value
was higher than the pH of 6.5 reported by Yang et al. (1985) and by
Linker and co-workers (Hovingh and Linker, (1965 and 1970)). Both
groups assayed their lyase preparations using time periods of up to
6 h where thermal instability might become a factor. The maximum
time period used in this study was only 3 min. The pH optimum of
heparin lyase II acting on heparan sulfate was 6.9. The pH optimum
for heparin lyase III was 7.6. Hovingh and Linker as well as
Dietrich and co-workers reported the pH optimum of between 6.0 and
7.0 for this enzyme. Again, the assay time intervals used by both
groups were up to 6 h, and the thermal instability might account
for the differences between these values.
The activity of heparin lyase I is slightly reduced by 1 mM zinc
and markedly reduced by 10 .mu.M and 1 mM mercury and 1 mM copper.
Calcium at 10 mM increased activity by 30%. The activity of heparin
lyase II acting on both heparin and heparan sulfate in the presence
of divalent metal ions showed inhibition by all of the metals
tested except for 10 .mu.M copper. Even calcium resulted in
dramatically reduced heparin lyase II activity. Heparin lyase III
was activated (20%) by calcium, unaffected by copper and mercury
(both at 10 .mu.M), and inhibited by zinc, mercury, and copper (all
at 1 mM). In general, the addition of divalent metal ions decreased
the activity of the heparin lyases. Optimal activity of heparin
lyase I was observed at an ionic strength of 100 mM. Heparin lyases
II and III activity decreases with increasing salt
concentrations.
Table III summaries the apparent Michaelis-Menten constants for
heparin lyases I-III acting on heparin and heparan sulfate.
Apparent K.sub.m values for heparin lyase I ranging from 0.3 to 42
.mu.M and a V.sub.max of 19.7 .mu.mol/min/mg protein have been
reported (Rice, et al., (1989); Yang, et al., (1985); Lindhardt,
(1984)). An apparent K.sub.m of 5.7 .mu.M and V.sub.max of
3.57.times.10.sup.-3 .mu.mol/min for a purified heparin lyase III
acting on heparin sulfate have been reported (Rice and Lindhardt,
(1989)).
Heparin lyase I and II act on both heparin and heparan sulfate
while heparin lyase III acts only on heparan sulfate. All three
enzymes act endolytically, however, all cleavable sites within the
polymer may not be equally susceptible (Cohen, D. M. and Linhardt,
R. J. (1990) Biopolymers 30, 733-741). The primary linkages within
these polymeric substrates that are cleaved by each enzyme were
deduced from oligosaccharide mapping experiments. The specificity
of pure heparin lyase I-III toward heparin and heparan sulfate were
identical to that previously reported for their partially purified,
commercially prepared counterparts. Oligosaccharide substrates
(i.e., tetrasaccharides and hexasaccharides) having equivalent
sites are poor substrates. The V.sub.max/K.sub.m observed for
heparin lyase I and III acting on tetrasaccharide substrates is
only 0.01 to 1% of the V.sub.max/K.sub.m measured for the polymer
substrates.
The action of heparin lyases I-III on dermatan and chondroitin
sulfates A-E was also studied. These substrates vary in position
and degree of sulfation as well as the chirality of their uronic
acid. The slight activity of these enzymes toward chondroitin
sulfate C and dermatan sulfate suggested that either the heparin
lyases are contaminated or that these substrates contained small
amounts of heparin or heparan sulfate. To distinguish between these
two possibilities, the reaction was followed for longer times. All
of the activity was observed initially, after which the substrate
became stable toward repeated challenges with fresh enzyme. This
confirmed that the small activity observed was the result of
contaminated substrate (approximately 1% heparin/heparan sulfate
contamination in chondroitin sulfate C and dermatan sulfate) and
not contaminated enzyme. None of the heparin lyases showed activity
on hyaluronic acid. The failure of the heparin lyases to act on
these other glycosaminoglycans clearly demonstrates both their
specificity for heparin/heparan sulfate and the lack of
contaminating lyase activity. No glycuronidase activity (Warnick,
C. T., and Linker, A. (1972) Biochemistry 11, 568-572) was observed
and less than 0.5% sulfatase activity (McLean, M. W., Bruce, J.s.,
Long, W. F., and Williamson, F. B. (1954) Eur. J. Biochem. 145,
607-615) was detected in the purified lyases.
II. Preparation of Monoclonal Antibodies to Heparinase I,
Heparinase II, and Heparinase III
Heparin lyase I was injected into mice and their B lymphocytes used
to form monoclonal antibody-producing hybridomas. The specificity
of the monoclonal antibodies (MAbs) for each of the three heparin
lyases was examined.
MATERIALS AND METHODS
Preparation of heparin lyases for antibody production
Heparin lyases I, II and III were isolated from Flavobacterium
heparinum and purified to homogeneity as described above. Heparin
lyase concentrations were determined using a Bio-Rad Protein Assay
Kit (Richmond, Calif., U.S.A.).
Preparation of monoclonal antibodies
Six monoclonal antibodies (mAbs) were prepared. Briefly, purified
heparin lyase I was injected into mice three times over a period of
70 days. The mouse spleens were harvested and lymphocytes were
isolated from the splenocyte mixture. The lymphocytes were fused
with mouse myeloma cells to produce hybridomas. The hybridomas were
cultured and screened for production of antibodies to heparin lyase
I. Six hybridomas found to produce mAbs to heparin lyase I were
designated M-1A, M2-B7, M2-A9, M-32, M-33, and M-34. Protein
concentrations of the mAb solutions were determined using BCA
Protein Assay Reagents from Pierce (Rockford, Ill., U.S.A.).
The concentration of each monoclonal antibody is shown in Table
IV.
TABLE-US-00004 TABLE IV MAb concentrations MAb (mg/mL).sup.a M-32
49 M-33 45 M-34 41 M-1A 42 M2-A9 44 M2-B7 48 MAb solution protein
concentration determined by BCA protein assay (Pierce).
Buffers for immunoassay procedures
Nitrocellulose membranes, Goat anti-Mouse IgG (H+L) Horseradish
Peroxidase (HRP) Conjugate, Tris {hydroxymethyl} aminomethane
(Tris), gelatin, Tween-20 and HRP Color Development Reagent
(4-chloro-1-naphthol) were purchased from Bio-Rad (Richmond,
Calif., U.S.A.). Tris buffered saline (TBS) was 20 mM Tris
containing 500 mM sodium chloride, pH 7.5. Blocking solution was
3.0% gelatin in TBS. Tween-20 wash solution diluted in TBS (TTBS)
was 0.05% Tween-20 in TBS. Antibody buffer was 1% gelatin in TBS.
HRP color development solution was made by mixing 60 mg HRP Color
Development Reagent in 100 mL methanol at 0.degree. C. with
0.015.degree. % H.sub.2O.sub.2 in TBS just prior to use.
Immunoassay analysis of heparin lyases using monoclonal
antibodies
Dot-blotting immunoassay techniques were conducted as recommended
in the Bio-Rad Immun-Blot Assay protocol (Bio-Rad, Richmond,
Calif., U.S.A.). Briefly, nitrocellulose membranes were cut to
2.times.3 cm pieces and 1.times.1 cm squares were drawn on the
membranes using a soft pencil. The membranes were soaked in TBS for
15 minutes and air dried on filter paper for 15 minutes. Various
concentrations of the heparin lyase (1 .mu.L in TBS) were placed in
the center of each square and the membrane was air dried for 15
minutes, then the membrane was immersed in blocking solution for 1
hour to coat the remaining hydrophobic sites. This was washed four
times in TTBS (two quick rinses, then two 5 minute agitations),
then soaked overnight in a solution of mAb 0.2% (V/V) in antibody
buffer, then the membranes were washed 4 times with TTBS and added
to a solution of Goat anti-Mouse-HRP (0.1% in antibody buffer) for
4 hours with gentle agitation. The membranes were washed 4 times
with TTBS, then twice with TBS. HRP color development solution was
added to the membranes and when the purple bands were clearly
visible, the development was stopped by placing the membranes in
distilled water. The membranes were then dried on filter paper for
15 minutes and covered with aluminum foil to protect from
light.
Electrophoresis
Materials
Electrophoresis was performed using a Mini-Protean II
electrophoresis cell from Bio-Rad (Richmond, Calif., U.S.A.).
Acrylamide and N,N'-methylene bisacrylamide were from International
Biotechnologics Inc. (New Haven, Conn., U.S.A.) or used as a
prepared 40% acrylamide solution that is 37.5 acrylamide:l
N,N'-methylene bisacrylamide (Fischer Scientific, Fairlawn, N.J.,
U.S.A.). Tris {hydroxymethyl} aminomethane (Tris) was from Bio-Rad
(Richmond, Calif., U.S.A.). N,N,N',N'-Tetramethylethylenediamine
(TEMED) was from Boehringer Mannheim Biochemicals (Indianapolis,
Ind., U.S.A.). Ammonium persulfate (APS) and glacial acetic acid
were from Mallinckrodt Inc. (Pads, Ky., U.S.A.). Urea and glycerol
were from Fischer Scientific (Fair Lawn, N.J., U.S.A.). Sodium
dodecyl sulfate (SDS) was from BDH Chemicals, Ltc. (Poole,
England). Naphthol red was from Sigma Chemical Co. (St. Louis, Mo.,
U.S.A.). 2-.beta.-mercaptoethanol was from EM Science (Gibbstown,
N.J., U.S.A.). Bromophenol blue was from MCB Manufacturing
Chemists, Inc. (Cincinnati, Ohio, U.S.A.). Molecular Weight
Standards and Rapid Coomassie Stain were from Diversified Biotech
(Newtown Centre, Mass., U.S.A.)
SDS-polyacrylamide gel electrophoresis (PAGE)
Heparin lyases I, II, III and Flavobacterium heparinum cell
homogenate were analyzed using SDS-PAGE as described above.
Separating gels (12% acrylamide, 10% SDS) were prepared by mixing
4.35 mL distilled water, 2.5 mL of 1.5M Tris, pH 8.8 and 3.0 mL of
a commercially prepared solution of 37.5 acrylamide:l
N,N'-methylene bisacrylamide (Fischer Scientific, Fairlawn, N.J.,
U.S.A.) as described above. This solution was degassed under vacuum
for at least 15 minutes. Next, 50 .mu.L of APS (10%) and 5 .mu.L of
TEMED were added to the monomer solution to initiate
polymerization. The gel solution was quickly poured between two
glass plates separated by 0.75 mm spacers, overlaid with distilled
water saturated gamma-butanol and allowed to polymerize at
25.degree. C. for 60 minutes.
Stacking gel was prepared by mixing 6.4 mL distilled water, 2.5 mL
0.5 M Tris, pH 6.8, 1.0 mL acrylamide/Bis solution (Fischer
Scientific), 50 .mu.L APS (10%) and 10 .mu.L TEMED. The
gamma-butanol was removed from the separating gel, the gel was
rinsed with distilled water and the stacking gel solution was
carefully added to the top of the separating gel. A well-forming
electrophoresis comb was inserted in the stacking gel prior to
polymerization. The stacking gel was allowed to polymerize for 60
minutes and the well-forming comb was removed just prior to loading
of the samples.
Sample buffer was prepared by mixing 4.0 mL distilled water, 1.0 mL
0.5M Tris, pH 6.8, 0.8 mL glycerol, 1.6 mL SDS (10%), 0.4 mL
2-.beta.-mercaptoethanol and 0.2 mL bromophenol blue (0.05% W/V).
Samples and molecular weight standard markers for electrophoresis
were diluted 1:4 in sample buffer and heated for 4 minutes at
100.degree. C. just prior to loading into the wells formed earlier
in the stacking gel. Running buffer (0.125M Tris, 0.1M glycine,
0.5% SDS, pH 8.3) was carefully overlaid on the stacking gel and
the electrophoresis was conducted at a constant voltage of 200 V
until the bromophenol blue marker moved to within 0.3 cm of the
bottom of the gel (typically about 45 minutes). Following
electrophoresis, the gels were either electro-transferred to
nitrocellulose membranes or were stained with Rapid Coomassie Stain
for 45 minutes followed by destaining with a 7.5% methanol/5%
acetic acid solution.
Urea/Acetic Acid-PAGE
In some experiments, an urea/acetic acid-PAGE system (Panyim, S.,
and Chalkley, R. (1969) High resolution acrylamide gel
electrophoresis of histories. Arch. Biochem. Biophys. 130, 337-346)
was used instead of SDS-PAGE to compare the effects of SDS on the
capacity of the mAbs to detect the heparin lyases in Western blots.
Stock solutions used in the preparation of the urea/acetic
acid-PAGE gels were prepared as follows. A 60% acrylamide solution
was prepared by dissolving 60 g acrylamide and 0.4 g N,N'-methylene
bisacrylamide in 1 00 mL of distilled water. A 43.2% acetic
acid/TEMED stock solution was prepared by mixing 43.2 mL acetic
acid, 4.0 mL TEMED and 52.8 mL distilled water. APS/urea was
prepared by dissolving 5 mg APS in 25 mL of 1 0M urea.
The urea/acetic acid-PAGE gels were formed by mixing 4.0 mL of 60%
acrylamide solution, 3.0 mL 43.2% acetic acid/TEMED and 2.0 mL
distilled water. This solution and the APS/urea solution were
degassed for 15 minutes, 15 mL of the APS/urea was added to the
acrylamide monomer solution, mixed and carefully poured between two
glass plates separated by two 0.75 mm spacers. A well-forming
electrophoresis comb was inserted into the top portion of the gel
and the gel was allowed to polymerize for 60 minutes.
The heparin lyases were diluted 1:4 in urea/acetic acid sample
buffer. This sample buffer was prepared by mixing 520 .mu.L acetic
acid, 1.0 mL glycerol, 1.0 mg naphthol red, and 6.0 g urea in
distilled water that was brought to a final volume of 10 mL. The
well-forming comb was removed and samples were loaded into wells
and overlaid with running buffer (0.9M acetic acid).
Electrophoresis was conducted at a constant current of 20 mA for 3
hours (prefocusing of the gel) and then at 10 mA until the naphthol
red moved to about 0.3 cm from the bottom of the gel (about 3
hours).
Electro-transfer of heparin lyases from acrylamide gels to
nitrocellulose membranes
Semi-dry transblotting was conducted using a SemiPhor Transfer Unit
(TE-70) from Hoefer Scientific Instruments (San Francisco, Calif.,
U.S.A.). Electro-transfer of the heparin lyases from the SDS-PAGE
or Urea/acetic acid-PAGE to nitrocellulose membranes was
accomplished using Semi-dry transblotting techniques as described
by Al -Hakim, A., and Linhardt, R. J. (1990) Isolation and recovery
of acidic oligosaccharides from polyacrylamide gels by semi-dry
electrotransfer. Electrophoresis 11, 23-28, except that 50 mM
sodium phosphate, pH 6.8 was used as the transfer buffer. Transfer
was accomplished in 40 minutes at 8 V.
Western blot detection of the heparin lyases using the monoclonal
antibodies
Heparinases on the nitrocellulose membranes were detected using
Western blotting techniques exactly as described above for
dot-blotting immunoassay procedures.
Effects of SDS on detection of monoclonal antibodies
The effects of SDS and 2-.beta.-mercaptoethanol on the
immunodetection of the heparin lyases by mAbs M-32 and M-33 were
examined. Dot-blotting immunoassays of heparin lyases I and II were
performed as described earlier except that the heparin lyases were
dissolved in solutions containing SDS and/or
2-.beta.-mercaptoethanol in the same proportions used in SDS-PAGE
analysis prior to blotting on the nitrocellulose membrane.
RESULTS
The reactivity of each of the six mAbs toward the three heparin
lyases was examined. Varying amounts of each of the three heparin
lyases were spotted on nitrocellulose membranes and detected using
the anti-heparin lyase mAbs followed by addition of Goat anti-Mouse
IGG-HRP and color development of the immune conjugates. Table V
summarizes the lowest levels of each heparin lyases that were
detected by immunoassay procedures. As seen in Table V, the mAbs
have a broad range of sensitives toward immunodetection of the
three heparin lyases. For instance, M2-A9 and M2-B7 can detect as
little as 10 pg of heparin lyase II, whereas M-32, M-33 and M-34
require the presence of 1 .mu.g of heparin lyase III in order to
detect that lyase.
TABLE-US-00005 TABLE V MAb detection of heparin lyases on
nitrocellulose membranes.sup.a MAb Heparin lyase I Heparin lyase II
Heparin lyase III M-32 10 ng 100 ng 1 .mu.g M-33 10 ng 10 ng 1
.mu.g M-34 10 ng 10 ng 1 .mu.g M-1A 100 pg 100 pg 10 ng M2-A9 100
pg 10 pg 10 ng M2-B7 100 pg 10 pg 10 ng .sup.aThe minimum amount of
each heparin lyase detectable by each of the six mAb using
dot-blotting immunodetection.
These data demonstrate that mAbs can be used to distinguish between
heparin lyases I and II when the two are present together, as in a
Flavobacterium heparinum cell homogenate. Specifically, M-32 can
detect levels of heparin lyase I that are ten times lower than
heparin lyase II. Conversely, M2-A9 and M2-B7 can detect levels of
heparin lyase II that are ten times lower than heparin lyase I.
M-33, M-34 and M-lA cannot be used to distinguish between heparin
lyases I and II. Furthermore, all six of the mAbs are able to
detect much lower levels of heparin lyases I and II than of heparin
lyase III, thus permitting distinction between heparin lyase III
and heparin lyases I or II. Distinction between heparin lyases I
and II is important because both enzymes can act on heparin and
heparan sulfate and therefore are not easily distinguished based on
their substrate specificity.
Western blot analysis of the heparin lyases
The three heparin lyases and Flavobacterium heparinum cell
homogenate samples were analyzed on SDS-PAGE followed by Western
blotting immunodetection, shown in FIG. 5a. FIG. 5b contains a
typical SDS-PAGE gel of the three heparin lyases stained with
Coomassie Blue along with molecular weight markers. The ability of
mAbs to detect heparin lyases was examined by running the three
heparin lyases and Flavobacterium heparinum cell homogenate through
six SDS-PAGE gels followed by Western blotting immunodetection of
the gel contents. Heparin lyase I (18 ng), heparin lyase II (570
ng), heparin lyase III (1.63 .mu.g) and cell homogenate (87 ng)
were loaded on each gel. The developing time used for detection on
the nitrocellulose membrane containing M-34, Ml-A, M2-A9 and M2-B7
were 20, 10, 15 and 40 minutes, respectively. Four of the mAbs
(M-34, M-lA, M2-A9 and M2-B7) were able to detect purified heparin
lyases I, II and III as well as heparin lyases present in the
Flavobacterium heparinum cell homogenate. Two mAbs (M-32 and M-33)
were not able to detect either the purified heparin lyases or
cellular proteins in the Western blots.
The reagent in the SDS-PAGE system that was responsible for
destroying the ability of M-32 and M-33 to immunodetect the heparin
lyases was determined. Dot-blotting immunoassays of the heparin
lyases using M-32 and M-33 were used to evaluate each component in
the SDS-PAGE system. Heparin lyases I and II, in the presence or
absence of SDS and/or 2-.beta.-mercaptoethanol, were blotted on
nitrocellulose membranes and examined using dot-blotting
immunoassay techniques. The mAbs were unable to detect the lyases
when SDS was present, demonstrating that SDS was responsible for
the reduction of sensitivity of these two MAbs during the Western
blotting procedures. This experiment suggests that M-32 and M-33
must be recognizing an epitope on the lyases that requires
secondary conformation such as a folded structure present in all
three heparin lyases that is destroyed by SDS denaturation.
To further demonstrate that the SDS was responsible for the
diminished reactivity of M-32 and M-33 toward the heparin lyases,
the three heparin lyases and Flavobacterium heparinum cell
homogenate were analyzed using the urea/acetic acid-PAGE followed
by Western blotting immunodetection with M-32 and M-33 to detect
the lyases in this system. The sensitivity of detection was
markedly reduced. Heparin lyase I (2.7 .mu.g), heparin lyase II
(3.4 .mu.g), heparin lyase III (4.7 .mu.g) and cell homogenate (7.7
.mu.g) were detectable. Thus, SDS is the agent primarily
responsible for the reduced reactivity of MAbs M-32 and M-33 toward
the heparin lyases. All six MAbs are able to detect all three
heparin lyases, in either the purified or the native form, when
analyzed using PAGE followed by Western blotting
immunodetection.
It was expected that at least one of the six MAbs would
specifically detect a single heparin lyase, enabling the detection
of that lyase in a complex mixture of heparin lyases such as a cell
homogenate. The dot-blotting and Western analyses revealed that all
of the mAbs are able to detect all three lyases. This observation
suggests that these three heparin lyases are remarkably similar in
structure since they share six common epitopes. Peptide mapping of
these three enzyme demonstrates a number of common peptide
fragments and suggests that these may be located at the highly
immunogenic regions within the three heparin lyases. The
sensitivities of individual mAbs toward each of the lyases in the
dot-blotting analyses varied greatly, thus offering the potential
to use the dot-blotting analysis to distinguish between the three
lyases.
Use of PAGE (SDS or urea/acetic acid) required much more protein
than dot-blotting procedures and the sensitivities of the mAbs
toward each of the lyases were different than those seen in the
dot-blotting analyses, probably due to alterations of secondary
structure during the PAGE and transfer steps. Thus, detection of
heparin lyases using mAbs is most efficiently conducted by use of
dot-blotting techniques as described here. Furthermore, all six
MAbs were able to detect all three lyases that were present in
Flavobacterium heparinum cell homogenate, thus offering the
potential that these mAbs could be used to rapidly demonstrate the
presence of heparin lyases in cell homogenate. To be beneficial in
lyase purification, these MAbs must first be immobilized and their
binding activity to the heparin lyases assessed. Methods and
materials for immobilization of antibodies are commercially
available and known to those skilled in the art.
In summary, the results described here demonstrate that mAbs can be
used to detect heparin lyases I, II and III in either their
purified state or when present together in a solution of
homogenized Flavobacterial cells. These mAbs can also be used in
dot-blotting analyses of the lyases to distinguish between the
three lyases based on their different sensitivity for each of the
three lyases.
Modifications and variations of the purified heparinases, method of
purification and monoclonal antibodies thereto will be obvious to
those skilled in the art from the foregoing detailed description.
Such modifications and variations are intended to come within the
scope of the appended claims.
* * * * *