Large Scale Preparation and Characterization of Mucopolysaccharase Contamination Free Heparinase

Overview

Journal Appl Biochem Biotechnol

Specialties Biochemistry
Biotechnology

Date 1987 Sep 1

PMID 3504129

Authors

V C Yang

H Bernstein

C L Cooney

R Langer

Affiliations

Soon will be listed here.

Abstract

By a combination of hydroxylapatite chromatography and negative adsorption on QAE-Sephadex at pH 8.3, heparinase (E.C.4.2.2.7) can be successfully isolated from all the other mucopolysaccharase contaminants present in Flavobacterium heparinum. Hydroxylapatite isolates heparinase primarily from chondroitinases, hyaluronidase, and most glycuronidases. QAE-Sephadex chromatography at pH 8.3 further separates heparinase from heparitinases, sulfatases, and the remaining glycuronidases. The heparinase preparation thus obtained contains no statistically significant levels of other contaminating mucopolysaccharases except for heparitinases that are present at an apparent maximum level of 3.4%. Owing to the presence of a crossreaction of heparinase on heparitin sulfate at conditions employed for the assay of heparitinase, the heparitinase level of 3.4% could be misleading because of the action of heparinase on heparitin sulfate. Characterization of this heparinase preparation shows that the enzyme has an optimum salt concentration of 0.08M NaCl, an optimum pH of 6.5, an activation energy of 5 kcal/mol, and a Km of 7.95 X 10(-6) M. These parameters are almost identical to those displayed by a homogeneous heparinase preparation. The method described here is suitable for scale-up purposes using batch chromatographic procedures.

References

Yang V, Langer R . pH-dependent binding analysis, a new and rapid method for isoelectric point estimation. Anal Biochem. 1985; 147(1):148-55. DOI: 10.1016/0003-2697(85)90021-1. View

Oike Y, Kimata K, Shinomura T, Nakazawa K, Suzuki S . Structural analysis of chick-embryo cartilage proteoglycan by selective degradation with chondroitin lyases (chondroitinases) and endo-beta-D-galactosidase (keratanase). Biochem J. 1980; 191(1):193-207. PMC: 1162197. DOI: 10.1042/bj1910193. View

Grant A, Linhardt R, Fitzgerald G, Park J, Langer R . Metachromatic activity of heparin and heparin fragments. Anal Biochem. 1984; 137(1):25-32. DOI: 10.1016/0003-2697(84)90341-5. View

Galliher P, Cooney C, Langer R, Linhardt R . Heparinase production by Flavobacterium heparinum. Appl Environ Microbiol. 1981; 41(2):360-5. PMC: 243699. DOI: 10.1128/aem.41.2.360-365.1981. View

Langer R, Linhardt R, Hoffberg S, Larsen A, Cooney C, Tapper D . An enzymatic system for removing heparin in extracorporeal therapy. Science. 1982; 217(4556):261-3. DOI: 10.1126/science.7089564. View

Korn E, PAYZA A . The degradation of heparin by bacterial enzymes. II. Acetone powder extracts. J Biol Chem. 1956; 223(2):859-64. View

Silva M, Dietrich C . Structure of heparin. Characterization of the products formed from heparin by the action of a heparinase and a heparitinase from Flavobacterium heparinum. J Biol Chem. 1975; 250(17):6841-6. View

Yang V, Linhardt R, Bernstein H, Cooney C, Langer R . Purification and characterization of heparinase from Flavobacterium heparinum. J Biol Chem. 1985; 260(3):1849-57. View

Linker A, Hovingh P . Isolation and characterization of oligosaccharides obtained from heparin by the action of heparinase. Biochemistry. 1972; 11(4):563-8. DOI: 10.1021/bi00754a013. View

10.

Linhardt R, Fitzgerald G, Cooney C, Langer R . Mode of action of heparin lyase on heparin. Biochim Biophys Acta. 1982; 702(2):197-203. DOI: 10.1016/0167-4838(82)90503-9. View

11.

Michelacci Y, Dietrich C . Chondroitinase C from Flavobacterium heparinum. J Biol Chem. 1976; 251(4):1154-8. View

12.

Yamagata T, Saito H, Habuchi O, Suzuki S . Purification and properties of bacterial chondroitinases and chondrosulfatases. J Biol Chem. 1968; 243(7):1523-35. View

13.

Dietrich C, Silva M, Michelacci Y . Sequential degradation of heparin in Flavobacterium heparinum. Purification and properties of five enzymes involved in heparin degradation. J Biol Chem. 1973; 248(18):6408-15. View

14.

Ototani N, Kikuchi M, YOSIZAWA Z . Purification of heparinase and heparitinase by affinity chromatography on glycosaminoglycan-bound AH-Sepharose 4B. Carbohydr Res. 1981; 88(2):291-303. DOI: 10.1016/s0008-6215(00)85542-1. View

15.

Lloyd A, Law B, Fowler L, Embery G . Bacterial carbohydrate sulphamidase induction and heparin degradation. Biochem J. 1968; 110(4):54P. PMC: 1187474. View

16.

Dietrich C . Enzymic degradation of heparin. A sulphamidase and a sulphoesterase from Flavobacterium heparinum. Biochem J. 1969; 111(1):91-5. PMC: 1187498. DOI: 10.1042/bj1110091. View

17.

Dietrich C . Enzymic degradation of heparin. A glucosaminidase and a glycuronidase from Flavobacterium heparinum. Biochemistry. 1969; 8(5):2089-94. DOI: 10.1021/bi00833a046. View

18.

Michelacci Y, Dietrich C . Isolation and partial characterization of an induced chondroitinase B from Flavobacterium heparinum. Biochem Biophys Res Commun. 1974; 56(4):973-80. DOI: 10.1016/s0006-291x(74)80284-6. View

19.

Bradford M . A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976; 72:248-54. DOI: 10.1016/0003-2697(76)90527-3. View

20.

LOWRY O, ROSEBROUGH N, FARR A, RANDALL R . Protein measurement with the Folin phenol reagent. J Biol Chem. 1951; 193(1):265-75. View