Purification and Characterization of Heparinase from Flavobacterium heparinum
AuthorYang, V.C.; Linhardt, Robert J.; Bernstein, H.; Cooney, C.L.; Langer, R.
SubjectBiology; Chemistry and chemical biology; Chemical and biological engineering; Biomedical engineering
Full CitationPurification and Characterization of Heparinase from Flavobacterium heparinum, V.C. Yang, R.J. Linhardt, H. Bernstein, C.L. Cooney, R. Langer, The Journal of Biological Chemistry, 260, 1849-1857 (1985).
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AbstractHeparinase (EC 220.127.116.11) isolated from Flavobacterium heparinum was purified to homogeneity by a combination of hydroxylapatite chromatography, repeated gel filtration chromatography, and chromatofocusing. Homogeneity was established by the presence of a single band on both sodium dodecyl sulfate and acid-urea gel electrophoretic systems. Amino acid analysis shows that the enzyme contains relatively high amounts of lysine residues (9%) consistent with its cationic nature (pI 8.5) but contains only 4 cysteine residues/polypeptide. The molecular weight of heparinase was estimated to be 42,900 +/- 1,000 daltons by gel filtration and 42,700 +/- 1,200 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is very specific, acting only on heparin and heparan monosulfate out of 12 similar polysaccharide substrates tested. It has an activity maximum at pH 6.5 and 0.1 M NaCl and a stability maximum at pH 7.0 and 0.15 M NaCl. The Arrhenius activation energy was found to be 6.3 kcal/mol. However, the enzyme is very sensitive to thermal denaturation and loses activity very rapidly at temperatures over 40 degrees C. Kinetic studies of the heparinase reaction at 37 degrees C gave a Km of 8.04 X 10(-6) M and a Vm of 9.85 X 10(-5) M/min at a protein concentration of 0.5 microgram/ml. By adapting batch procedures of hydroxylapatite and QAE (quaternary aminoethyl)-Sephadex chromatography, gram quantities of heparinase that is nearly free of catalytic enzyme contaminants can be purified in 4-5 h.;
DescriptionThe Journal of Biological Chemistry, 260, 1849-1857; Note : if this item contains full text it may be a preprint, author manuscript, or a Gold OA copy that permits redistribution with a license such as CC BY. The final version is available through the publisher’s platform.
DepartmentThe Linhardt Research Labs.; The Shirley Ann Jackson, Ph.D. Center for Biotechnology and Interdisciplinary Studies (CBIS);
RelationshipsThe Linhardt Research Labs Online Collection; Rensselaer Polytechnic Institute, Troy, NY; https://harc.rpi.edu/;
AccessCC BY — Creative Commons Attribution; A full text version is available in DSpace@RPI; Open Access;
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