title: Purification and characterization of a novel heparinase.
abstract: A unique heparinase was isolated from a recently discovered Gram-negative soil bacterium. The enzyme (heparinase III) was purified by hydroxylapatite chromatography, chromatofocusing, and gel permeation chromatography. The enrichment was 48x, and the specific activity of catalytically pure heparinase was 127 IU/mg of protein. Similar to the heparinase I from Flavobacterium heparinum, heparinase III also degrades heparin to mainly disaccharide fragments. It is specific for heparin and also breaks down heparan sulfate, but not hyaluronic acid and chondroitin sulfate. Heparinase III, however, differs markedly from heparinase I in several other aspects: it has a higher molecular mass (94 versus 43 kDa), pI (9.2 versus 8.5), its Km and kcat are different, and it has a higher energy of activation (15.6 versus 6.3 kcal/mol). Optimal activity was also found at higher pH (7.6 versus 6.5) and temperature (45 versus 37 degrees C). Furthermore, the amino acid composition of heparinase III is quite different from that of heparinase I.
authors.affiliation: Department of Physiology, University of Pretoria, South Africa.
authors.forename: L H
authors.initials: LH
authors.lastname: Böhmer
authors.forename: M J
authors.initials: MJ
authors.lastname: Pitout
authors.forename: P L
authors.initials: PL
authors.lastname: Steyn
authors.forename: L
authors.initials: L
authors.lastname: Visser
mesh_terms: D000596:Amino Acids / Q000032:analysis; D002845:Chromatography; D002850:Chromatography, Gel; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D005417:Flavobacterium / Q000201:enzymology*; D019763:Heparin Lyase; D006882:Hydroxyapatites; D005779:Immunodiffusion; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D009994:Osmolar Concentration; D011133:Polysaccharide-Lyases / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D000596:Amino Acids; D006882:Hydroxyapatites; D007527:Isoenzymes; D017886:Durapatite; D011133:Polysaccharide-Lyases; D019763:Heparin Lyase