Heparin-sepharose affinity chromatography for purification of bull seminal-plasma hyaluronidase.
Bull seminal-plasma hyaluronidase was purified 180-fold by chromatography on concanvalin A-Sepharose, heparin Sepharose, Sephadex G-200 and Sephacryl S-200. With hyaluronic acid as the substrate, the specific activity and turnover number of purified hyaluronidase were 3.63 mumol/min per mg (104000 National Formulary units/mg of protein) and 214 min-1 (mol of product formed/mol of enzyme per min) respectively. Polyacrylamide-gel electrophoresis indicated that the purified enzyme migrated as a single band on 7.5 and 10% (w/v) gels at pH 4.3 and 5.3. Bull seminal-plasma hyaluronidase was markedly inhibited by hydroxylamine, phenylhydrazine and semicarbazide. Purified hyaluronidase (1.25 munits; 1 unit = 1 mumol of N-acetylglucosamine liberated/min at 37 degrees C) dispersed the cumulus clot of rabbit ova in 1 h at 22 degrees C.
D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D005260:Female; D006493:Heparin; D006821:Hyaluronoglucosaminidase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000494:pharmacology; D006898:Hydroxylamines / Q000494:pharmacology; D008297:Male; D008722:Methods; D010063:Ovum / Q000187:drug effects; D010659:Phenylhydrazines / Q000494:pharmacology; D011817:Rabbits; D012661:Semen / Q000201:enzymology*; D012663:Semicarbazides / Q000494:pharmacology; D012685:Sepharose
D006898:Hydroxylamines; D010659:Phenylhydrazines; D012663:Semicarbazides; D006493:Heparin; D012685:Sepharose; D006821:Hyaluronoglucosaminidase