Purification of bull sperm hyaluronidase by concanavalin-A affinity chromatography.
A new method for obtaining highly purified hyaluronidase (hyaluronate glycanohydrolase EC 3.2.1.25) in high yield is described. Bull seminal plasma was fractionated with (NH4)2 SO4 and the 30 to 65% saturation fractions  were applied to a DEAE-cellulose column. The first protein peak contained hyaluronidase, beta-N-acetylglucosaminidase and beta-glucuronidase. The latter two enzymes were separated by gel filtration on Sephadex G-200. The hyaluronidase was further purified by a Concanavalin-A Sepharose 4B affinity column. By gradient elution with alpha-methyl-D-glucoside a fraction which had a specific activity of 1998 units/mg protein (57 942 National Formulary Standard units/mg protein) was obtained. The highly purified enzyme showed one major protein band on acrylamide gel electrophoresis at pH 4.3. The purified hyaluronidase did not show any beta-glucuronidase or beta-N-acetylglucosaminidase activities. The percent yield of purified hyaluronidase calculated on the basis of total activity was ten times higher than by any pervious method [Yang, C.H. and Srivastava, P.N. (1975) J. Biol. Chem. 250, 79-83].
D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003208:Concanavalin A; D004589:Electrophoresis, Disc; D006821:Hyaluronoglucosaminidase / Q000302:isolation & purification* / Q000378:metabolism; D008297:Male; D008970:Molecular Weight; D013094:Spermatozoa / Q000201:enzymology*
D003208:Concanavalin A; D006821:Hyaluronoglucosaminidase