A rapid purification of bovine testicular hyaluronidase by chromatography on dermatan sulphate-substituted 1,6-diaminohexane--sepharose 4B.
The binding of bovine testicular hyaluronidase to AH-Sepharose (1,6-diaminohexane--Sepharose) gels substituted with (1) dermatan sulphate, (2) desulphated dermatan sulphate, (3) heparin and (4) de-N/O-sulphated, re-N-acetylated heparin was investigated. Hyaluronidase was found to bind to (1) and (3), but not (2) and (4). On the basis of these observations a preparative scheme for the purification of testicular hyaluronidase was developed. This consisted of two steps: (i) chromatography on dermatan sulphate-substituted AH-Sepharose 4B; (ii) chromatography on acetylated AH-Sepharose 4B. This procedure gave hyaluronidase with a specific activity of 19.1 units (mumol/min)/mg in high yield. Polyacrylamide-gel electrophoresis at pH 4.3 revealed two components, both possessing hyaluronidase activity. Sodium dodecyl sulphate polyacrylamide-gel electrophoresis likewise revealed two close bands with approximate molecular weights of 61000 and 67200.
D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods*; D003871:Dermatan Sulfate; D003959:Diamines; D004591:Electrophoresis, Polyacrylamide Gel; D006025:Glycosaminoglycans; D006821:Hyaluronoglucosaminidase / Q000302:isolation & purification*; D008297:Male; D012685:Sepharose / Q000031:analogs & derivatives; D013737:Testis / Q000201:enzymology*
D003959:Diamines; D006025:Glycosaminoglycans; D003871:Dermatan Sulfate; C024903:aminohexyl-sepharose; D012685:Sepharose; D006821:Hyaluronoglucosaminidase; C007297:1,6-diaminohexane