Preliminary characterization of the multiple forms of ram sperm hyaluronidase

Abstract

An investigation was made of the inter-relationships and characteristics of various hyaluronidase forms isolated from ram spermatozoa. They were shown to be members of an oligomeric series, apparently formed by intermolecular disulphide cross-linking. Two monomer species were detected, alpha (Mr 89,600) and beta (Mr 81,200). Although the alpha species predominated, the two were evenly distributed throughout the oligomer population, and they shared antigenic determinants; the beta species did not arise from the alpha species as a result of catabolism following cell disruption. The oligomeric series was of the form [Hyal]n, where n = 1, 2, 4, 5, 6, 7 etc.; no trimer was detectable. Though essentially cationic, part of the hyaluronidase population also had anionic characteristics, probably due to oxidation of free thiol groups. In the anionic subpopulation tetramers and higher oligomers predominated, whereas the non-anionic subpopulation was composed of monomers, dimers and tetramers. The pH optimum of the monomer was 4.3 in 0.2 M-NaCl/0.1 M-sodium citrate, whereas that of the anionic oligomers was 4.9. Both serum albumin and polylysine stimulated enzyme activity at pH 4.0 in the absence of NaCl; polylysine was particularly effective. NaCl diminished the stimulatory effects, and essentially suppressed them above the pH optimum. The specific activities of different oligomer populations were the same as that of the monomer, and conversion of oligomers into monomer by reduction had likewise no effect upon the specific activity. Low concentrations of poly(vinyl alcohol), poly(ethylene glycol) or polyvinylpyrrolidone stabilized soluble hyaluronidase activity by preventing the enzyme's binding to surfaces; solutions of anionic oligomers were further stabilized by NaCl. Enzyme preparations were stable for several months frozen in the presence of poly(vinyl alcohol) and salt.