OPTIMIZATION OF THE USP ASSAY FOR HYALURONIDASE

The current USP XXII assay for hyaluronidase (EC 3.2.1.35, HAse) deter mines activity indirectly by measuring the amount of undegraded hyalur onic acid (HA) substrate remaining after the enzyme is allowed to reac t with the HA for 30 min at 37-degrees-C. To be acceptable as a substr ate, the HA must pass a USP suitability test. In this study, seven HA samples, which differed in their anatomical origin, their commercial s upplier, and their chondroitin sulphate content, were tested as substr ates. One of these did not pass the USP suitability test and therefore would not be an officially acceptable substrate; however, it was carr ied through the investigation along with the others in order to demons trate its effect on the analysis. All seven HAs were used as substrate s to assay testicular hyaluronidases from three different suppliers. T he standard by which the other hyaluronidase activities were measured was USP hyaluronidase reference standard. The activity values calculat ed for a particular hyaluronidase differed significantly depending on which HA was used as substrate in its assay. Optimal results, as judge d on the bases of initial purity, suitability for the assay, linearity of the standard curve, and per cent relative standard deviation of th e measured activity, were obtained with a HA substrate derived from vi treous humour.