A new affinity method for purification of bovine testicular hyaluronidase enzyme and an investigation of the effects of some compounds on this enzyme

Abstract

In this study, a new affinity gel for the purification of bovine tes-ticular hyaluronidase (BTH) was synthesized. L-Tyrosine wasadded as the extension arm to the Sepharose-4B activated withcyanogen bromide. m-Anisidine is a specific inhibitor of BTHenzyme. m-Anisidine was clamped to the newly formed Sepha-rose-4B-L-tyrosine as a ligand. As a result, an affinity gel havingthe chemical structure of Sepharose-4B-L-tyrosine-m-anisidinewas obtained. BTH purified by ammonium sulfate precipitationand affinity chromatography was obtained with a 16.95% yieldand 881.78 degree of purity. The kinetic constants KMand VMaxfor BTH were determined by using hyaluronic acid as a sub-strate. KMand VMaxvalues obtained from the Lineweaver-Burkgraph were found to be 2.23 mMand 19.85 U/ml, respectively.Invitroeffects of some chemicals were determined on purified BTHenzyme. Some chemically active ingredients were 1,1-dimethylpiperidinium chloride,b-naphthoxyacetic acid and gibberellicacid. Gibberellic acid showed the best inhibition effect on BTH.