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Abstract
In this study, a new affinity gel for the purification of bovine testicular hyaluronidase (BTH) was synthesized. l-Tyrosine was added as the extension arm to the Sepharose-4B activated with cyanogen bromide. m-Anisidine is a specific inhibitor of BTH enzyme. m-Anisidine was clamped to the newly formed Sepharose-4B-l-tyrosine as a ligand. As a result, an affinity gel having the chemical structure of Sepharose-4B-l-tyrosine-m-anisidine was obtained. BTH purified by ammonium sulfate precipitation and affinity chromatography was obtained with a 16.95% yield and 881.78 degree of purity. The kinetic constants KM and VMax for BTH were determined by using hyaluronic acid as a substrate. KM and VMax values obtained from the Lineweaver–Burk graph were found to be 2.23 mM and 19.85 U/mL, respectively. In vitro effects of some chemicals were determined on purified BTH enzyme. Some chemically active ingredients were 1,1-dimethyl piperidinium chloride, β-naphthoxyacetic acid and gibberellic acid. Gibberellic acid showed the best inhibition effect on BTH.
Acknowledgements
The authors thank Dr. Malcolm Lyon for his invaluable contribution on this paper.
Declaration of interest
The authors do not report any declaration of interest for this study.
This work was supported by a Balikesir University Research Project (2012/38) and carried out at the Balikesir University Research Center of Applied Sciences (BURCAS).