A new affinity method for purification of bovine testicular hyaluronidase enzyme and an investigation of the effects of some compounds on this enzyme.
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 K(M) and V(Max) for BTH were determined by using hyaluronic acid as a substrate. K(M) and V(Max) 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.
Division of Basic Sciences, Biochemistry Department, Faculty of Veterinary Medicine, Siirt University , Siirt , Turkey .
Mustafa Oguzhan
MO
Kaya
Oktay
O
Arslan
Ozen Ozensoy
OO
Guler
D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006821:Hyaluronoglucosaminidase / Q000187:drug effects / Q000302:isolation & purification*; D008297:Male; D013737:Testis / Q000201:enzymology*
D006821:Hyaluronoglucosaminidase
Affinity chromatography; bovine testicular hyaluronidase; inhibition