ISOLATION AND CHARACTERIZATION OF THE CATALASE-PEROXIDASE ENZYME OF BACILLE CALMETTE-GUERIN - EFFECT OF ISONIAZID

A haem-containing enzyme with both catalase and peroxidase activities was extracted from BCG by sonication, DNase I digestion and solubilization with the nonionic detergent, 1-0-n-octyl-beta-D-glucopyranoside. Further isolation was achieved by ammonium sulphate precipitation followed by chromatography on hydroxyapatite and Sephacryl S-1000 columns. After non-denaturing polyacrylamide gel electrophoresis of the purified enzyme, a single protein band revealed peroxidase activity when the gel was stained with ABTS [2,2'-azino-di-(3-ethylbenzthiazoline-6-sulphonic acid)]. Gel chromatography on Sephacryl S-1000 revealed a relative molecular weight of about 141 kDa for the enzyme. The haem absorbance, catalase and peroxidase activities co-eluted from the Sephacryl S-1000 column. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis under reducing conditions indicated two major polypeptides with M(r) 98.7 kDa and 37.9 kDa, respectively. During reaction with isoniazid (INH) in the presence of a glucose/glucose oxidase H2O2-generating system, haem loss of the enzyme occurred. The ability of the enzyme to catalyse the one-electron oxidation of ABTS to form the ABTS.+ cation radical also decreased after reaction with INH. Enzyme kinetic experiments showed that at low INH concentrations, inhibition of catalase activity was non-competitive, while at higher INH concentrations, it was non-competitive-uncompetitive.