ISOLATION, PURIFICATION AND PROPERTIES OF CATHEPSIN-B FROM BUFFALO LIVER

Cathepsin B was isolated from buffalo liver by salt fractionation, ion -exchange resin treatment, gel filtration and repeated ion-exchange ch romatography using a linear salt gradient. The enzyme showed activity, against denatured hemoglobin (or ovalbumin), alpha-N-benzoyl-DL-argini ne p-nitroanilide (BAPNA), and alpha-benzoyl-DL-arginine-naphthylamine (BANA). It inactivated buffalo muscle aldolase with a half life perio d of 21 min. The pH-activity profiles obtained for the digestion of he moglobin (or ovalbumin) and aldolase inactivation by the enzyme were f ound to be different. The enzyme (mol wt 27,800 by SDS-PAGE) eluted in gel filtration with a molecular weight of 27,000 and a Stokes radius of 2.31 nm. The results showed buffalo cathepsin B to be a single-chai n molecule. The N- and C-terminal amino acids of the enzyme were found to be leucine and aspartic acid, respectively. It contained 0.7% conc anavalin A reactive neutral carbohydrate. The amino acid composition o f buffalo cathepsin B was found to be similar to that of human liver c athepsin B. The optical properties of the buffalo enzyme were found co nsistent with its aromatic amino acid content. The isoionic pH of the enzyme was found to be 5.70 and the intrinsic viscosity was 3.48 ml/g whence the frictional ratio, f/f(0) was computed to be 1.10 suggesting that the native enzyme conformation is compact and is globular in sol ution.