PURIFICATION AND SOME PROPERTIES OF CALPAIN-II FROM TILAPIA MUSCLE (TILAPIA-NILOTICA X TILAPIA-AUREA)

Calpain II (EC 3.4.22.17), calcium-activated neutral proteinase, from tilapia muscle (Tilapia nilotica x Tilapia aurea) was purified by using hydrophobic chromatography with phenyl-Sepharose CL-4B and substrate affinity chromatography with alpha-casein agarose. Electrophoretic analysis and inhibitor specificity studies indicated that the calpain II was a monomer and purified to homogeneity. The molecular weight was electrophoretically determined to be 80 000. The requirement of Ca2+ concentration was 0.1 mM for half-maximal and 1 mM for maximum activation. This enzyme was fully activated by 10 mM 2-mercaptoethanol. The inhibitory ability of calpain inhibitor I was the highest, followed by leupeptin, calpain inhibitor II, antipain, iodoacetate, PCMB, and PMSF in decreasing order. Accordingly, the active site of this proteinase might contain cysteine and histidine for catalysis. The caseinolytic activity of this purified calpain was 480 units/mg at pH 7.5. The recovery was 35%.