Purification of cat submaxillary kallikrein

The cat submaxillary gland contains 1,000–2,400 kallikrein units (KU)/g of tissue. The submaxillary kallikrein was purified to homogeneity by acetone fractionation, DEAE-Sephadex A-50 chromatography, Sephadex G-75 gel filtration, and Ampholine isoelectric focusing. The kallikrein was separated by isoelectric focusing into 6–7 forms with pl's between 4·2 and 5·1. One mg of the purified kallikrein contained 930–1,260 KU in the dog vasodilator assay, and hydrolyzed 15–25 and 9–12 μmol of N-α-benzoyl-L-arginine ethyl ester (BAEE) and N-α-toluenesulfonyl-L-arginine methyl ester (TAME), respectively, in 1 min at 25°C and pH 8·0. The Km'S of the purified kallikrein with BAEE and TAME were 0·67 and 0·34 mM respectively. Hydrolysis of N-α-benzoyl-L-tyrosine ethyl ester (BTEE), N-α-benzoyl arginine-p-nitroanilide (BApNA), and casein was small or negligible. The apparent molecular weight of the kallikrein was estimated to be 5 × l04 by Sephadex G-100 gel filtration and 4·7 × 104 by polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS). The kallikrein was found to contain 18·5% carbohydrate by weight. Trasylol and soybean trypsin inhibitor were not specific inhibitors of this kallikrein.