Studies on Urinary Kallikreins: I. Purification and Characterization of Human Urinary Kallikreins

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Abstract
Human urinary kallikrein [EC 3.4.21.8] (HUK) was purified about 200-fold with an overall yield of 40 percent from crude powder by DEAE-cellulose chromatography, acetone fractionation, Sephadex G-100 gel filtration and DEAE-Sephadex A–50 chromatography. Its activity was 200 kallikrein units (KU) per A280. HUK from active fractions obtained by DEAE-Sephadex A–50 chromatography was separated into three active components showing isoelectric points of 3.9 (HUK-1), 4.0 (HUK-2), and 4.2 (HUK-3) by isoelectric focusing: each HUK component was homogeneous on disc electrophoresis. The approximate molecular weights of HUK-1, -2, and -3 were estimated to be 2.7×104, 2.7×104, and 2.9×104, respectively, by gel filtration on a Sephadex G–100 column. The optimum pH's of HUK–1, -2, and -3 in esterolytic action were found to be 8.0, 8.3, and 7.5, respectively, and they were fairly heat stable in comparison with other glandular kallikreins. The three components of HUK were weakly inhibited by Trasylol, but were not affected by soybean and ovomucoid trypsin inhibitors. They were strongly resistant to treatment with urea and weakly resistant to treatment with guanidine. The activation energies of HUK-1, -2, and -3 were found to be 1.17×104, 5.1×103, and 1.45×104 cal per mole, respectively. The Km values were estimated toward N-α-tosyl-L-arginine methyl ester (TAME), N-α-benzoyl-L-arginine ethyl ester (BAEE), and N-α-benzoyl-L-arginine methyl ester (BAME).