Structural studies of human urinary kallikrein (urokallikrein).

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Abstract
Human urinary kallikrein (urokallikrein) was purified by affinity chromatography with aprotinin coupled to CH-Sepharose and by gel filtration. The isolation procedure, which was performed under mild conditions, was completed in a 36-h period and yielded an overall recovery of more than 75% and a purification of 1727-fold. Homogeneity of the urokallikrein was demonstrated by 3 criteria: the coincidence of the stained protein band and functional urokallikrein in duplicate gels after alkaline disc gel electrophoresis; the appearance of a single stained band of MW 48,000 on sodium dodecyl sulfate/polyacrylamide gel electrophoresis of reduced and unreduced enzyme; and the finding of a single amino-terminal residue, namely alanine, after dansylation and acid hydrolysis of purified enzyme. The Km of urokallikrein on N.alpha.-p-tosyl-L-arginine methyl ester was 400 .mu.M, and the Vmax was 194 .mu.mol/min per mg of protein, which is higher than that observed with any previous preparations. The MW of 48,700 determined on gel filtration and the MW of 48,000 observed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis are in good agreement with the MW of 48,213 calculated from the amino acid composition. The finding of a MW higher than those previously reported, namely 27,000-43,500, the increased functional activity on tosylarginine methyl ester, and the detection of a single amino-terminal residue are consistent with the isolation of a more native protein by the procedure described.