A New Fluorogenic Substrate Method for the Estimation of Kallikrein in Urine1

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Abstract
Kallikrein in human urine was measured using a fluorogenic peptide substrate, prolyl-phenyl-alanyl-arginine-4-methylcoumaryl-7-amide (Pro-Phe-Arg-MCA). Using 20 μ1 of normal urine, kallikrein activity was quantitatively assayed by incubation with a final concentration of 10−4m Pro-Phe-Arg-MCA at 37°C for 90 min. The reaction was terminated by adding 50 units of TrasylolR or 20 μ1 of 10% acetic acid. Using this method, kallikrein activity was measured in urine from normal subjects and patients with glomerulonephritis and Burtter's syndrome. These values were comparable with the values obtained by radioimmunoassay using bovine low-molecular-weight kininogen. When normal urine was applied to a column of arginine-Sepharose 4B, two activities, Pro-Phe-Arg-MCA hydrolyzing activity and kinin-releasing activity toward bovine low-molecular-weight kininogen, were eluted in the same fraction. These results indicate that the present method is useful for the estimation of kallikrein in urine, in terms of specificity, sensitivity, and simplicity.

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