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J. Biochem, 1980, Vol. 87, No. 4 1127-1132
© 1980 Japanese Biochemical Society

research-article

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

Hisao KATO*,2, Noriaki ADACHI**, Sadaaki IWANAGA*,2, Keishi ABE***, Katsumi TAKADA****, Terutoshi KIMURA**** and Shumpei SAKAKIBARA****

*Institute for Protein Research, Osaka University Suita, Osaka 565
**Department of Medicine (Neurology), Shinshu University, School of Medicine Matsumoto, Nagano 390
***Department of Internal Medicine, Tohoku University School of Medicine Sendai, Miyagi 980
****Peptide Institute, Protein Research Foundation Minoh, Osaka 562

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 10–4 M 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.

1This study was supported in part by a grant (487025) from the Scientific Research Fund of the Ministry of Education, Science and Culture of Japan.

2Present address: Department of Biology, Faculty of Science, Kyushu University, Fukuoka, Fukuoka 812.


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