Practical -Plasma Kallikrein Assay Method Using Fluorogenic Substrate

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A practical method for the assay of piasma kallikrein using a fluorogenic substrate (Carbobenzoxy-phenylalanyl-arginine-7-methylcoumarin amide) was described.Prekallikrein was activated with kaolin or dextran sulfate under various conditions at 0°.Treatment of kaolin suspension (6.25mg/ml tris-Hcl buffer, volume ratio to plasma 20: 1) showed highest activation during 20minutes to 40minutes. Optimal conditlons for assay of plasma prekailikrein and plasma spontaneous kallikrein were as follows: substrate concentration 0.1mM and 0.1mM, incubation time 10minutes and 30minutes, plasma volume 100μl and 50μl, respectivility. Heparin used as anticoaguiant affected to plasma kallikrein activity, however sodium citrate and EDTA-2Na did not affected. intra-assay coefficient of variance was 11.2% in plasma spontaneous kalikrein and 3.6% in plasma prekallikrein. A good correation was obtained in values of plasma prekallikrein between chromogenic substrate (H-D-prolyl-phenyialanyl-arginine-P-nitroanilide) method and fluorogenic substrate method. A survey in 26 heaithy sublects showed mean activities of 0.39±0.22nmoie AMC/min/ml at spontaneous kallikrein and 193.4±55.6nmole AMC/min/ml at prekailikrein in plasma, and both values in essential hypertensive patients were not significant by different from those in healthy subject.