螢光基質を用いたヒト血漿中プレカリクレインの測定法とその応用

抄録

In order to elucidate the involvement of kallikrein-kinin system in the body, it is necessary to measure its components accurately. Plasma kallikrein once activated is readily inhibited by proteinase inhibitors in plasma, thus making it difficult to estimate its activity in biological fluids. Therefore, a residual activity of a proenzyme, prekallikrein, should be determined. In this report, we describe an assay method for prekallikrein in human plasma using peptidylfluorogenic substrate, Z-phe-arg-MCA. The method consists of two steps: 1) The first step involves activation of prekallikrein with acetone and kaolin. The procedure used was similar to Imanari's method. Normal human plasma, 50μl, was mixed with 850μl tris buffer-acetone mixture. Then, 100μl kaolin suspension (10mg/ml) was added and mixed vigorously. After incubation for 30 or 60min at room temperature, a 20μl aliquot of the mixture was taken and assayed for its amidolytic activity as follows: 2) Each aliquot was taken into Tube A or Tube B. Tube A contains soy bean trypsin inhibitor and 1ml of Z-phe-arg-MCA solution, and Tube B contains lima bean trypsin inhibitor and the substrate solution. Each tube was incubated at 37°C for 10min and the reaction was terminated by addition of 17% AcOH solution. The fluorescence generated was then read at 380 (excitation) and 460 (emission) nm. The difference in values between Tube A and B was calculated to give the prekallikrein activity.
The enzyme activity generated was stable upto 60min. Ten % of the normal level of Hageman factor and high molecular weight kininogen were required for the full activation of prekallikrein by this method. The results obtained suggest that this assay system can be applied to the measurement of prekallikrein in deficient plasmas and also to samples from pathological conditions provided that these samples are examined in the presence and absence of normal plasma.