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Objective: To determine the Km, Vmax, and Ki of BOC-Leu-Ser-Thr-Arg-pNA (B3644) in the determination of kallikrein, and then to establish the procedure for plasma prekallikrein/kallikrein determination. Method Chromogenic substrate assays on a semi-auto spectrophotometer were employed.
Results: The apparent activities of seven substrates: B3644, S2238, S2251, ChromozymPCa, T6140, ChromozymPK, and T1637, for porcine kallikrein were 0.788, 0.691, 0.659, 0.636, 0.438, 0.396, and 0.145, respectively, that of B3644 being the highest. Amidolysis caused by kaolin activated human normal plasma was independent of coagulation factors II, X, XI, and protein C, but dependent on prekallikrein and to some extent on factor XII. The kallikrein’s, thrombin’s, and plasmin’s Km and Vmax for the B3644 were determined and calculated to be 235μmol/L and 337nmol.s^-1.U^-1, 469μmol/L and 63nmol.s^-1.U^-1, and 70μmol/L and 358nmol.s^-1.U^-1, respectively. At the same time, the kallikrein, thrombin, and plasmin Ki values for antithrombin III(AT-III) or limabean trypsin inhibitor(LBTI) were detected to be 840 or 2.50. 32 or 0.32, and, 108 or 1.55U/L, [d1]respectively. With B3644, the recovery percentages of kallikrein and kaolin activated plasma were 100%. Intra-assay CV values ranged from 2.3% to 4.6%. By using B3644 and introducing AT-III or LBLI into the determination system, an optimized procedure for prekallikrein/kallikrein determination was obtained. With this procedure, the mean percentage values of plasma prekallikrein in normal subjects, pregnant women (gestation≥24 weeks), and in patients with hypertension, hepatic failure, or with advanced cancers were determined to be 113.5, 151.6(P<0.01), 173.2(P<0.01), 43.5(P<0.005), and 88.7(P<0.05), respectively.
Conclusion: B3644 was identified as a suitable substrate for the determination of plasma prekallkrein/kallikrein activity. A sensitive and reliable chromogenic substrate assay for human plasma prekallikrein was developed by using this novel substrate.
DOI: Clin. Lab. 2009;55:106-113
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