Abstract
An amidolytic assay using a new substrate, H-D-Pro-Phe-Arg-ρ-acetylanilide (KL-12), was designed to measure prekallikrein in plasma, Prekallikrein was converted to kallikrein with Pseudomonas aeruginosa elastase, and the amidolytic response of kallikrein to KL-12 was subsequently measured.
The kinetic parameters of plasma kallikrein as a function of KL-12 or of the ordinary substrate, H-D-Pro-Phe-Arg-ρ-nitroanilide (S-2302), were Km=2.8×10-4M and kcat=208s-1, or Km=2.5×10-4M and kcat=176s-1, respectively, A comparative study of the automated methods employing plasma samples showed good correlation between the assays using KL-12 and S-2302 (r=0.997, n=30, y=0.95x+4.5). The coefficients of variation for multiple measurements with pseudomonal elastase and KL-12 of a plasma sample with low level prekallikrein (106nM, 30% of the normal pooled plasma) within a run (n=10) and between runs (n=10) were CV=2.51% and CV=4.24%, respectively. Prekallikrein concentrations in plasma samples from 80 healthy individuals (M=40, F=40) were measured with the automated method and the normal range obtained by the parametric method was 270-426nM (77-121% of the normal pooled plasma). We have reported that the determination of plasma prekallikrein can be easily automated by the activation of prekallikrein with Pseudomonas aeruginosa elastase and by using a new chromogenic substrate of kallikrein.
