| Research Abstract |
The utility of the synthetic chromogenic and fluorogenic peptide substrates for specific assay of various proteases in body fluid has been established, because of their high sensitivities. The specific substrates for alpha-thrombin, factor Xa, plasma kallikrein, protein Ca, urokinase, factor XIIa, factor XIa, plasmin and limulus clotting enzyme developed by our group are now commercially available. The purpose of this project is to kind specific and sensitive substrates for factors VIIa and IXa and glandular kallikreins and to realize these synthetic substrates for determinations of various proteases in body fluids. The results are as follows:
1. Boc-Met-Ala-Arg4-methylcoumaryl-7-amide was found to be the most sensitive substrate for mouse glandular (submaxillary) kallikrein. the substrate comprised the amino acid sequence adjacent to the NH_2- and COOH-terminal regions of the kinin moiety found in mouse low molecular weight kininogen. The mouse kallikrein cleaved this substrate (Km = 2
… More
30 muM, kcat = 1.4s^<-1>) approximately 80-fold faster than hog glandular kallikreins.
2. We newly synthesized a chromogenic substrate of Boc-Leu-Thr-arg-p-nitrobenzylester for factor VIIa. This substrate was found to be most sensitive one for factor VIIa among the previously eynthesized 74 peptide substrates. This finding made it possible to investigate the molecular interaction between factro VII and tissue factor participated in the extrinsic coagulation system.
3. A murine monoclonal antibody (designated VII-M31) directed against bovine factor VII was prepared and characterized as the first step for specific assay of factor VII in body fluid. The antibody VII-M31 possessed a strong affinity only for factor VII in the presence of Ca^<2+> without any reactivities to prothrombin, factor X, factor IX, protein C, protein S and protein Z. Denaturation of factor VII by urea and SDS all its reduction with 2-mercaptoethanol did not destroy the antigenic site, measured by immunoblotting method. Moreover, the antibody bound specifically to the Gla-containing peptide corresponding to the NH_2-terminal 23-50 residues of factor VII. These results indicated that VII-M31 is available for specific enzyme-linked immunoassay of factor VII, in combination with the synthetic peptide substrate described above. Less
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