Development of Synthetic Fluorogenic Peptide Substrates for Blood Clotting Proteases

由于人工合成的发色底物和荧光肽底物具有很高的灵敏度，已经建立了用于体液中各种酶的特异性分析的实用方法。本课题组开发的α-凝血酶、凝血因子Xa、血浆激肽释放酶、蛋白钙、尿激活酶、凝血因子XIIa、凝血因子Xia、纤溶酶和凝血酶的特异性底物现已上市。本项目的目的是分类因子VIIa和IXa以及腺激肽释放酶的特定和敏感的底物，并实现这些用于测定体液中各种蛋白酶的合成底物。结果如下：1.BOC-Met-Ala-Arg4-甲基香豆基-7-酰胺是小鼠腺激肽释放酶最敏感的底物。底物含有与小鼠低分子激肽原激肽部分的NH_2-和COOH-末端相邻的氨基酸序列。小鼠激肽释放酶可切割该底物(Km=2…超过30um，kcat=1.4s；-1；-1；比猪腺激肽释放酶快80倍。我们新合成了因子VIIa的显色底物Boc-Leu-Thr-Arg-对-硝基苯甲酸酯。在已合成的74种多肽底物中，该底物对VIIa因子最敏感。这一发现使得研究凝血因子VII和参与外源性凝血系统的组织因子之间的分子相互作用成为可能。制备了一种针对牛凝血因子VII的鼠单抗(命名为VII-M31)，并对其进行了鉴定，为体液中凝血因子VII的特异性检测奠定了基础。该抗体对凝血酶原、凝血因子X、凝血因子IX、蛋白C、S蛋白和蛋白Z均无反应。用尿素和十二烷基硫代十二烷基硫酸钠变性，用2-巯基乙醇还原后，免疫印迹法均不破坏抗原性。此外，该抗体可与凝血因子VII的NH_2端23-50个残基对应的含GLA的多肽特异结合。这些结果表明，VII-M31可与上述合成肽底物结合用于凝血因子VII的特异性酶联免疫分析。较少

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