Development of Synthetic Fluorogenic Peptide Substrates for Blood Clotting Proteases

合成显色和荧光肽底物用于体液中各种蛋白酶的特异性测定已经建立，因为它们的高灵敏度。我们团队开发的α -凝血酶、Xa因子、血浆钾激肽肽、Ca蛋白、尿激酶、XIIa因子、XIa因子、纤溶蛋白和鲎凝血酶的特异性底物现已上市。本项目的目的是为VIIa、IXa因子和腺体钾化酶寻找特异性和敏感性底物，并实现这些合成底物用于体液中各种蛋白酶的测定。研究结果如下：1。boc -met - ala - arg4 -甲基香豆醇-7-酰胺是小鼠腺（颌下）激肽肽最敏感的底物。底物由小鼠低分子量激肽原中激肽片段NH_2-和cooh末端邻近的氨基酸序列组成。小鼠的缓动因子（kcat = 1.4s^<-1>）切割底物的速度比猪的快约80倍。我们为VIIa因子合成了一个Boc-Leu-Thr-arg-p-nitrobenzylester的显色底物。结果表明，该底物对VIIa因子最敏感。这一发现为研究体外凝血系统中VII因子与组织因子之间的分子相互作用提供了可能。制备了一种针对牛血清VII因子的小鼠单克隆抗体（编号为VII- m31），并对其进行了鉴定，作为牛体液中VII因子特异性测定的第一步。抗体VII- m31仅在Ca^<2+>存在的情况下对因子VII具有较强的亲和力，对凝血酶原、因子X、因子IX、蛋白C、蛋白S和蛋白z无反应性。通过免疫印迹法测定，因子VII经尿素和SDS变性后，其全部被2-巯基乙醇还原，未破坏抗原位点。此外，该抗体特异性结合因子VII的nh_2末端23-50残基对应的含gla肽。这些结果表明，与上述合成肽底物结合，VII- m31可用于因子VII的特异性酶联免疫测定。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