Cherkasov A V, Nedospasov V A, Iakhimovich A D, Nedospasov A A
Biokhimiia. 1994 Oct;59(10):1574-88.
A new accurate method (ANSA-analysis) is used for studying the interactions of proteases with their inhibitors or other proteases. The method is based on the cleavage by proteases of mixtures of competing chromogenic substrates containing aminonaphthalenesulfonamide (ANSA) detectable groups. Each substrate contained a specifically substituted ANSA group which showed its specific retention time during chromatographic separation. For the analysis of blood coagulation, mixtures of blood-clotting factor substrates were used. Hydrolysis of the substrate mixture catalyzed by blood samples gave characteristic chromatograms (ANSA spectra) for each sample. The activation time before injection of the blood sample into the substrate mixture and the pool of clotting factors and inhibitors both had influence upon the ANSA spectrum. The ANSA spectra of mixtures of trypsin and/or chymotrypsin with snake venoms are described as A x (the ANSA spectrum of a protease) + B x (the ANSA spectrum of a venom) + C x (the ANSA spectrum of catalytically active interaction products). They are additive (A = B = 1, C = 0), if no proteolysis, inhibition or activation takes place. ANSA spectra analysis shows deviations from additivity for some mixtures of Viperidae, (including E. carinatus), Naja naja, Agkistrodon contortrix and A. halys venoms. Explanations for the inability to detect inhibitors in venoms having a high protease activity by previously used methods are given.
一种新的精确方法（ANSA分析）被用于研究蛋白酶与其抑制剂或其他蛋白酶之间的相互作用。该方法基于蛋白酶对含有可检测氨基萘磺酰胺（ANSA）基团的竞争性显色底物混合物的切割。每个底物都含有一个特定取代的ANSA基团，该基团在色谱分离过程中显示出其特定的保留时间。为了分析血液凝固，使用了凝血因子底物的混合物。血样催化的底物混合物水解为每个样品给出了特征色谱图（ANSA光谱）。将血样注入底物混合物之前的活化时间以及凝血因子和抑制剂的库都对ANSA光谱有影响。胰蛋白酶和/或胰凝乳蛋白酶与蛇毒混合物的ANSA光谱被描述为A×（蛋白酶的ANSA光谱）+B×（毒液的ANSA光谱）+C×（催化活性相互作用产物的ANSA光谱）。如果不发生蛋白水解、抑制或活化，它们是可加性的（A = B = 1，C = 0）。ANSA光谱分析表明，蝰蛇科（包括锯鳞蝰）、眼镜蛇、美洲水蛇和蝮蛇毒液的某些混合物存在与加性的偏差。给出了用先前使用的方法无法检测具有高蛋白酶活性的毒液中抑制剂的解释。
