ABSTRACT
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.
