[ANSA analysis. IV. Specificity spectra in characterizing proteases and mixtures of them]

ANSA-analysis was used for characterization of proteases and their mixtures, such as snake venoms. The method is based on the cleavage by proteases of mixtures of competing chromogenic substrates containing substituted aminonaphtalenesulfonamide (ANSA) detectable groups. All detectable ANSA groups in the substrate mixtures have non-identical modifiers, one or two substituents in the sulfonamide fragment and can be determined by chromatographic methods. To identify venoms, a mixture of six peptide substrates cleaved at the Arg-ANSA bond was proposed. Hydrolysis of this substrate mixture catalyzed by the venoms of different Crotalidae and Viperidae species gave characteristic chromatograms (ANSA spectra) for each tested sample. A method for quantitative description of differences in ANSA spectra has been proposed. Each ANSA spectrum can be presented as a vector going from the origin of the coordinated axes to a point in an n-dimensional space (n is the number of assayed ANSA products of proteolysis) with peak squares of corresponding ANSA as coordinates. The similarity between two ANSA spectra will then be characterized by angle between their vectors.