New chromogenic substrates for thrombin with increased specificity

Chromogenic substrates for thrombin with high specificity are necessary for several functional assays, especially for the performance of photometric PT and APTT. A new approach to improve the specificity of chromogenic peptide substrates is made coupling tripeptide sequences selective for thrombin to derivatives of 5-amino-2-nitro benzoic acid (ANBA). Especially when the chromophore's side chain is substituted by amines or amino acids hydrolysis rates by other enzymes like kallikrein, plasmin or factor Xa are decreased significantly compared to corresponding para-nitroanilides of the same amino acid sequence. On the other hand, most of these compounds are still sensitive thrombin substrates. KM-values for thrombin and other enzymes are in the same order of magnitude as corresponding pNA-peptides. ANBA peptide substrates may be useful to measure thrombin selectively in a mixture of other proteases like plasmin, factor Xa or kallikrein and for the colorimetric determination of PT and APTT.