Prospects For Chromogenic And Fluorogenic Substrate Use In Biochemistry And Blood Protease Assay

Kinetic parameters describing the hydrolysis of peptide p-nitroanilide substrates by thrombin, Factor Xa, and activated Protein C indicate that a high degree of selectivity for each of these proteases can be achieved by using appropriate substrates. Determination of kcat (the maximum velocity per mole of enzyme) for peptide p-nitroanilide substrates indicates that sensitivity sufficient to detect pM concentrations of these proteases can be obtained. The use of fluorogenic substrates should increase sensitivity, although the absence of data for maximum velocities of hydrolysis of peptide fluorogenic substrates precludes quantitative statements about the extent of increase in sensitivity that may actually be obtained. The use of more than one substrate provides an opportunity to selectively assay individual enzymes present in a mixture of proteases. The selectivity of the various assays can be enhanced by use of competitive inhibitors such as those developed in several laboratories for use as potential antithrombotic agents. Although manual methods may be too tedious to be practical in complex situations, the automated methods which handle multiple samples can make such multiple-substrate based assay methods practical. The fact that a single function of the protease coagulation factors, namely the peptide bond hydrolysis function is being assessed by peptide chromogenic and fluorogenic substrates will permit more specific information to be obtained about these multifunctional molecules.