The inhibition of trypsin, plasmin, and thrombin by benzyl 4-guanidinobenzoate and 4′-nitrobenzyl 4-guanidinobenzoate

AbstractThe inhibition of the enzymes trypsin, plasmin, and thrombin by benzyl 4-guanidinobenzoate and 4′-nitrobenzyl 4-guanidinobenzoate is caused by acylation of the active site. Second order rate constants were determined

Development of a countergradient parking system for gradient liquid chromatography with online biochemical detection of serine protease inhibitors

A gradient HPLC approach in combination with a countergradient system for online biochemical detection (BCD) to screen for inhibitors of serine proteases is described. For gradient separations, this novel countergradient system was developed to produce a biocompatible constant solvent composition in the BCD. The countergradient system is based on retaining complete gradients in an additional preparative HPLC column, followed by subsequent and reversible elution to the separation column effluent. Major advantages compared with existing countergradient systems are that no additional LC pumps are needed and enhanced stability. The developed countergradient system was systematically characterized applying different gradient programs. Inhibitors eluting in a post-column continuous flow analysis interfere with the enzymatic release of fluorescent 7-amino-4-methylcoumarin (AMC) from an AMC-labeled peptide. The inhibitory activity of eluting substances is sensitively detected as the degree of reduced fluorescence intensity. This biochemical detection system (BCD) for proteases was validated with three known inhibitors of the benzamidine type. Their I