Carbomer inhibits tryptic proteolysis of luteinizing hormone-releasing hormone and N-alpha-benzoyl-L-arginine ethyl ester by binding the enzyme

Purpose. To determine the mechanism by which Carbomer inhibits the enzymati c activity of trypsin in hydrolysis of N-alpha-benzoyl-L-arginine ethyl est er (BAEE) and luteinizing hormone-releasing hormone (LHRH). Methods. Inhibition of enzymatic activity was studied by measuring the form ation of metabolites from LHRH and BAEE. Binding of trypsin and substrates to 0.35% (w/v) Carbomer at pH 7.0 was studied by centrifugal filtration. Ge l filtration and reverse phase HPLC was used to determine the stability of trypsin. Results. Carbomer reduced the rate of hydrolysis of BABE and LHRH by trypsi n to 34% and 28% of the control activity, respectively. The rate of metabol ite formation for both substrates followed pseudo-zero order kinetics in th e presence and absence of carbomer. Binding studies showed that 68% of the trypsin protein and 10% of BABE was bound to carbomer, but no LHRH was boun d. No low molecular weight autolysis products of trypsin could be identifie d by gel filtration. Reverse phase HPLC analysis of the unbound carbo mer-t reated-trypsin suggests a number of conformational forms of trypsin. The eq uilibrium binding capacity was 30 mu g of trypsin to 1000 mu g of carbomer. Conclusions. Decreased hydrolysis of LHRH and BABE by trypsin in the presen ce of carbomer is due to enzyme-polymer interaction.