63] Inactivation of trypsin-like enzymes with peptides of arginine chloromethyl ketone

Abstract

The value of affinity labels in the identification and characterization of proteases in their physiological environments has been clearly demonstrated by the use of tosyl phenylalanyl chloromethyl ketone, tosyllysine chloromethylketone, and alanyl peptide chloromethyl ketones. This chapter presents the use of arginine chloromethyl ketone in the development of selective-affinity labels for individual trypsin-like proteases that function in blood coagulation, fibrinolysis, and hormone processing. This group of enzymes functions by the hydrolysis of either one or two bonds of their physiological substrates, usually at arginyl residues. The chapter uses the approach of incorporating part of the sequence of the physiological substrate in the reagent, thus taking advantage of binding selectivity in both primary and secondary sites. The reactivity of the chloromethyl ketones with proteases is determined by incubating the protease with the affinity label, removing timed aliquots, and measuring residual enzymatic activity. The initial concentration ofprotease was selected so that the concentration of affinity label was at least 10 times greater than that of the enzyme.