Zymogen/Enzyme Discrimination Using Peptide Chloromethyl

Glutamylglycinylarginyl chloromethyl ketone, tyrosylglycinylarginyl chloromethyl ketone, and phenylalanylprolylarginyl chloromethyl ketone have been labeled at their amino termini using fluorescein, rhodamine-X, lissamine-rhodamine, pyrene, and the 1,5-, 2,5-, and 2,6-dimethylaminonaphthalene-l-sulfonyl moieties. These peptidyl chloromethyl ketones have also been modified by incorporation of biotin and tamino caproyl biotin. The ability of these various chloromethyl ketones to be incorporated into a collection of zymogen-enzyme pairs has been evaluated using a variety of coagulation and fibrinolytic proteins. All labeled chloromethyl ketones were efficiently incorporated into the proteases tested, with the exception of urokinase which was refractory to inhibition by phenylalanylprolylarginyl chloromethyl ketone derivatives. N o modification of any zymogen species was observed even under conditions designed to detect minimal reactivity. When enzymes were modified using chloromethyl ketones labeled with t-amino caproyl bi tin, the modified proteins readily reacted with avidin under a variety of different conditions. The observed reactivity with avidin was used in enzyme “blotting” following electrophoretic resolution of polypeptide chains and to remove active enzyme present in enzymezymogen mixtures. These reagents have been used to evaluate the potential for active site expression by the single-chain human factor VI1 molecule. Studies conducted with tissue factor, phospholipids, and calcium using factor X as substrate demonstrate that no activity can be obtained without initial activation of either factor X to factor Xa or factor VI1 to factor VIIa by an external source. We thus conclude that factor VI1 is a true zymogen, inert in the blood clotting process prior to its cleavage to factor VIIa.