Exo-site Affinity Labeling of Human Thrombins

Human cu-thrombin, the two-polypeptide chain form with high specific clotting and esterolytic activities, was selectively and irreversibly inactivated with m-lo-Gchloro-5fluorosulfonylphenylureido)phenoxybutoxylbenzamidine (mCP(PBA)-F). The time course of inactivation followed pseudo-first order kinetics (k, = 3.7 x lo-” s-l, 23”), while initial velocities followed a rectangular hyperbolic function predicted for affinity labeling kinetics (KL = 72.7 pM, k, = 3.01 x lo-” s-l). The hydrolyzed compound with a sulfonic acid replacing the reactive sulfonylfluoride group was a strong competitive inhibitor of thrombin (K, = 3.65 pM versus 7.2 mM for benzamidine), implicating active site binding. The analogue o-(2-chloro&fluorosulfonylphenylureido)methoxybenzene, which lacks the active site-directed benzamidine cation but possesses the corresponding reactive fluoride group, only slowly inactivated the enzyme (14 uersus >90% with mCP(PBA)-F in 30 min). Both L3HlmCP(PBA)-F and LWldiisopropylphosphorofluoridate (iPr,P-F) were incorporated stoichiometrically when added separately to ry-thrombin (1.07 [“HlmCP(PBA) label/enzyme versus 1.15 [‘“C]iPr,P label/enzyme), while either compound prevented the incorporation of the other when added sequentially (0.00 L3HlmCP(PBA) label/ [14CliPr,P enzyme versus 0.03 [ “‘C]iPrsP label/ V’HlmCP(PBA) enzyme). In contrast to [“CliPr,P-F, which was found to label only the B chain (M, = 32,000) of (Ythrombin, the IRHlmCP(PBA)-F label was distributed 20.2 + 0.07 (N = 14) on the A chain (M, = 4,600) and 79.8 + 0.07 (N = 14) on the B chain when labeled cu-thrombin was reduced and electrophoresed in sodium dodecyl sulfate, 10% acrylamide gels containing 6 M urea. A mixture of 78% y-, 20% p-, and 2% cu-thrombin (four-, three-, and two-chain forms) which retained 84% of the esterolytic but only 0.5% of the clotting activity of the parental cu-thrombin was made by limited tryptic digestion.