Proflavine binding within the fibrinopeptide groove adjacent to the catalytic site of human .alpha.-thrombin

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Abstract
Human .alpha.-thrombin with high fibrinogen-clotting activity binds proflavin at a single specific site (n = 0.996 site/.alpha.-thrombin, Kd = 22.0 .mu.M) with the same affinity as the bovine enzyme (Kd = 22 .+-. 3 vs. 24 .+-. 3 .mu.M, respectively, at pH 7.4, .apprx. 23.degree. C). This human enzyme form further displayed no significant difference in its ability to bind the dye over a broad NaCl concentration range (0.15-3 .mu.M), and its hydrolysis of Bz-Arg-OEt was inhibited by the dye in a simple competitive manner (Ki = 30 .+-. 3 .mu.M). Conversion of the human .alpha.- to .gamma.-thrombin by controlled tryptic digestion essentially destroyed clotting activity without appreciably altering synthetic substrate activities and caused only .apprx. 2-fold reduction in proflavin binding. Chemical modification of .apprx. 4 tryptophans or .apprx. 4 tyrosines per enzyme also caused analogous differential losses of clotting vs. synthetic substrate activities and reduced proflavin binding .apprx. 5- and .apprx. 10-fold, respectively. Inactivation of the enzyme by conjugation at the catalytic serine (Ser-195, chymotrypsin numbering) with MeSO2-F, PhMeSO2-F or i-Pr2P-F decreased binding .apprx. 4-, 26- and 55-fold, respectively, following the increasing size the steric hindrance properties of the conjugated group. Conjugation of the catalytic histidine (His-57) with Tos-Lys-CH2-Cl decreased binding only .apprx. 10-fold, suggesting partial displacement by the dye. Such partial displacement appeared to occur to a slightly greater extent with the conjugate of a large exo site affinity-labeling reagent, which convalently attaches to the enzyme within the fibrinopeptide groove distal to the catalytic site. D-Phe-Pro-Arg-CH2-Cl, which specifically binds within the fibrinopeptide groove and covalently reacts at or adjacent to the catalytic site, reduced proflavin binding .apprx. 40-fold. Evidently the proflavin binding site resides in an apolar active-site region, which is next to (or slightly overlaps) the catalytic site (His-57 and Ser-195) and extends into the fibrinopeptide groove. With the least sterically hindered inactivated form, MeSO2-.alpha.-thrombin, hirudin displaced proflavin, while antithrombin III in the presence of heparin could not, indicating major differences in the active-site regions required for either of these protein inhibitors of .alpha.-thrombin.

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