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

Human alpha-thrombin with high fibrinogen-clotting activity binds proflavin at a single specific site (n = 0.996 site/alpha-thrombin, Kd = 22.0 microM) with the same affinity as the bovine enzyme (Kd = 22 +/- 3 vs. 24 +/- 3 microM, respectively, at pH 7.4, approximately 23 degrees 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 microM), and its hydrolysis of Bz-Arg-OEt was inhibited by the dye in a simple competitive manner (Ki = 30 +/- 3 microM). 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 approximately 2-fold reduction in proflavin binding. Chemical modification of approximately four tryptophans or approximately four tyrosines per enzyme also caused analogous differential losses of clotting vs. synthetic substrate activities and reduced proflavin binding approximately 5- and approximately 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 approximately 4-, 26-, and 55-fold, respectively, following the increasing size and steric hindrance properties of the conjugated group. Conjugation of the catalytic histidine (His-57) with Tos-Lys-CH2-Cl decreased binding only approximately 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 covalently attaches to the enzyme within the fibrino-peptide groove distal to the catalytic site.(ABSTRACT TRUNCATED AT 250 WORDS)