Affinity Labeling of Human Thrombins with Extended Benzamidine Sulfonylfluoride Exo-Site Reagents

The reagent m[o-(2-chloro-5-fluorosulfonylphenylureido)phenoxybutoxy]benzamidine [mCP(PBA)-F] is an exo-site affinity labeling reagent for human thrombins. To delineate the sites of the reacticn of mCP(PBA)-F on thrombin (Th), experiments were done with three structurally related variants of mCP(PBA)-F in which the distijnce from the cationic amidino portion to the reactive sulfonylfluoride was varied by 5-6 A. In clotting inhibition assays the reagent m[o-(2-chloro-5-fluorosulfonylphenylamido)phenoxypropoxy]benzamidine (t½ = 99.4 ± 7.8 sec, k_lst = 6970 ± 550 × 106 sec^-l n=5) was 2-fold more inhibitory than mCP(PBA)-F (t½ = 200 ± 10.1 sec, k_lst = 3460 ± 150 x 12^-6 sec^-1 n=5). Minimal energy models of the compounds show the optimum length is 14-17 Å. In other experiments hirudin (9600 μ/mg, gift F. Markwardt) mixed with 13.1 μM α (clotting) or 12.1 ±M γ/β (nonclotting) Th in 1- to 3-fold molar excess did not prevent incorporation of [³H]mCP( PBA)-F (0.961-0.794 mole label/mole Th) but did inhibit [³H]diisopropylfluorophosphate (iPr₂P-F) uptake (.096-.005 mole label/mole Th). mCP(PBA)-Th had reduced binding for proflavin (Kd ≈ 9-fold that of Th) but not as low as iPr₂P-Th (Kd : 38-fold that of Th) and further suggests that mCP(PBA)-F is attached to Th at points distal to the catalytic site. These new data show that mCP(PBA)-F reacts at nonclotting related but specific secondary sites on Th and that mCP(PBA)-F can be used to identify the structural features associated with the uniq.ue proteolytic specificity of thrombin. (Supported by NIH and AHA)