INTERACTIONS OF A FLUORESCENT ACTIVE-SITE-DIRECTED INHIBITOR OF THROMBIN - DANSYLARGININE N-(3-ETHYL-1,5-PENTANEDIYL)AMIDE

Dansylarginine N-(3-ethyl-1, 5-pentanediyl)amide is a specific and potent inhibitor of thrombin. The apparent avidity and specificity of the inhibitor coupled with the fluorescence properties of the dansyl moiety suggested to us that the compound could be extremely useful as a probe for the study of thrombin and its interactions. The synthesis of the inhibitor was accomplished by coupling 4-ethylpiperidine to dansylarginine previously activated with N, N'-carbonyldiimidazole. Although the compound is not susceptible to hydrolysis catalyzed by thrombin, it competitively inhibits the thrombin-catalyzed hydrolysis of either synthetic substrates or fibrinogen with an apparent Ki of about 10-7 M. When the inhibitor is bound to thrombin, marked changes in the fluorescence properties of the dansyl moiety are observed, including a threefold increase in intensity and lifetime and a decrease in the depolarization of the excitation signal. Using measurements of fluorescence intensity and polarization to study binding, a dissociation constant of 4.3X10-8 M and a stoichiometry of 1 mol of inhibitor per mol of thrombin were found. The enhancement of fluorescence intensity concomitant with binding provides a continuous monitor of the conversion of prothrombin to thrombin as catalyzed by the “prothrombinase” complex. This permits the precise analysis of the kinetics of thrombin formation. In addition, thrombincatalyzed feedback reactions which can occur during prothrombin activation are inhibited, and therefore the interpretation of the kinetics of the activation process are greatly simplified. In general the present results indicate that dansylarginine N-(3-ethyl-1, 5-pentanediyl)amide is an ideal probe of the catalytic function and numerous interactions of thrombin. © 1979, American Chemical Society. All rights reserved.