Preparation and properties of active dansyl-α- and -γ-thrombins

Abstract

Human α- and γ-thrombins were labeled with a fluorescent dansyl group, and tested for their utility in polarization of fluorescence experiments. The enzyme carbohydrate groups were oxidized with sodium periodate, labeled with dansyl-hydrazine, and reduced with sodium borohydride. Dansyl-α-thrombin showed 75% of the clotting activity of the unlabeled enzyme. The γ form (a proteolyzed derivative of α-thrombin with essentially no clotting activity) retained 90% of its esterase activity, and 60% of its amidase activity. Steady-state and polarization of fluorescence measurements of the two dansyl-thrombins were essentially the same indicating a similarity in gross structural properties despite the covalent modifications which convert the α to the γ form. Both dansyl-thrombins reacted with antithrombin and the expected decrease in tumbling time was observed in the fluorescence polarization measurements. High-affinity heparin had no effect on the fluorescence parameters for the dansyl-thrombin-antithrombin complex. Dansyl-α-thrombin also reacted with α 2-macroglobulin; the polarization measurements indicated substantial immobilization suggesting the enzyme is not bound to subregions of high-rotational freedom.