PREPARATION AND CHARACTERIZATION OF ANHYDROTHROMBIN

Anhydrothrombin, a catalytically inactive derivative of thrombin in wh ich dehydroalanine replaces the active-site serine, was prepared by a novel method. The active-site serine of thrombin was modified to dehyd roalanine by promoting the beta-elimination of phenylmethylsulfonic ac id from phenylmethylsulfonyl fluoride-inactivated thrombin under condi tions in which the enzyme is unfolded. After the elimination reaction was quenched, the resulting anhydrothrombin was folded by diluting the denaturant, Gdn . HCl, to nondenaturing concentrations. Anhydrothromb in was purified by PAB affinity chromatography. Both native thrombin a nd anhydrothrombin were digested by cyanogen bromide, and the peptides from the region of the active-site serine (S205) were isolated by rev erse-phase high-pressure liquid chromatography. Serine was present in the native thrombin peptide but absent from the anhydrothrombin peptid e, as shown by amino acid analysis. This anhydrothrombin peptide was f ound to be 18.7 +/- 1.6 lower in mass units than the native peptide by electrospray mass spectrometry, in accord with the elimination of a w ater molecule. The anhydrothrombin preparation was monomeric, as deter mined by sedimentation equilibrium. Anhydrothrombin was used in a comp etitive titration of the complex of native thrombin with the leech sal iva protein hirudin, a potent thrombin inhibitor, as measured by the r ecovery of thrombin amidolytic activity. This demonstrated that anhydr othrombin is capable of nativelike binding interactions with macromole cular ligands.