Preparation and characterization of anhydrothrombin.
Anhydrothrombin, a catalytically inactive derivative of thrombin in which dehydroalanine replaces the active-site serine, was prepared by a novel method. The active-site serine of thrombin was modified to dehydroalanine by promoting the beta-elimination of phenylmethylsulfonic acid from phenylmethylsulfonyl fluoride-inactivated thrombin under conditions 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. Anhydrothrombin was purified by PAB affinity chromatography. Both native thrombin and anhydrothrombin were digested by cyanogen bromide, and the peptides from the region of the active-site serine (S205) were isolated by reverse-phase high-pressure liquid chromatography. Serine was present in the native thrombin peptide but absent from the anhydrothrombin peptide, as shown by amino acid analysis. This anhydrothrombin peptide was found 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 water molecule. The anhydrothrombin preparation was monomeric, as determined by sedimentation equilibrium. Anhydrothrombin was used in a competitive titration of the complex of native thrombin with the leech saliva protein hirudin, a potent thrombin inhibitor, as measured by the recovery of thrombin amidolytic activity. This demonstrated that anhydrothrombin is capable of nativelike binding interactions with macromolecular ligands.
Ashton
R W
RW
Baker Laboratory of Chemistry, Cornell University, Ithaca, New York 14853-1301, USA.
Scheraga
H A
HA
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D006629:Hirudins / Q000378:metabolism; D013058:Mass Spectrometry; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D011485:Protein Binding; D012694:Serine / Q000031:analogs & derivatives; D013917:Thrombin / Q000737:chemistry* / Q000378:metabolism; D014867:Water
D006629:Hirudins; D010446:Peptide Fragments; D014867:Water; D012694:Serine; D013917:Thrombin