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.