Stability differences between high coagulant (alpha) and noncoagulant (gamma) human thrombins. Denaturation.
Human alpha-thrombin, the two (covalently linked)-chain, highly coagulant blood-clotting enzyme was compared with its noncoagulant, yet estero/amidolytically active derivative, gamma-thrombin, a three (noncovalently associated)-domain enzyme which results from two proteolytic cleavages of the coagulant a form. Studies of their denaturation behavior by Tos-Arg-OMe esterase activity, by intrinsic fluorescence, by fluorescence of active serine-directed dansyl labels, and by monitoring the ESR of a fluorosulfonylphenyl spin-labeled inhibitor clearly demonstrated the reduced stability of the noncovalently associated gamma-thrombin form. At pH 6.5, 0.75 M NaCl, gamma-thrombin unfolds in approximately 2. 1 M urea while the more stable a form denatures at approximately 4 M urea. By monitoring active serine probes (spin label or fluorescent labels), these transitions were slightly lower, 1.0 +/- 0.1 and 2.8 +/- 0.2 M urea for spin-labeled gamma- and alpha-thrombins, respectively. Similar behavior was found for the same spin-labeled derivatives in guanidine HCl with unfolding transitions of 0.4 M and 1.0 M for spin-labeled gamma- and alpha-thrombin, respectively. These differences in structural stabilization serve as a good physical diagnostic for the two thrombin species.
D001777:Blood Coagulation; D004355:Drug Stability; D004578:Electron Spin Resonance Spectroscopy; D004950:Esterases / Q000378:metabolism; D006146:Guanidines; D006801:Humans; D046911:Macromolecular Substances; D011487:Protein Conformation; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D013917:Thrombin / Q000378:metabolism; D014508:Urea
D006146:Guanidines; D046911:Macromolecular Substances; D014508:Urea; D004950:Esterases; D013917:Thrombin