Stability differences between high coagulant (alpha) and noncoagulant (gamma) human thrombins. Denaturation

Area of Science:

• Biochemistry
• Enzymology
• Protein Chemistry

Background:

• Human alpha-thrombin is a key enzyme in blood coagulation.
• Gamma-thrombin is a noncoagulant derivative of alpha-thrombin, differing in its domain association and proteolytic cleavage.
• Understanding the structural stability of these thrombin forms is crucial for their characterization.

Purpose of the Study:

• To compare the structural stability of human alpha-thrombin and gamma-thrombin.
• To identify physical diagnostics for differentiating between the two thrombin species.

Main Methods:

• Denaturation studies using urea and guanidine hydrochloride.
• Assays for Tos-Arg-OMe esterase activity.
• Intrinsic fluorescence spectroscopy.
• Fluorescence of active serine-directed dansyl labels.
• Electron spin resonance (ESR) of a fluorosulfonylphenyl spin-labeled inhibitor.

Main Results:

• Gamma-thrombin exhibits significantly reduced stability compared to alpha-thrombin.
• At pH 6.5, 0.75 M NaCl, gamma-thrombin unfolds at ~2.1 M urea, while alpha-thrombin denatures at ~4 M urea.
• Spin-labeled probes indicated lower unfolding transitions for gamma-thrombin (1.0 M urea) versus alpha-thrombin (2.8 M urea).
• Similar stability differences were observed using guanidine HCl.

Conclusions:

• The noncovalently associated gamma-thrombin form is less stable than the covalently linked alpha-thrombin.
• The observed differences in structural stabilization provide a reliable physical method for distinguishing between alpha- and gamma-thrombin.