Denaturation behavior of antithrombin in guanidinium chloride. Irreversibility of unfolding caused by aggregation.
The structural stability of the protease inhibitor antithrombin from bovine plasma was examined as a function of the concentration of guanidinium chloride (GdmCl). A biphasic unfolding curve at pH 7.4, with midpoints for the two phases at 0.8 and 2.8 M GdmCl, was measured by far-ultraviolet circular dichroism. Spectroscopic and hydrodynamic analyses suggest that the intermediate state which exists at 1.5 M GdmCl involves a partial unfolding of the antithrombin molecule that exposes regions of the polypeptide chain through which slow, intermolecular association subsequently takes place. The partially unfolded molecule can be reversed to its fully functional state only before the aggregation occurs. Upon return of the aggregated state to dilute buffer, the partially unfolded antithrombin remains aggregated and does not regain the spectroscopic properties, thrombin-inhibitory activity, or heparin affinity of the native inhibitor. This behavior indicates that the loss of the functional properties of the proteins is caused by the macromolecular association. Comparative experiments gave similar results for the human inhibitor. Analyses of bovine antithrombin in 6 M GdmCl indicated that the second transition reflects the total unfolding of the protein to a disulfide-cross-linked random coil. This transition is spectroscopically reversible; however, on further reversal to dilute buffer, the molecules apparently are trapped in the partially unfolded, aggregated, intermediate state. The results are consistent with the existence of two separate domains in antithrombin which unfold at different concentrations of GdmCl but do not support the contention that the thrombin-binding and heparin-binding regions of the protein are located in different domains [Villanueva, G. B., & Allen, N. (1983) J. Biol. Chem. 258, 14048-14053].
D000991:Antithrombins / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D019791:Guanidine; D006146:Guanidines; D007700:Kinetics; D008433:Mathematics; D011489:Protein Denaturation
D000991:Antithrombins; D006146:Guanidines; D019791:Guanidine