Effects of the reduction and S-carbamidomethylation on the conformation, activity and heparin-binding capacity of human antithrombin III.

Antithrombin III has been shown to contain three disulphide bridges. They can be broken by reduction under non-denaturing conditions. The completely reduced and S-carbamidomethylated protein is devoid of thrombin-inhibiting activity and does not bind heparin. However, the immunological reactivity as determined by electroimmunoassay is almost unchanged, indicating that no large conformational changes occur upon cleavage of the disulphide bonds. This is also supported by the observation that the reduced and S-carbamidomethylated protein has the same fluorescence properties as the native protein dichroic spectrum of the native protein shows a pronounced pattern in the near-ultraviolet region, indicating restricted rotation of the aromatic amino acid side chains. This is drastically changed by the reduction and carbamidomethylation. The character of the far-ultraviolet circular dichroic spectrum is similar in the native and reduced S-carbamidomethylated protein. The reduction and S-carbamidomethylation of antithrombin III induces some local changes in the tertiary structure affecting the inhibitor activity, heparin binding and near-ultraviolet circular dichroic spectrum, but do not seem to induce any substantial general conformation change.