Method for Selection of Antithrombin Disulfide-Bonded Subsets.

Studies of coagulation proteins have demonstrated that they are frequently produced in multiple partially disulfide-bonded states, with each state potentially influencing the protein's function. The ability to isolate distinct disulfide-bonded forms could be valuable for functionally characterizing these protein subsets. Here, we describe protocols for the selection of a disulfide-bonded subset of antithrombin, characterized by its distinct biophysical properties, using a limiting quantity of heparin-Sepharose. Antithrombin is a key anticoagulant protein that regulates the proteolytic activity of several procoagulant proteases, and its deficiency is associated with pathological thrombosis. Antithrombin contains three disulfide bonds, which exist in both formed and unformed states in human plasma, demonstrating that antithrombin circulates in blood in multiple disulfide-bonded states. This method provides a practical approach for isolating and studying partially disulfide-bonded subsets of antithrombin and can be extended to other disulfide-bonded protein states.