Separation between the α and β forms of human antithrombin by hydroxyapatite high-performance liquid chromatography

Human antithrombin (AT) inhibits several proteases in the coagulation system, including thrombin and factor Xa, and thus, plays an important role in the regulation of blood coagulation. The predominant form of AT in plasma is ATα, which contains four glycosylated asparagine residues, and the minor form is ATβ, which lacks the Asn-135 glycosylation. In this study, hydroxyapatite high-performance liquid chromatography, using a segmented sodium phosphate gradient, was utilized for the high-resolution separation of ATα and ATβ. The detection limit (signal-to-noise ratio of 3) for ATβ was 30 μg/mL, corresponding to 0.5% of the injected concentration of AT. Two analyzed commercial AT products both contained about 2% ATβ. This method is suitable for the determination of ATβ in pure samples of native AT.