Characterization of Protein-Protein and Protein-Ligand Interactions by High Performance Size Exclusion Chromatography

Abstract

Abstract HPLC has been used in our laboratory to characterize a wide range of protein-protein and protein-ligand interactions. In a study of the dissociation and recombination of human chorionic gonadotropin subunits, HPLC provided a fast and sensitive method for directly observing the state of association of samples equilibrated under various conditions. The α subunit (15 Kd) was easily resolved from the β subunit (23 Kd) using a Toyo Soda type SW 3000 column (0.8 × 60 cm) eluting at 1 ml/min. The subunit was poorly resolved from the intact hormone (38 Kd) in agreement with results obtained using conventional exclusion media. In another study, the same column was used to assess the degree of aggregation of various protease inhibitors (antithrombin III (AT III), C1-inactivator and α1-proteinase inhibitor) after heating, as part of an effort to determine conditions under which these potentially therapeutic proteins might withstand pasteurization to reduce the risk of transfusion hepatitis. The ability of AT III (65 Kd) to bind heparin (5–20 Kd) and thrombin (37 Kd) was also readily ascertained by HPLC. When native inhibitor was premixed with excess heparin, its elution shifted toward the void and became broader due to the polydispersity of the mucopolysaccharide. By contrast, formation of a complex with thrombin only slightly increased the rate of elution of AT III. Nevertheless, the extent of complex formation could be determined from the depletion of the much slower moving thrombin peak. The latter approach proved useful for characterizing thrombin after covalent attachment of fluorescent probes.