[Reversed-phase high performance liquid chromatographic analysis of the unfolding procedure of bovine insulin]

A method for measurement of the dynamic unfolding procedure of bovine insulin by reversed-phase HPLC has been established. Insulin contains 51 amino acids and two intrachain disulfide bridges. The denaturation of bovine insulin was carried in dithiothreitol solution at 100 degrees C, and the equilibrium products were examined by HPLC at different reaction time. The results show that the conformation of insulin has changed before cleavage of the disulfide bonds to A and B chains. Bovine insulin, two intermediates and the reduction products A and B chains were well separated on a C18 column (4.6 mm x 150 mm) with a linear gradient elution of acetonitrile containing 0.1% trifluoroacetic acid. The conformation of the unfolding intermediates of insulin was indicated by chromatographic method, and the results were verified by matrix-assisted laser desorption ionization time of flight mass spectrometry. The method is helpful to reveal the conformation changes in the procedures of protein unfolding.