CHROMATOGRAPHY OF THE INTERFERON-GAMMA AND THE ANALOG-II-FTIR ANALYSIS

Although reversed-phase liquid chromatography has been widely used to separate proteins, the retention mechanisms remain often unpredictable . To evidence the influence of the protein structure on these mechanis ms, the conformations, of the recombinant human interferon gamma ((25) ASN) and one of its analogues (Analogue II (25)ASP), were Studied in r elation with their chromatographic behavior. Despite their closely rel ated primary structures, these molecules can be separated by gradient elution in acidic medium. For the solutions, but also when the protein s are adsorbed on a reversed phase RP-C6 support, the present FTIR stu dy delivers an estimation of their secondary structures and also infor mation on the capacity for solvent molecules to accede to the correspo nding polypeptide backbones. During the chromatographic procedure, the acetonitrile in solution and the solid phase upon adsorption, induce different structural changes for the r-hu IFN gamma and Analogue II. T his should generate their different retention times. (C) 1996 John Wil ey & Sons, Inc.