Ion-exchange chromatography of proteins: modulation of selectivity by addition of organic solvents to mobile phase: Application to single-step purification of a proteinase inhibitor from corn and study of the mechanism of selectivity modulation

• Published in: Journal of Chromatography A 1998-10, Vol.825 (1), p.17-27
• Main Authors: Tauc, Patrick, Cochet, Sylvie, Algiman, Etienne, Callebaut, Isabelle, Cartron, Jean-Pierre, Claude Brochon, Jean, Bertrand, Olivier
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Enzyme inhibitors; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods; Pop corn inhibitor; Proteinase inhibitors; Proteins
• ISSN: 0021-9673
• DOI: 10.1016/S0021-9673(98)00688-8

Chromatography on a SP-Spherodex column was tried to purify pop corn inhibitor, a proteinase inhibitor from corn. Addition of organic solvent to mobile phase (acetonitrile, isopropanol or ethylene glycol) was tested as a method of improving selectivity. Thirty percent acetonitrile gave optimal results and allowed one to design a single-step purification procedure for the inhibitor. The efficacy of acetonitrile addition, most likely, is not due to an influence on eventual hydrophobic (or hydrophilic) interactions of the chromatographic support with proteins but to the induction of some subtle protein conformation changes of the proteins retained on the column. The effect of acetonitrile on protein conformation was studied by the sensitive technique of time-resolved fluorescence spectroscopy.