Denaturant-gradient chromatography for the study of protein denaturation: principle and procedure

The particle volume of a protein which represents the expansion of the molecular chain is a useful probe to measure its conformation change in solution. To study the details of the conformation transition, we will report a novel device to trace the particle-volume change against increasing denaturant concentration. According to the denaturant gradient in the eluant of high-performance gel-permeation chromatography, information concerning the elution volume of proteins in the course of denaturation can be successively accessed. When a protein is injected into a denaturant solution, its effective molecular volume increases by the unfolding of a native compact structure, and the elution peak goes forward to separate from that of the native protein. Typical elution patterns were computer-simulated in an extended algorithm of the plate theory. The kinetic feature of denaturation and the effect of the denaturant gradient are discussed. The denaturation schemes of lysozyme, cytochrome c, and phosphoglycerate kinase were studied.