ESTIMATION OF HYDRODYNAMIC VOLUME OF PROTEINS USING HIGH-PERFORMANCE SIZE-EXCLUSION CHROMATOGRAPHY AND INTRINSIC-VISCOSITY MEASUREMENT - AN ATTEMPT AT UNIVERSAL CALIBRATION

Hydrodynamic volume was estimated by using high-performance size-exclusion chromatography (SEC) with a silica-based column and intrinsic viscosity ([eta]) measurement for native proteins and those reduced in sodium dodecyl sulphate (SDS) or guanidine hydrochloride (Gu-HCl). In solutions of 6M Gu-HCl, proteins in their reduced state behaved hydrodynamically as randomly coiled linear homopolymers and exhibited good linearity between an equivalent hydrodynamic radius (R(e)) obtained from [eta] data against erf-1(1-k(d)) obtained from SEC data. However, [eta] values of SDS-reduced protein complex in the SEC solvent were considerably lower than those reported by Reynolds and Tanford, and their SEC behavior demonstrated several undesirable phenomena. The proteins reduced in 6M Gu-HCl solution are recommended as polymer standards for the universal calibration of proteins and polypeptides.