Conformational transformations of the fusion protein BR96 sFv-PE40 as monitored by micellar electrokinetic capillary chromatography and circular dichroism

Three isoforms of BR96 sFv-PE40, a single-chain fusion protein, were separated by micellar capillary electrokinetic chromatography (MECC) using cholic acid as a micelle-forming surfactant. Two well-known modifiers of protein structure guanidine hydrochloride and trifluoroethanol altered the separation profile in a concentration-dependent manner. In parallel, conformational changes of BR96 sFv-PE40 were monitored by near- and far-UV circular dichroism measurements. In general, a good correlation was found between structural transformations and alterations in the electrophoretic profile. It was suggested that differences in the electrophoretic mobilities of closely-related species, such as isoforms and/or conformers, may result from differences in exposure of polar and hydrophobic elements on the exterior of the protein globule via formation of distinct complexes between the micelles and protein molecules. Application of MECC to protein analysis offers a unique tool for separation and quantitation of closely related species such as isoform and conformers.