Discrimination between peak spreading in capillary zone electrophoresis of proteins due to interaction with the capillary wall and due to protein microheterogeneity.

Abstract

Our study attempts to find an approach to distinguishing between the contribution to peak spreading in capillary zone electrophoresis (CZE) due to protein microheterogeneity and that due to interaction with the capillary wall, by analyzing correlations between observed peak spreading and peak asymmetry. The peak asymmetry was measured as ln[(tm-t1)/(t2-tm)] where tm, t1, and t2 are migration times at the mode of the peak and at the intersection of the peak width at half-height with the ascending and descending limbs, respectively. Two isoforms of recombinant green fluorescent protein (GFP-1 and GFP-2, 27 kDa molecular mass), glucose-6-phosphate dehydrogenase (GPD, 104 kDa), and the naturally fluorescent protein R-phycoerythrin (PHYCO, 240 kDa) were subjected to CZE in polyacrylamide-coated fused-silica capillaries of 50 and 100 microns diameters under varying conditions of protein concentration, field strength, and the initial zone length. Under conditions such that contributions to peak spreading from axial diffusion, thermal effects, and electrophoretic dispersion are negligible, the analysis of the interrelations between peak width and peak asymmetry was found to allow a conclusion as to the cause of peak spreading in CZE of protein. It appears that the peak width of GFP-2 originates mostly in protein microheterogeneity while that of GFP-1 is due to protein-capillary wall interactions. For PHYCO, both microheterogeneity and protein-capillary wall interactions contribute to peak spreading. GPD exhibits relatively little microheterogeneity or interaction with capillary walls. Thus, its peak width appears to be mostly affected by an extracolumn source of spreading such as the initial zone length.