VLDL receptor fragments of different lengths bind to human rhinovirus HRV2with different stoichiometry - An analysis of virus-receptor complexes by capillary electrophoresis

The formation of complexes between the minor receptor group human rhinoviru s HRV2 and two recombinant soluble receptor fragments derived from the huma n very low density lipoprotein receptor (VLDLR) and containing ligand-bindi ng repeats 1-3 (MBP.VLDLR1-3) or 1-8 (MBP.VLDLR1-8) fused to the carboxyl t erminus of the maltose binding protein was analyzed by affinity capillary e lectrophoresis. At low molar ratios of receptor/virus, the peaks correspond ing to substoichiometric complexes were broad indicating heterogeneity. Whe n the receptors were present in molar excess with respect to the virus, the peaks were sharp, suggesting saturation of all binding sites. For the dete rmination of the stoichiometry, constant amounts of receptor were incubated with increasing amounts of virus, and the peak areas corresponding to free receptor were measured and plotted versus total virus concentration. Extra polation of the linear part of the resulting curve to zero concentration of free receptor enabled quantitation of the molar ratios of the components p resent in the complex. Using this method, we determined that about 60 molec ules of MBP.VLDLR1-3 but only about 30 molecules of MBP.VLDLR1-8 were bound per virion.