Cell-based kinetic studies of ligand or candidate drug binding to membrane proteins have produced affinity and kinetic values that are different from measurements using purified proteins. However, ligand binding to fixated cells whose membrane constituents (e.g., proteins and their glycosylated forms) are partially connected by a cross-linking reagent has not been compared to that to live cells. Under the same experimental conditions for the LigandTracer method, we measured the interactions of fluorophore-labeled lectins and antibody molecules with glycans at HFF cells and the human epithelial growth receptor 2 at SKBR3 cells, respectively. In conjunction with surface plasmon resonance microscopy, the effects of labels and cell/sub-cell heterogeneity on binding kinetics were investigated. Our results revealed that, for cell constituents whose structures and functions are not closely dependent on cell viability, the ligand binding kinetics at fixated cells is only slightly different from that at live cells. The altered kinetics is explained on the basis of a less mobile receptor confined in a local environment created by partially interconnected protein molecules. We show that cell/sub-cell heterogeneity and labels on the ligands can alter the binding reaction more significantly. Thus, fixating cells not only simplifies experimental procedures for drug screening and renders assays more robust but also provides reliable kinetic information about drug binding to cell constituents whose structures are not changed by chemical fixation.
Keywords: LigandTracer; SPRM; drug binding; fixated cells; kinetics; live cells.