Effect of Fluorescently Labeling Protein Probes on Kinetics of Protein−Ligand Reactions

Abstract

We studied the effect of fluorescently labeling proteins on protein−ligand reactions. Unlabeled ligands (streptavidin-binding peptides and rabbit immunoglobulin G (IgG) as antigen targets) are immobilized on epoxy-functionalized glass slides. Unlabeled and Cy3-labeled protein probes from the same batch (streptavidin and goat antibodies) subsequently react with the surface-immobilized targets. By monitoring in situ the surface mass density change using an oblique-incidence reflectivity difference scanning microscope (a label-free detector), we measured k_on and k_off for streptavidin−peptide reactions and antibody−antigen reaction. We found that (1) equilibrium dissociation constants, defined as K_D = k_off/k_on, for streptavidin−peptide reactions increases by a factor of 3−4 when the solution-phase streptavidin is labeled with Cy3 dye and (2) K_D for reactions of solution-phase goat anti-rabbit antibodies with rabbit IgG targets also change significantly when the goat antibodies are labeled with Cy3 dye.