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

Abstract: We studied the effect of fluorescently labeling proteins on proteinligand 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 kon and koff for streptavidinpeptide reactions and antibodyantigen reaction. We found that (1) equilibrium dissociation constants, defined as KD = koff/kon, for streptavidinpeptide reactions increases by a factor of 34 when the solution-phase streptavidin is labeled with Cy3 dye and (2) KD 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.