Effect of fluorescently labeling protein probes on kinetics of protein-ligand reactions

Westudied the effect of fluorescently labeling proteins on protein-ligand reactions. Unlabeled ligands (streptaVidinbinding peptides and rabbit immunoglobulinG(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 obliqueincidence 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 KD ) 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.