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.

Fig. 1

Sketch of an oblique-incidence reflectivity difference (OI-RD) scanning microscope for detection of association and dissociation of solution-phase protein with ligand microarrays immobilized on a glass slide surface. It employs the total internal reflection geometry for illumination and a combination of a 152-element photodiode array (PDA) for y-scan and a mechanical translation stage for x-scan. PEM: photoelastic modulator that changes the polarization of the illumination laser beam at 50 kHz. PS: variable phase-shifter. L1: cylindrical lens that focuses the illumination laser beam to a line segment on the sample surface. L2: 10× Objective lens that forms an enlarged image of the illuminated line segment across PDA. A: polarization analyzer.

Fig. 2

Real-time association-dissociation curves of solution-phase streptavidin (SA) with immobilized peptide-BSA conjugates on an epoxy-coated glass slide. The concentration of streptavidin is 1 µM in both measurements. Panel (a): reactions of streptavidin with linear SAWSHPQFEK-BSA conjugates. The curve near zero is the association-dissociation curve of streptavidin with a BSA control spot, which shows no evidence of non-specific reaction between BSA and streptavidin. Panel (b): reactions of streptavidin with disulfide-bridged cyclic CHPQGPPC-BSA. The optical signal Im{Δ_p–Δ_s} is related to the captured streptavidin through Eq. (2) and Eq. (4).

Fig. 3

Association-dissociation curves of (a) unlabeled and (b) Cy3-labeled streptavidin with surface-immobilized linear SAWSHPQFEK-BSA conjugates at different streptavidin concentrations. It should be noted that the probe concentrations in Part (b) are 4 times larger than those in Part (a). The curves are globally fitted (dot lines) to a two-site Langmuir reaction model (see main text for details) with the global fitting parameters listed in Table 1.

Fig. 4

Association-dissociation curves of (a) unlabeled and (b) Cy3-labeled streptavidin with surface-immobilized disulfide-bridged cyclic CHPQGPPC-BSA conjugates at different streptavidin concentrations. It should be noted that the probe concentrations in Part (b) are 4 times larger than those in Part (a). The dot lines are global fits to a two-site Langmuir reaction model (see main text for details) with the global fitting parameters listed in Table 2.

Fig. 5

Association-dissociation curves of (a) unlabeled and (b) Cy3-labeled monovalent F_ab fragment of goat IgG against the (H+L) domain of rabbit IgG with surface-immobilized rabbit IgG at different goat IgG concentrations. The curves are globally fitted (dot lines) to a two-site Langmuir reaction model (see main text for details) with the global fitting parameters listed in Table 3.

Fig. 6

Association-dissociation curves of (a) unlabeled and (b) Cy3-labeled goat anti-rabbit whole IgG against the F_c domain of rabbit IgG with surface-immobilized rabbit IgG at different goat IgG concentrations. The dot lines are global fits to a two-site Langmuir reaction model (see main text for details) with the global fitting parameters listed in Table 4.