Effect of fluorescently labeling protein probes on kinetics of protein-ligand reactions.
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.
Sun
Y S
YS
Department of Physics, University of California, Davis, California 95616, USA.
Landry
J P
JP
Fei
Y Y
YY
Zhu
X D
XD
Luo
J T
JT
Wang
X B
XB
Lam
K S
KS
D000818:Animals; D002232:Carbocyanines / Q000737:chemistry*; D004852:Epoxy Compounds / Q000737:chemistry; D005898:Glass / Q000737:chemistry; D007074:Immunoglobulin G / Q000737:chemistry*; D007700:Kinetics; D008024:Ligands; D009842:Oligopeptides / Q000737:chemistry*; D011817:Rabbits; D019809:Streptavidin / Q000737:chemistry*; D013499:Surface Properties; D013997:Time Factors
D002232:Carbocyanines; D004852:Epoxy Compounds; D007074:Immunoglobulin G; D008024:Ligands; D009842:Oligopeptides; C089187:cyanine dye 3; D019809:Streptavidin