Using nonfluorescent Förster resonance energy transfer acceptors in protein binding studies.
The purpose of this article is to highlight the versatility of nonfluorescent Förster resonance energy transfer (FRET) acceptors in determination of protein equilibrium dissociation constants and kinetic rates. Using a nonfluorescent acceptor eliminates the necessity to spectrally isolate the donor fluorescence when performing binding titrations covering a broad range of reagent concentrations. Moreover, random distribution of the donor and acceptor chromophores on the surface of proteins increases the probability of FRET occurring on their interaction. Three high-affinity antibodies are presented in this study as characteristic protein systems. Monoclonal antibody (mAb) 106.3 binds brain natriuretic peptide (BNP)5-13(C10A) and full-length BNP1-32 with the dissociation constants 0.26+/-0.01 and 0.05+/-0.02 nM, respectively, which was confirmed by kinetic measurements. For anti-hCG (human chorionic gonadotropin) mAb 8F11, studied at two incorporation ratios (IRs=1.9 and 3.8) of the nonfluorescent FRET acceptor, K(D) values of 0.04+/-0.02 and 0.059(-0.004)(+0.006) nM, respectively, were obtained. Likewise, the binding of goat anti-hamster immunoglobulin G (IgG) antibody was not affected by conjugation and yielded K(D) values of 1.26+/-0.04, 1.25+/-0.05, and 1.14+/-0.04 nM at IRs of 1.7, 4.7, and 8.1, respectively. We conclude that this FRET-based method offers high sensitivity, practical simplicity, and versatility in protein binding studies.
Ruan
Qiaoqiao
Q
Diagnostic Research, Abbott Diagnostics Division, Abbott Park, IL 60064, USA.
Skinner
Joseph P
JP
Tetin
Sergey Y
SY
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D000915:Antibody Affinity; D002264:Carboxylic Acids / Q000378:metabolism; D006063:Chorionic Gonadotropin / Q000276:immunology; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes; D007074:Immunoglobulin G / Q000276:immunology / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D020097:Natriuretic Peptide, Brain / Q000276:immunology; D011485:Protein Binding; D013388:Succinimides / Q000378:metabolism; D017186:Titrimetry
C492694:Alexa Fluor 488 carboxylic acid succinimidyl ester; D000911:Antibodies, Monoclonal; D002264:Carboxylic Acids; D006063:Chorionic Gonadotropin; D005456:Fluorescent Dyes; D007074:Immunoglobulin G; D008024:Ligands; D013388:Succinimides; D020097:Natriuretic Peptide, Brain