Study of binding and denaturation dynamics of IgG and anti-IgG using dual color fluorescence correlation spectroscopy.
In this article, we present a systematic study on IgG and Fab fragment of anti-IgG molecules using fluorescence auto- and cross-correlation spectroscopy to investigate their diffusion characteristics, binding kinetics, and the effect of small organic molecule, urea on their binding. Through our analysis, we found that the diffusion coefficient for IgG and Fab fragment of anti-IgG molecules were 37+/-2 microm2s(-1) and 56+/-2 microm2s(-1), respectively. From the binding kinetics study, the respective forward (k(a)) and backward (k(d)) reaction rates were (5.25+/-0.25)x10(6)M(-1)s(-1) and 0.08+/-0.005 s(-1), respectively and the corresponding dissociation binding constant (K(D)) was 15+/-2 nM. We also found that urea inhibits the binding of these molecules at 4M concentration due to denaturation.
Varghese
Leo Tom
LT
School of Electrical and Computer Engineering, Purdue University, West Lafayette, IN 47907, United States.
Sinha
Rajeev K
RK
Irudayaraj
Joseph
J
D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000737:chemistry* / Q000276:immunology*; D000937:Antigen-Antibody Reactions / Q000187:drug effects; D003116:Color; D004058:Diffusion; D006041:Goats / Q000378:metabolism; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D007074:Immunoglobulin G / Q000737:chemistry* / Q000276:immunology*; D007700:Kinetics; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014508:Urea / Q000494:pharmacology
D000888:Antibodies, Anti-Idiotypic; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; C048844:anti-IgG; D014508:Urea