Analysis of coupled bimolecular reaction kinetics and diffusion by two-color fluorescence correlation spectroscopy: Enhanced resolution of kinetics by resonance energy transfer

In two-color fluorescence correlation spectroscopy (TCFCS), the fluorescence intensities of two fluorescently-labeled species are cross-correlated over time and can be used to identify static and dynamic interactions. Generally, fluorophore labels are chosen that do not undergo Forster resonance energy transfer (FRET). Here, a general TCFCS theory is presented that accounts for the possibility of FRET between reactants in the reversible bimolecular reaction, A + B <---->(kf)(kb) C, where k(f) and k(b) are forward and reverse rate constants, respectively (dissociation constant K-d = k(b)/k(f)). Using this theory, we systematically investigated the influence on the correlation function of FRET, reaction rates, reactant concentrations, diffusion, and component visibility. For reactants of comparable size and an energy-transfer efficiency of similar to90%, experimentally measurable cross-correlation functions should be sensitive to reaction kinetics for K-d > 10(-8) M and k(f) greater than or equal to similar to10(7) M-1 s(-1). Measured auto-correlation functions corresponding to donor and acceptor labels are generally less sensitive to reaction kinetics, although for the acceptor, this sensitivity increases as the visibility of the donor increases relative to the acceptor. In the absence of FRET or a significant hydrodynamic difference between reactant species, there is little effect of reaction kinetics on the shape of auto- and cross-correlation functions. Our results suggest that a subset of biologically relevant association-dissociation kinetics can be measured by TCFCS and that FRET can be advantageous in enhancing these effects.