Determination of equilibrium and rate constants for complex formation by fluorescence correlation spectroscopy supplemented by dynamic light scattering and Taylor dispersion analysis.

Xuzhu Zhang,Anna Zagożdżon,Andrzej Poniewierski,Robert Hołyst,Agnieszka Wisniewska,Aldona Jelińska,Sen Hou

doi:10.1039/c6sm01791f

Abstract

The equilibrium and rate constants of molecular complex formation are of great interest both in the field of chemistry and biology. Here, we use fluorescence correlation spectroscopy (FCS), supplemented by dynamic light scattering (DLS) and Taylor dispersion analysis (TDA), to study the complex formation in model systems of dye-micelle interactions. In our case, dyes rhodamine 110 and ATTO-488 interact with three differently charged surfactant micelles: octaethylene glycol monododecyl ether C12E8 (neutral), cetyltrimethylammonium chloride CTAC (positive) and sodium dodecyl sulfate SDS (negative). To determine the rate constants for the dye-micelle complex formation we fit the experimental data obtained by FCS with a new form of the autocorrelation function, derived in the accompanying paper. Our results show that the association rate constants for the model systems are roughly two orders of magnitude smaller than those in the case of the diffusion-controlled limit. Because the complex stability is determined by the dissociation rate constant, a two-step reaction mechanism, including the diffusion-controlled and reaction-controlled rates, is used to explain the dye-micelle interaction. In the limit of fast reaction, we apply FCS to determine the equilibrium constant from the effective diffusion coefficient of the fluorescent components. Depending on the value of the equilibrium constant, we distinguish three types of interaction in the studied systems: weak, intermediate and strong. The values of the equilibrium constant obtained from the FCS and TDA experiments are very close to each other, which supports the theoretical model used to interpret the FCS data.

Highlights

Determination of the equilibrium and rate constants of reagents that form noncovalent complexes is crucial for the understanding of various biochemical processes and chemical reactions,[1,2,3] such as drug activities in vivo,[4,5,6] formation of supermolecular structures and their dynamics,[7,8,9] etc
Our results show that the association rate constants for the model systems are roughly two orders of magnitude smaller than those in the case of the diffusion-controlled limit
We study the interactions between fluorescent dyes and surfactant micelles by fluorescence correlation spectroscopy (FCS), in particular, the kinetics of the dye–micelle complex formation

Summary

Introduction

Determination of the equilibrium and rate constants of reagents that form noncovalent complexes is crucial for the understanding of various biochemical processes and chemical reactions,[1,2,3] such as drug activities in vivo,[4,5,6] formation of supermolecular structures and their dynamics,[7,8,9] etc. Fluorescence correlation spectroscopy (FCS) was first introduced by Magde and Elson in the early 1970s to determine the chemical kinetic constants of the interaction between DNA and ethidium bromide.[14,15,16] Since the advent of confocal microscopy illumination, FCS experienced a renaissance in the 1990s.17–19. Recent advances in theoretical studies make it possible to determine the equilibrium and rate constants of noncovalent interactions by fitting the experimental autocorrelation function of FCS to a theoretical model.[21] For example, McNally et al proposed a new model of the autocorrelation function for analyzing the association and dissociation rates of DNA binding in live cells.[1] Al-Soufi et al introduced a general correlation

Methods

Results

Conclusion