Published: April 13, 2011 r2011 American Chemical Society 5842 dx.doi.org/10.1021/la200580z | Langmuir 2011, 27, 5842–5849 ARTICLE pubs.acs.org/Langmuir Effects of Arginine and Other Solution Additives on the Self-Association of Different Surfactants: An Investigation at Single-Molecule Resolution Shubhasis Haldar and Krishnananda Chattopadhyay* Structural Biology and Bioinformatics Division, Indian Institute of Chemical Biology, Council of Scientific and Industrial Research, 4 Raja S. C. Mullick Road, Kolkata 700032, India b S Supporting Information ’ INTRODUCTION The self-association of biological molecules has been studied extensively because of their relevance to a number of biological questions. For example, the self-association of proteins may lead to the formation of amyloids, which has been implicated in several human diseases. Additionally, the self-association of therapeutic proteins could lead to immunogeneicity and other undesirable complications in their manufacturing and formula- tion development. The self-association of surfactants is consid- ered to be important because they offer a suitable model system for biological membranes. 1À5 Encapsulation by surfactants has been used as an efficient method of drug delivery. 6 The self- association of surfactants leads to the formation of micelles and other aggregated forms that may play crucial roles in their interaction with proteins or other biomolecules. 7À11 We have been studying protein self-association using fluores- cence correlation spectroscopy (FCS) and other biophysical meth- ods. Fluorescence correlation spectroscopy (FCS) is an important single-molecule technique in the study of the diffusional and conformational properties of labeled molecules. 12À20 In an FCS experiment, the sample is kept in a small observation volume at thermodynamic equilibrium. Fluorescence fluctuations, which oc- cur because of the molecular diffusion in and out of the observation volume, are analyzed by measuring the correlation functions providing a measure of the hydrodynamic radius (r H ). FCS can be used in combination with other fluorescence techniques to study chemical kinetics and dynamics on the microsecond timescale. 13,21,22 Using FCS, we have shown recently that arginine, an amino acid, can be used to inhibit protein self-association. 23 We have shown further by measuring the microsecond conformational dynamics using FCS that arginine inhibits the formation of misfolded intermediate states in the unfolding transition of proteins. 18 Our data and other results available in the literature suggest that the ability of arginine to suppress protein self-association may arise from its interaction with the side chains of the native and nativelike states of proteins. 24À27 This property would be in contrast with other protein stabilizers and osmolites that interact only with the unfolded state of proteins without affecting their native states. 18 In this article, we have carried out FCS measurements to study the effect of different stabilizers, including arginine, on the self- association of two surfactants with the same chain length and opposite charge. We show that the use of a high concentration of urea or glycerol affects the process of self-association presumably by affecting the interaction of water with the surfactant molecules. We observe that self-association is favorable in the presence of salt. The behavior of arginine, however, depends on the nature of the surfactants. Arginine favors the self-association of SDS, a negatively Received: February 14, 2011 Revised: March 28, 2011 ABSTRACT: Fluorescence correlation spectroscopy is used to monitor the self-association of SDS and DTAB monomers at single-molecule resolution. Tetramethylrhodamine-5-maleimide (TMR) has been chosen as a probe because rhodamine dyes have been shown to bind surfactant micelles. Correlation functions obtained by FCS experiments have been fit using conventional discrete diffu- sional component analysis as well as the more recent maximum entropy method (MEM). Hydrodynamic radii calculated from the diffusion time values increase with surfactant concentration as the monomers self-associate. Effects of several solution additives on the self-association property of the surfactants have been studied. Urea and glycerol inhibit self-association, and arginine shows a dual nature. With SDS, arginine favors self-association, and with DTAB, it inhibits micelle formation. We propose surfactant self-association to be a “supersimplified” model of protein aggregation.