Photophysical studies on binding of curcumin to bovine serum albumin[para]

Photochemistry and Photobiology, Jun 2003 by Barik, A, Priyadarsini, K I, Mohan, Hari

ABSTRACT

The excited-state photophysical properties of curcumin in the presence of bovine serum albumin (BSA) have been studied. The absorption and fluorescence changes in curcumin on binding to BSA have been followed at varying concentrations of either curcumin or BSA to determine the binding constant, which has been found to be 10^sup 4^ to 10^sup 5^ M^sup -1^. Stopped-flow kinetics studies suggested at least two distinct kinetic steps for the binding of curcumin to BSA. The photophysical properties of the singlet-excited state of the curcumin-BSA complex have also been studied. Whereas the absorption spectrum of curcumin is redshifted, the fluorescence spectrum of curcumin was blueshifted in the presence of BSA. The fluorescence quantum yield of curcumin on complexing with BSA was 0.05. Steady-state fluorescence anisotropy studies showed a significant increase in the anisotropy value of 0.37 in BSA-bound curcumin. The fluorescence decay of the curcumin-BSA complex followed a biexponential decay with fluorescence lifetimes of 413 ps (33%) and 120 ps (67%). On the basis of these complementary results, it has been concluded that curcumin shows very high binding to BSA, probably at the hydrophobic cavities inside the protein.

INTRODUCTION

Serum albumins such as bovine serum albumin (BSA) and human serum albumins are plasma proteins contributing significantly to physiological functions and act as carrier proteins (1-3). They aid in the transport, distribution and metabolism of many exogenous ligands. The ligands include fatty acids, amino acids, metals, drugs and pharmaceuticals (4-19). There are a number of reports in the literature, where binding of metabolites, drugs, dyes, fatty acids, etc. have been studied in detail. BSA has conformational adaptability while binding to the ligands of great varieties. X-ray diffraction studies have shown that the principal binding sites in serum albumin are located in the hydrophobic cavities. The tertiary structure of the protein is composed of three domains, and at least six binding sites (3,12) are available for the binding of the ligands. Hence, the nature of binding of a ligand with BSA is different for different ligands (1-3).

Curcumin (1,7-bis[4-hydroxy-3-methoxyphenyl]-1,6-heptadiene-3,5-dione; structure given below) is a natural polyphenol found as a major pigment in the Indian spice turmeric (20). It shows remarkable pharmacological activity, including anti-inflammatory, anticarcinogenic and antioxidant activity (21-25). Curcumin acts as a lipoxygenase substrate and also as an inhibitor of cyclooxygenase enzymes (21,26). It is considered as a potential chemopreventive agent, and the clinical trials in this direction are in different stages (21,22,24). The most important properties responsible for all the activity of curcumin are its ability to scavenge reactive oxygen and nitrogen free radicals and its lipid soluble nature (23,25,27,28). Being a lipophillic compound, it can inhibit or bind to several regulatory enzymes and proteins. To understand the transport and binding of curcumin to proteins, we have studied the spectroscopic properties of curcumin in the presence of BSA because spectroscopic methods are more sensitive and are easy to use. Molecules, where the chromophores exhibit large differences in excited-state properties with solvents, can be used to understand the local polarity of the biological molecule (9,16). Earlier, we have studied the excited-state properties of curcumin in detail (29). The photophysical properties and fluorescence spectra of curcumin are highly sensitive to the solvent environment (29-32). Therefore, this particular property was used to study the binding of curcumin to BSA. In this article, the excited-state photophysical properties of curcumin on binding to BSA have been studied and presented in detail, using different spectroscopic methods such as steady-state absorption and fluorescence spectroscopy, time-resolved fluorescence and single-mixing stopped-flow spectrometric studies.

MATERIALS AND METHODS

BSA from E. Merck (Mumbai, India; purity >98% by agarose gel electrophoresis) and curcumin from Sigma (St. Louis, MO) were used as received. Solutions were prepared in nanopure water from a Millipore Milli-Q system. Wherever necessary spectrograde solvents from Spectra Chem. (Mumbai, India) were used.

Absorption spectra were recorded on a JASCO V-530 spectrophotometer, and fluorescence spectra were recorded on a Hitachi F-4010 fluorimeter.

Stopped-flow experiments were carried out using an SX. 18MV stopped-flow reaction analyzer (from Applied Photo Physics Ltd., UK) with an absorption detector to study the kinetics of binding between BSA and curcumin. In brief, equal volumes of two solutions containing curcumin and BSA were mixed simultaneously with the help of a pneumatic drive, and the changes in absorption with time were monitored by an absorption detector. The kinetic data were fitted to an appropriate function. At least three independent runs were used to get the observed rate constant. Each independent run is an average of four shots.