Deconvolution of the fluorescence spectra into its component Gaussians: A method to analyze the binding of coumarin to bovine serum albumin

Abstract

7-N,N-Diethylamino-4-methylcoumarin, (cou-I), a readily available laser dye binding to bovine serum albumin (BSA). at room temperature has been studied by steady state fluorescence spectroscopy. Existing methods of analysis of the binding to obtain the binding parameters are based on the change in fluorescence intensity at a particular wavelength. These methods are not convenient when there is a gradual shift in the emission maxima for increasing protein concentration. In this paper we present a method to obtain the binding constants of cou-I to BSA using a Windows '95 based package to deconvolute the asymmetrical spectrum (fluorescence intensity <i>versus </i>wave number curve) into two Gaussians, each corresponding to the binding of the fluorophore to a particular site. This method is convenient to analyze the binding constant data and obtain the binding parameters of each binding site, and can also provide information about the microenvironment of each site, relating micropolarity and microviscosity.