Identical independent sites for dye ligand on bovine serum albumin demonstrated by multivariate analysis

The most fundamental parameters concerning an interaction between a ligand and a protein are equilibrium constants and the number of binding sites. Th e Scatchard plot has for a long time been widely used to obtain those param eters. However, controversy in 1982-1983 over the reliability of this plot (the graphical estimation of the number of identical independent sites from the x-intercept) indicated that some methodologies other than the Scatchar d plot are expected. Over the past decade, we have developed a method for a pplying multivariate analysis to the problem of determining spectral featur es of a ligand associated with a protein molecule. In principle, this metho d is based mainly on the computer-assisted adjustment of dissociation const ants to an assumed reaction model. We discovered in this process that an n- parameter, introduced into an equation for calculating the amount of dye li gand bound to a protein, coincided with the number of identical independent sites, under a certain condition in principal factor analysis calculation. In this study, we established a new methodology for determining the number of identical independent sites using synthesized spectral series, and we t hen applied this method to a simple reaction system composed of bovine seru m albumin (BSA) and bromocresol purple (BCP) anions. BSA was found to have two identical independent sites for BCP anions at pH 8.8. (C) 2000 Elsevier Science B.V. All rights reserved.