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

Abstract

The most fundamental parameters concerning an interaction between a ligand and a protein are equilibrium constants and the number of binding sites. The Scatchard plot has for a long time been widely used to obtain those parameters. 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 Scatchard plot are expected. Over the past decade, we have developed a method for applying multivariate analysis to the problem of determining spectral features of a ligand associated with a protein molecule. In principle, this method is based mainly on the computer-assisted adjustment of dissociation constants to an assumed reaction model. We discovered in this process that an n-parameter, introduced into an equation for calculating the amount of dye ligand 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 then applied this method to a simple reaction system composed of bovine serum albumin (BSA) and bromocresol purple (BCP) anions. BSA was found to have two identical independent sites for BCP anions at pH 8.8.