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. 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.
Nakamaru
Y
Y
Biological Institute, Graduate School of Science, Tohoku University, 980-8578, Sendai, Japan. ynak@mail.cc.tohoku.ac.jp
Sato
C
C
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004396:Coloring Agents / Q000378:metabolism*; D005163:Factor Analysis, Statistical; D008024:Ligands; D015999:Multivariate Analysis; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet
D004396:Coloring Agents; D008024:Ligands; D012710:Serum Albumin, Bovine