A SHIFT IN PROTOLYTIC EQUILIBRIUM UPON BINDING OF PROTEIN-SENSING DYES TO PROTEINS IS THE BASIC MECHANISM OF ABSORNANCE RESPONSE

Absorbance changes accompanying dye binding to proteins have been shown to result from a shift in dye protolytic equilibria rather than from a solvatochromic effect. The shift is quantitatively characterized by parameter r, which is defined as the ratio of the protolytic constants for the bound; and free dye. A model of protein interaction with pH-sensitive dyes has been devised, which justifies the empirical choice of the optimum pH for the reaction: 0.5-1.0 units below the pK(H) for the dye in solution. A theoretical basis is provided for the selection of optimal ranges of dye and protein concentrations in quantitative assays of proteins and detecting their structural rearrangements.