The binding of phenol red by serum and by bovine serum albumin

Abstract

Serum proteins from four species were shown to interact with the anions of phenol red. The extent of indicator-protein interaction, studied by a combination of spectrophotometry and ultrafiltration, was found to be highly species dependent. Reaction of crystallized bovine serum albumin with the indicator produced changes in absorption spectrum of the indicator similar to those caused by ox serum. The acid (univalent) form of phenol red is bound about 1.5 times as tightly by bovine albumin as is the alkaline (bivalent) form. The calculated association constants for both forms decrease with increasing pH. The capacity of bovine albumin to bind phenol red, measured in 0.04 M phosphate buffer at pH 7.38, and resulting from the competition of the two anions of the indicator for albumin, may be explained in terms of two classes of binding sites. One molecule of phenol red is bound extremely tightly (association constant = 1.1 × 10 5). An additional six molecules of phenol red per molecule of bovine albumin (association constant = 1.2 × 10 3) are bound at high concentrations of indicator.