Differences in serum albumins reflected in absorption spectra of a bound dye

Abstract

Binding of the anionic dye 2-(4′-hydroxyphenylazo)-benzoic acid (HABA) by human and other serum albumins in phosphate buffer, pH 6.2, was studied by using equilibrium dialysis. Absorption spectra of the bound dye were determined. Free HABA has an absorption maximum at 348 mμ. When the dye is bound by albumin, its 348 mμ absorption band alters, and an impressive new band develops at about 480 mμ. The changes vary in a characteristic manner depending on the type of albumin employed. The new absorption band of albumin-bound HABA is pH dependent, reaching maximum intensity at about pH 6. The band does not develop if dyes differing slightly from HABA are used, indicating that the structural requirements of the dye are highly specific. The changes in absorption of HABA induced by albumin-binding may be closely simulated by dissolving HABA, but not related dyes, in certain organic solvents or detergent solutions. Thus it appears that the changes depend on certain environmental conditions not uniquely associated with albumins. Differences in absorption of HABA that is bound to different albumins are attributed to differences in binding-site structure of the albumins.