Hydroxyphenylazo Benzoic Acid Research Articles

Spectroscopic studies on molecular interactions. VI. Mechanism of metachromasy of 2-(4'-hydroxyphenylazo)benzoic acid by serum albumin.

Mechanism of the albumin-induced metachromasy of 2-(4'-hydroxyphenylazo) benzoic acid (HABA) has been investigated with regard to its interactions with monomeric amino acids and some dipeptides, spectral effect of polarity of the dye environment, and azohydrazone tautomerism of HABA. The metachromasy is concerned not with the monomeric amino acids or the primary structure of serum albumin, but with its intramolecular environment of lower polarity corresponding to that of isopropanol. HABA, once bound to serum albumin by electrostatic forces etc., is buried in such an environment of lower polarity in the interior of the protein molecule, and is converted into the hydrazone form which has an absorption maximum at about 480 mμ. Thus, the metachromasy may occur. The azo-hydrazone tautomerism is supported by the infrared spectra, and is compatible with the results of quantum chemical calculations on 4-hydroxyazobenzene.

Protein bindings. I. Binding of 2-(4'-hydroxyphenylazo)benzoic acid to bovine serum albumin.

Binding of anionic dye 2-(4'-hydroxyphenylazo) benzoic acid (HABA) to bovine serum albumin at pH 7.4 was studied spectrophotometrically. The relationship between albuminbound ratio of HABA and concentration of the unbound dye was expressed by a modified Langmuir-type equation involving a repulsing interaction. The binding becomes more difficult with the increasing bonds. The binding constant and the number of binding sites on albumin were evaluated in 0.05 M phosphate buffer solution and in 0.15 M tris. buffer solution at 25°and 37°.

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

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.