Interaction of serum albumin with a sulphonated azo dye in acidic solution.
The uptake of the sulphonated azo dye [1-(tetrazolylazo)-2-hydroxynaphthalene 3,6-disulphonic acid, or T-azo-R] by bovine serum albumin (BSA) in the pH range 1-4 was investigated by a spectrophotometric method, based on the fact that the absorption spectra of the free and bound dye are different. The effect of increasing concentration of sodium chloride and sodium perchlorate was considered, as well as the influence of the BSA on the protonation equilibria of the dye. The Scatchard model, which has been widely used previously to describe the interaction between small substances and proteins, was not helpful in the treatment of data obtained in the acidity range considered here. Instead a phase distribution model allowed a good quantitative treatment of the experimental findings. The distribution constant of the monoprotonated T-azo-R between aqueous solution and the albumin microphase was found to be log K(d(HL)/gamma'HL) = 6.3, while the biprotonated form does not bind to BSA. As a consequence, the protonation constant of T-azo-R is decreased in the presence of BSA, particularly at low salt concentrations in aqueous solution. An equation relating the observed protonation constant to the protonation constant of the dye in solution and to the ionic strength is proposed. It has been found that the effect of salts on the uptake of T-azo-R by BSA can be explained simply by considering the variation of the activity coefficients of the ionic species involved in aqueous solution.
Pesavento
M
M
Dipartimento di Chimica Generale, Università di Pavia, Viale Taramelli 12, 27100 Pavia, Italy.
Profumo
A
A