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The Binding of Indole Compounds to Bovine Plasma Albumin

EFFECTS OF POTASSIUM CHLORIDE, UREA, DIOXANE, AND GLYCINE

From the ¹ From the Departments of Biochemistry and Surgery, the Medical School, State University of New York at Buffalo, and the Edward J. Meyer Memorial Hospital, Buffalo, New York 14215

By the use of a rapid dialysis equilibrium technique, the binding of skatole and acetyl-l-tryptophan with bovine albumin has been studied at various concentrations of KCl, dioxane, urea, and glycine, at different pH values, and at different temperatures. Binding was found to be principally at one site with an association constant much higher than at any secondary sites. The primary association constant, k'₁, for skatole at pH 6.3, 0.1 m KCl, was 4.2 x 10⁵ m^-1 (4°) and 1.9 x 10⁵ m^-1 (18°); for acetyl-l-tryptophan at the same conditions, k'₁ was 3.5 x 10⁵ m^-1 (4°) and 1.8 x 10⁵ m^-1 (18°). A decrease in KCl concentration from 0.1 to 0 decreased k'₁ for skatole to a value 1/12 and 1/15 that in 0.1 m KCl. A decrease in KCl concentration from 0.1 to 0.002 m, on the other hand, increased k'₁ for acetyl-l-tryptophan approximately 3-fold. Chloride was a competitive inhibitor of acetyl-l-tryptophan. The addition of either dioxane or urea reduced k'₁ for skatole and acetyl-l-tryptophan, whereas the addition of glycine had no effect on k'₁. When added to urea solutions, glycine increased k'₁. The activity coefficient of skatole, , in urea, dioxane, or glycine-water mixtures and urea-glycine-water mixtures was determined by solubility measurements. The changes in k'₁ for skatole in dioxane at all concentrations studied and in urea up to 2 m could be approximately reconciled by the assignment 1/² for the ratio of the activity coefficients of the protein-skatole complex and the unassociated protein. The similarities in the effects of dioxane and urea (up to 2 m) on the association of the ligand have been taken to indicate that the primary influence of these solutes is on the apolar surfaces of the associating molecules. At approximately 4 m urea, k'₁ became larger at 18° than at 4°, resulting in a crossing over of the temperature curves of k'₁ plotted as a function of urea concentration.

Thermodynamic changes for the association were also estimated. Under the same conditions, S° was 5 to 15 calories per mol degree more negative for skatole than for acetyl-l-tryptophan. S° increased markedly at urea concentrations above 2 m urea and also increased, but at a somewhat slower rate, as KCl concentrations were reduced from 0.1 to 0.002 m.

The pH binding profiles of the indole ligands show a sharp change in binding at the position of the N-F transition, and also a change in binding between pH 7.5 and 9.5 consistent with a simple ionization process on the protein. The fatty acid content of several albumin preparations was determined, and the binding of the indole compounds was found to decrease in the presence of fatty acid. A molecular weight of 64,300 ± 300 was obtained for bovine albumin on the basis of a 1:1 stoichiometric relationship of the primary site ligand and albumin.

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