Effects of Various Salts on the Binding of Indole Compounds with Bovine Albumin

R H Mcmenamy,M I Madeja,F Watson

doi:10.1016/s0021-9258(18)93418-6

R H Mcmenamy, M I Madeja + Show 1 more

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https://doi.org/10.1016/s0021-9258(18)93418-6

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Abstract

Abstract Influences of different concentrations of salts (0.002 to 1.25 eq per liter) on the binding of skatole and acetyl-l-tryptophan at the primary binding site of bovine plasma albumin have been studied by equilibrium dialysis. With the exception of Na2-ethylenediaminetetraacetate and ethylenediammonium aspartate, which have little effect, all salts in the concentration range of 0.005 to 0.1 eq per liter inhibit the binding of acetyl-l-tryptophan; their order of effectiveness, which is invariant with salt concentration, is KClO4 g KSCN g KI g LiCl g CaCl2 g KF g KCl g NaCl g ethylenediammonium chloride g K2SO4. With skatole the addition of halide-like salts (except KF) in the concentration range of 0 to 0.05 eq per liter increases the binding markedly. The order at 0.002 eq per liter solute is KClO4 g KSCN g KI g ethylenediammonium chloride g KCl g LiCl g CaCl2 (this order is consistent with increased skatole binding with increased salt anion binding). Between 0.05 and 0.25 eq per liter binding of skatole decreases with further addition of KClO4 and KSCN, remains approximately constant with further addition of KI, but increases with further addition of the Cl- salts. Other solutes have no effect (Na2-ethylenediaminetetraacetate, ethylenediammonium aspartate, K2SO4, and glycine), or a small enhancing effect (KF) on skatole binding. Between 0.25 and 1.25 eq per liter the addition of most salts increases the binding of skatole. The salt concentration binding profile of β-3-indolylethanol in KSCN solutions is similar to that of acetyl-l-tryptophan, implying that the charge of the acetyl-l-tryptophan is not the element which leads to the differences between its binding and that of skatole. Effects of SCN- and Cl- on acetyl-l-tryptophan-albumin binding show direct competitive inhibition as well as anomalous ionic strength effects. It is concluded that the primary influence of salts is against the polar elements of the binding center.

Highlights

Tryptophan at the primary binding site of bovine plasma albumin have been studied by equilibrium dialysis
0.05 and 0.25 eq per liter binding of skatole decreases with further addition of KC104 and KSCN, remains approximately constant with further addition of KI, but increases with further addition of the Cl- salts
At least duplicate saturation experiments were conducted at each salt concentration

Summary

Methods

Bovine plasma albumin (Armour, Lot Y20907) was the same preparation as used in other studies [8]. It was dialyzed extensively against 1.0 mM EDTA and deionized by passage through a mixed bed resin column. By electrophoresis it contained less than 2% nonalbumin, and by ultracentrifugation it contained less than 2% dimer. Skatole and acetyl-L-tryptophan were obtained from Mann. An example of a low binding condition is that of the binding of skatole in 0.05 M K,SO, (Fig. 1). With tryptophol the primary binding site was always distinguishable, secondary site binding was usually appreciable. The units of k’ are always moles per liter (M-l)

Results

Discussion

Conclusion