Using Difference Spectrophotometry to Study the Influence of Different Ions and Buffer Systems on Drug Protein Binding

Abstract

Difference spectrophotometry was used to investigate the effect of different ions and buffer systems on the binding of the anti-inflammatory drug tenoxicam to human serum albumin (HSA). Chloride anions, as well as sodium cations, were found to decrease the binding affinity. The effect of chloride ions was greater on the primary binding constant K₁, while sodium ions had a greater effect on the secondary binding constant K₂. The number of binding sites n₁ n₂ were not affected except at 0.12% HSA, for which the presence of sodium ions halved n₂. Potassium ions significantly increased K₁. The presence of potassium instead of sodium ions increased binding affinity at lower HSA concentrations. The number of binding sites n₁ and n₂ were fewer in presence of potassium than in the presence of sodium ions except at 0.12% HSA. The divalent calcium and magnesium cations increased the binding affinity of HSA to tenoxicam, with a greater effect on K₁. The effect of magnesium ions on K₁ occurred when the MgCl₂ concentration was increased to 3 and 9 mM, with the former seeming to be a critical concentration. The number of primary binding sites n₁ was not affected by calcium ions, but was halved by 1 mM MgCl₂. Both calcium and magnesium cations decreased n₂, which was halved when the concentration of either cation was increased to 9 mM. The effect of buffer systems on tenoxicam binding to HSA was dependent on HSA concentration. The value of K₁ was higher in Sorensen's phosphate buffer than in Tris [tris(hydroxymethyl) aminomethane HCl] buffer when the HSA concentration was 0.04% and 0.16%, while the reverse was observed at 0.08% and 0.12% HSA. The other binding parameters (K₂, n₁, and n₂) were higher in Sorensen's phosphate than in Tris buffer. However; at certain HSA concentrations, the values of such parameters were comparable in both buffer systems.