Influence of Blood Proteins on Biomedical Analysis. I. Interaction of Xanthurenic Acid with Bovine Serum Albumin

Abstract

In order to characterize the amplifying effect of bovine serum albumin (BSA) on the optical density in the color reaction for the determination of xanthurenic acid (XA) using 4-aminoantipyrine (4-AAP), the interactions of XA with native and modified BSA were examined by equilibrium dialysis. BSA was modified with hydrogen peroxide (H₂O₂), p-chloromercuribenzoic acid (PCMB) and 2-hydroxy-5-nitrobenzyl bromide (HNBB). Binding of XA with BSA was confirmed by gel filtration of a mixture containing BSA and XA on a Sephadex G-150 column. The amount of bound XA increased dose-dependently in proportion to the BSA concentration in the mixture. Scatchard plots for the interaction of XA with BSA at pH 4.0, 5.7, 7.4 and 10.0 were obtained. The plots indicated two classes of XA-binding sites on BSA at pH 5.7 and 7.4, but gave a simple straight line with disappearance of the primary binding site at pH 4.0 and 10.0. The binding capacity (n₂K₂) in the secondary binding site decreased linearly with increasing pH of the solution from 4.0 to 10.0, suggesting that the binding force was electrostatic. The standard free energy change (ΔG^o₂), standard enthalpy change (ΔH^o₂) and entropy change (ΔS^o₂) were-5.3 kcal/mol, -4.2 kcal/mol and 3.6 entropy units at pH 7.4, respectively. The temperature dependence of the secondary binding affinity (K₂) was large, suggesting that the association was not ionic. consequently, these results indicate that the secondary association may involve both electrostatic and hydrophobic interactions. Any modifications of cysteine, methionine and tryptophan residues on the BSA molecule destroyed the primary binding site, and the secondary binding site was also slightly affected ; thus, it is speculated that both binding sites may be associated with regions around these three amino acid residues on the BSA molecule. In conclusion, although the relationship between the coloration and the interaction of XA with BSA must be examined more closely, the present results suggest that the binding of XA to BSA contributes substantially to the amplification of the optical density in the color reaction of XA using 4-AAP.