A study on the allosteric interaction between the major binding sites of human serum albumin using microcalorimetry

Abstract

The binding of warfarin and oxyphenbutazone to albumin has been studied at pH 6.8 and pH 9.2 by measuring the heat of binding of these ligands to their high-affinity binding sites on albumin ( ΔH 1 o ′). The − ΔH 1 o ′ values for the binding of warfarin at pH 6.8 and 9.2 and oxyphenbutazone at pH 6.8 and 9.2 were found to be 16.9(±0.6), 28.8(±0.6), 10.5(±0.4) and 17.4(±0.6) kJmol −1, respectively. The Gibbs energies ( ΔG 1 o ′) corresponding to these ΔH 1 o ′ values cover a much smaller range. The pH dependences of ΔG 1 o ′ an ΔH 1 o ′ are explained in terms of p K shifts in the albumin upon binding warfarin or oxyphenbutazone. Diazepam, which binds to a site on albumin which is different from the warfarin-oxyphenbutazone binding site, increases − ΔH 1 o ′ for the binding of warfarin and oxyphenbutazone to albumin at pH 6.8, but it does not influence the − ΔH 1 o ′ at pH 9.2. This phenomenon may be attributed to an allosteric interaction between the diazepam binding site and the warfarin binding site. This allosteric interaction must have its origin in a phenomenon other than the N-B transition.