Interaction of human serum albumin with uremic toxins: a thermodynamic study

Abstract

We present a comprehensive study of the interaction of human serum albumin (HSA) with two uremic toxins, namely phenylacetic acid (PhAA) and indoxyl sulfate (IDS) in aqueous solution. The interaction of HSA with PhAA is studied for a series of salt concentrations (20–150 mM) and temperature (25, 30 and 37 °C). The effect of in vitro urea modification of HSA upon its binding affinity towards the uremic toxins, is studied under the highest and lowest salt concentrations and the different temperatures. Isothermal titration calorimetry (ITC) is used to study the interaction by analyzing binding affinities and related thermodynamic data. It is found that two PhAA molecules bind to HSA in a sequential binding process with a binding constant k_b in the order of ≈10⁴ and ≈10³ for the first and second binding respectively. In contrast, IDS binds much stronger to HSA with a total of ≈3 molecules to a high and low affinity binding site in the order of ≈10⁵ and ≈10³. Binding of uremic toxins to HSA in all cases show a decreasing binding affinity trend with increasing temperature and higher ionic strength. Thus binding of a second uremic toxin is strongly weakened at 37 °C and 150 mM. Urea induced HSA modification have only minor effect on the binding interaction of the uremic toxins.