Indoxyl and p-cresol sulfate binding with human serum albumin

Abstract

Protein bound uremic toxins are of intense interest because they accumulate in the blood compartment of patients suffering from kidney dysfunction, are poorly cleared by hemodialysis, and are associated with poor patient outcomes. In particular, high blood concentrations of indoxyl sulfate (IS) and p-cresol sulfate (PCS) are associated with both cardiovascular disease and increased inflammation. Limited work has been done to understand the interaction between HSA and IS or PCS, where the binding mechanism, preferred binding sites, and resulting conformation of HSA remain ill-defined. Using isothermal titration calorimetry, it was observed that both IS (Ka=10.06 ×103) and PCS (Ka=1.39 ×103) binding to HSA are enthalpy driven, with electrostatic interactions playing an important role. Saturation transfer difference NMR was used to verify IS or PCS interactions with HSA and to confirm, using warfarin and ibuprofen, that IS binds to Sudlow’s site I and site II whereas PCS only partially binds to site II. Circular dichroism and fluorescence spectroscopy results indicated that changes in the secondary and tertiary conformation of HSA occurred upon binding of IS or PCS. This fundamental physiochemical analysis of IS or PCS binding to HSA provides key parameters that will help in the development of materials that can directly compete with HSA for binding IS or PCS from blood, or compete for the albumin binding sites so as to displace IS or PCS, to enhance their clearance using commonly employed dialysis methods and perhaps lead to better patient outcomes.