4‐Ethyl phenyl sulfate binding to human serum albumin

Abstract

Protein‐bound uremic toxins (PBUTs) such as 4‐ethylphenyl sulfate (4‐EPS) challenge kidney failure management due to strong binding to human serum albumin (HSA), thus limiting dialysis clearance. This study examined 4‐EPS–HSA interactions using saturation transfer difference nuclear magnetic resonance spectroscopy (STD‐NMR), isothermal titration calorimetry (ITC), in silico analysis via molecular docking, molecular dynamics (MD), and molecular mechanics Generalized Born surface area (MM/GBSA) simulations. STD‐NMR qualitatively showed competitive binding with the site‐specific ligands, warfarin and ibuprofen, indicating interaction with both Sudlow Sites I and II; competitive ITC results also showed competition between these molecules. ITC analysis using a two‐component model indicated two distinct binding interactions with association constants K _a1  = 6.62 × 10 ⁵  M ⁻¹ (entropically unfavorable) and K _a2  = 2.71 × 10 ⁴  M ⁻¹ (enthalpically favorable), respectively. Docking models were used to analyze representative binding poses at Sudlow Sites I and II, while MD revealed hydrophobic stabilization at Site I and polar interactions at Site II. MM/GBSA per‐residue decomposition identified key stabilizing residues. Circular dichroism measurements showed that the observed binding effects are not accompanied by gross protein unfolding. Comparisons with other PBUTs, including indoxyl sulfate and p ‐cresyl sulfate, indicated the role of electrostatic and hydrophobic forces involved in binding. These findings advance understanding of PBUT–HSA interactions and inform strategies for improved toxin removal in kidney failure treatments.