Analysis of competitive binding of ligands to human serum albumin using NMR relaxation measurements

Abstract

The competitive binding of two ligands, ibuprofen (IBP) and salicylic acid (SAL), to human serum albumin (HSA) was studied by using nuclear magnetic resonance (NMR) relaxation measurements. When the concentration of one ligand was increased in the solution containing IBP, SAL and HSA, the fractions of free IBP and SAL were increased because of the competitive binding. The 1 H relaxation rates ( R 1) of both ligands were subsequently decreased. If a ligand is in fast exchanging between the free and bound forms, the observed 1 H relaxation rate is a weighted average of that for the free ligand and the protein–ligand complex. The concentrations of the free and bound ligands can be quantitatively derived from the relaxation rates. The results presented in this work revealed that IBP and SAL shared certain low-affinity binding sites on the HSA molecule, in addition to the same high-affinity binding site of AIII.