Investigating Quinclorac Binding to Human Serum Albumin using Spectroscopy and Molecular Docking

Abstract Human serum albumin (HSA) is the most abundant plasma protein and serves as a major transporter and reservoir for a wide range of endogenous ligands, including fatty acids, heme, bilirubin, prostaglandins, and metal ions, as well as numerous exogenous compounds, such as pharmaceuticals. HSA is a 66 kDa monomeric, multidomain protein containing two principal ligand-binding sites, Sudlow sites I and II. Sudlow site I contains Trp214, the sole tryptophan residue in HSA, whose intrinsic fluorescence provides a sensitive probe for studying ligand–protein interactions. In this study, the interaction between HSA and the persistent organochlorine herbicide quinclorac (QNC) was investigated using steady-state fluorescence spectroscopy and molecular docking. The binding of QNC to HSA is characterized by concentration-dependent quenching of HSA’s intrinsic tryptophan fluorescence. Quantitative analysis revealed a high affinity of QNC for HSA, with ∼ 1 μM dissociation constant ( K d ). Temperature-dependent fluorescence quenching experiments indicate a static quenching mechanism. Moreover, a competitive displacement assay using site-specific markers identified Sudlow site I as the primary binding site for QNC in HSA. Induced-fit docking further demonstrated that QNC is accommodated and stabilized by polar interactions, π-stacking, and/or hydrophobic interactions within the Sudlow I binding pocket. Together, these results provide a biochemical and molecular understanding of how QNC, a persistent pesticide, binds to HSA.