Spectroscopic and in silico insight into the influence of substituent position on the molecular interaction mechanism between methoxychlor and human serum albumin

P,p’-Methoxychlor (4-4MXC), an insecticide banned under the Stockholm Convention, exhibits significant environmental persistence and poses health risks. However, the interaction mechanisms of 4-4MXC and its isomer (2-4MXC) with transport proteins remain poorly understood. This study investigated the influence of structural differences on their functionality by evaluating the toxicity variations between 2-4MXC and 4-4MXC and their interactions with HSA. ProTox 3.0 analysis revealed similar toxicity properties for both isomers. They spontaneously bound to HSA at site I via hydrophobic interactions mediated by Arg218 and Trp214. In fluorescence experiments, the binding constants of 4-4MXC were consistently higher than those of 2-4MXC at three temperatures (298 K, 303 K, and 310 K). This can be attributed to its larger negative surface area, which facilitates stronger binding to HSA. 4-4MXC induced a greater reduction in α-helix content (5.0% compared to 3.5% for 2-4MXC) and an increased radius of gyration, indicating more pronounced structural relaxation. Functional assays demonstrated isomer-specific effects: 4-4MXC significantly activates the activity, while 2-4MXC shows slight inhibition at concentrations below 10 × 10⁻⁶ M⁻¹, transitioning to activation at higher concentrations. These differences stemmed from distinct effects on the accessible surface areas of catalytic residues (Lys199/Lys195). These findings highlight the structure-dependent binding of isomers to HSA, leading to variations in their functional properties.