Interactions between food additive butylated Hydroxytoluene (BHT)/its metabolites and pepsin: Mechanisms and biological implications

BHT and its metabolites (BHT-OH, BHT-COOH) form stable complexes with pepsin via static quenching. • Hydrogen bonding and van der Waals forces are the primary driving forces for BHT derivatives-pepsin binding. • BHT derivatives induce conformational changes in pepsin and inhibit its activity in a dose-dependent manner. • Functional groups differentially regulate binding affinity and inhibitory potency: hydrophilic groups (-OH, -COOH) enhance binding affinity but reduce inhibitory potency, whereas hydrophobic groups (-CH₃, tert-butyl) weaken binding affinity but strengthen inhibitory potency. Butylated hydroxytoluene (BHT) is a synthetic phenolic antioxidant extensively used in food. Given the increasing biosafety concerns regarding BHT and its metabolites (BHT-OH and BHT-COOH), their interactions with pepsin were systematically investigated using multispectral techniques, molecular docking, and simulation methods. Fluorescence analysis indicated static quenching and stable complex formation, with binding affinities following the order BHT-OH > BHT-COOH > BHT. Hydrogen bonding and van der Waals forces dominated the interactions, and the carboxyl group of BHT-COOH contributed additional hydrogen bonds. Binding induced conformational changes in pepsin, altered the microenvironment of tryptophan residues, and resulted in dose-dependent inhibition of enzymatic activity. Meanwhile, the binding also significantly reduced the radical scavenging and reducing capacity of BHT and its metabolites. These findings highlight the critical role of functional group substitution in modulating gastrointestinal interactions and potential health risks of these compounds.