Fluorescence spectroscopy combined with molecular docking to study the interaction between folic acid derivatives from wheat grains and bovine trypsin

Folic acid is an essential substance for the growth and reproduction of human cells. This study employs fluorescence spectroscopy and molecular docking to investigate the interactions between trypsin and four primary folic acid derivatives present in wheat grains: tetrahydrofolic acid (THF), 5-methyl-tetrahydrofolic acid (5-CH₃-THF), 5-formyl-tetrahydrofolic acid (5-CHO-THF), and 5,10-methylene-tetrahydrofolic acid (5,10-CH₂-THF). All derivatives effectively quenched trypsin's intrinsic fluorescence via distinct mechanisms: THF and 5-CH₃-THF followed dynamic quenching, while 5-CHO-THF and 5,10-CH₂-THF exhibited static-dynamic combined quenching. Notably, 5-CHO-THF demonstrated the strongest binding affinity across all tested temperatures. Thermodynamic analysis revealed that the binding of THF and 5-CH₃-THF (ΔH > 0, ΔS > 0) was primarily driven by hydrophobic interactions, whereas 5-CHO-THF and 5,10-CH₂-THF (ΔH < 0, ΔS < 0) interacted mainly through hydrogen bonding and van der Waals forces. Molecular docking confirmed these findings, identifying specific binding residues and interaction types. These results provide crucial insights for developing folic acid-fortified staple crops.