Multiple spectroscopic insights into the interaction mechanisms between proteins and humic acid

Abstract

Proteins are important constituents of dissolved organic matter (DOM) in aqueous environments, and their interaction with humic acid (HA), another key component of DOM, substantially affects the environmental behaviors of DOM. In this work, the interaction mechanisms between tryptophan-containing proteins and HA were systematically investigated using multiple molecular spectroscopic approaches. The fluorescence quenching tests indicate that bovine serum albumin (BSA) was more readily quenched by HA and the coexisting phenolic, carboxyl, and quinone groups in HA contributed to this process significantly. By comparison, the fluorescence of L-tryptophan (L-Trp) was more stable under the same conditions. Furthermore, with multiple groups in HA, static quenching with the binding constants and the number of sites were calculated in the protein-HA and L-Trp-HA mixtures. In addition, the differential fluorescence spectra, UV‒Vis spectra, and two-dimensional correlation spectroscopy results confirmed that L-tryptophan amino acid could indeed form a complex with HA, while did not lead to fluorescence quenching. Finally, the molecular docking and density functional theory (DFT) simulations highlighted the contribution of multiple residues surrounding the HA groups to their interactions. The direct interaction between the tryptophan residue and HA might not be the prerequisite for the fluorescence response. Therefore, our work provides further insights into protein-HA interactions and implies other reasonable elucidations for further explanation.