In this study, 2,2 0 -(4,4 0 -biphenylylenebisvinylene)bisbenzenesulfonicacid (CBS-X) and its disodiumsalt (CBS) were used as model compounds to investigate the interaction mechanism between 4,4 0 -distyrylbiphenyl based fluorescent whitening agents (DSBP-FWAs) and human serum albumin (HSA) through various techniques, including 1 H saturation transfer difference nuclear magnetic resonance ( 1 H STD-NMR), fluorescence studies, UV-vis absorption, Fourier transform infrared (FT-IR) spectroscopy, circular dichroism (CD) spectroscopy, and molecular docking. The 1 H STD-NMR analyses indicated that CBS and CBS-X can bind to HSA at the favored Sudlow's sites II and I, respectively. Fluorescence emission spectra showed that CBS and CBS-X quenches HSA fluorescence through a dynamic mechanism, and this was further verified by fluorescence lifetime experiments and UV-vis absorption. Moreover, the effective binding constant values of the two compounds at the same temperature decreased in the order CBS > CBS-X. Furthermore, the energy transfer efficiency for CBS and CBS-X were 50.5% and 40.6%, respectively. Thermodynamic analyses indicated that the binding of CBS and CBS-X with HSA are both primarily controlled by hydrophobic forces. FT-IR and CD spectroscopy provided complementary information on the micro-environmental and conformational changes of HSA with the additions of CBS and CBS-X. Molecular docking further confirmed the NMR and spectroscopic results. Overall, the comparative studies on the interaction mechanism of CBS and CBS-X when binding to HSA may provide useful information for evaluating their effects on the human body. Fig. 7 (a) Spectral overlap between the UV-vis absorption spectrum of CBS and the fluorescence spectrum of HSA, (b) spectral overlap between the UV-vis absorption spectrum of CBS-X, and the fluorescence spectrum of HSA. (c) FT-IR spectra of free HSA (I) and spectra of the CBS-HSA system (II) and CBS-X-HSA system (III), with a molar concentration ratio of HSA to CBS (or CBS-X) of 1 : 0.7 in pH 7.4 PBS, [HSA] ¼ 0.2 mM. (d) Circular dichroism spectra of 2 mM HSA in the absence or presence of CBS and CBS-X. Fig. 8 Three-dimensional docking representations for CBS-HSA and CBS-X-HSA interactions. The sites of the interaction of CBS and CBS-X are magnified on the right panel along with the labeling of the HSA residues near the ligands at the interaction site. The H-bonds (green dotted line) are surrounded by a green coil.This journal is