Molecular characterization of the β-lactoglobulin conjugated with fluorescein isothiocyanate: Binding sites and structure changes as function of pH.

Protein conjugated with dyes is a method which can be used for analyzing food components. For example β-lactoglobulin (βlg) can be conjugated with amine-reactive dyes to form βlg-dye conjugates. In this study, the effect of pH on the conjugation of βlg with fluorescein isothiocyanate (FITC) was investigated using MALDI-TOF MS, LC-MS, dynamic light scattering (DLS) and fourier transform infrared spectroscopy (FTIR). The results showed that the binding numbers increased with the increase in pH, which leading to a greater change in the zeta-potential and the secondary structure of βlg after dye conjugation. In particular, the degree of labelling (DOL) was 94.9 ± 7.9%, and the conjugation was mono-labelled at pH 8, indicating no significant changes in the physicochemical properties of βlg. Furthermore, LC-MS revealed that the most probable conjugated lysine is located at position 100, 47 and 77 of βlg.