The influence of zinc ions concentration on β-lactoglobulin structure – physicochemical properties of Zn–β-lactoglobulin complexes

The study of metal-ions binding to whey proteins is the first step for their application in nutraceuticals. This study is focused on the investigation of the zinc ions concentration (6, 60 and 600 mg/L) effect on the metallocomplexes formation (ZnβLG3, ZnβLG30, ZnβLG300) with the β-lactoglobulin and their characterization by a series of instrumental and computational techniques. Detailed surface morphology of the metallocomplexes was investigated by transmission and scanning electron microscopy combined with energy dispersive X-ray analysis, and X-ray diffraction. Moreover, metallocomplex formation was monitored by attenuated total reflectance infrared spectroscopy and Raman spectroscopy. Experimental results were complemented by molecular dynamics (MD) studies indicating the precise binding sites of βLG to Zn2+(aq) ions and quantum mechanics (QM) simulation combined with density functional theory (DFT). Electron microscopy studies indicated that with increasing concentrations of Zn2+(aq) ions, the surface morphology of βLG changes, and the formed metallocomplexes exhibit higher porosity. Meanwhile, spectroscopic studies indicated the formation of metallocomplexes through the binding of Zn2+(aq) ions to amino acids (AAs) such as glutamic acid (Glu), aspartic acid (Asp), tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe). Strong positive correlations between the increasing concentration of Zn2+(aq) ions and the number of binding sites were determined based on the MD results. DFT calculations confirm the strong binding affinity of Asp− and Glu− to Zn2+(aq) ions.