Functionalization of Alpha-Lactalbumin by Zinc Ions

Abstract

Alpha-lactalbumin (α-LA) and binding of zinc cations to protein were studied. Molecular characteristics of protein was determined by MALDI-TOF/MS and electrophoresis SDS-PAGE, and also, for complexes, it was determined by spectroscopic techniques (ATR-FT-IR and Raman) and microscopic techniques (SEM along with an EDX detector and also TEM). The pH dependence of zeta potential of α-LA was determined in saline solution. The zinc binding to the protein mechanism was investigated; zinc binding to protein kinetics, the molecular modeling by the DFT method, and electron microscopy (SEM and TEM) for microstructure observation were performed. The experiments performed indicate a quick binding process (equilibrium takes place after 2 min of incubation) which occurs onto the surface of α-LA. Zinc cations change the conformation of the protein and create spherical particles from the morphological point of view. DFT studies indicate the participation of acidic functional groups of the protein (aspartic acid and glutamic acid residues), and these have a decisive influence on the interaction with zinc cations. Application studies of general toxicity and cytotoxicity and bioavailability were conducted.

Figure 1

Zinc kinetics of binding to α-LA.

Figure 2

(A) ATR-IR spectrum of α-LA (blue line) and α-LA complexes with zinc (red line); (B) Raman spectrum of α-LA (blue line) and α-LA complexes with zinc (red line).

Figure 3

(A,B) SEM pictures of the α-LA protein with different magnifications of the sample place; (C–E) SEM pictures of the Zn−α-LA complex with different sample place magnifications; (F) SEM–EDX imaging, photograph of the sample; (G) spectrum from the selected image; (G) list of characterized elements with the content of individual elements in the material; and (H–J) TEM pictures of the Zn−α-LA complex with different bars.

Figure 4

Modeled structures: α-LA with its apo form (A) and modeled complex with zinc cations (with a concentration of 30 mg Zn/L in the reagent mixture) (B).

Figure 5

Flexibility analysis of α-LA–Zn complexes.

Figure 6

Optimized geometries of various conformations of Zn²⁺–Asp^– (ZnAsp1–ZnAsp5) and Zn²⁺–Glu^– (ZnGlu1–ZnGlu5) complexes. Interaction distances are in Å, and binding free energies (ΔG₂₉₈) are in kJ/mol.

Figure 7

SDS-PAGE presenting peptic digestion kinetics, where bands of native α-LA are presented on 2, 4, 6, 8, and 10 and the bands of the complex of α-LA with zinc are presented on 3, 5, 7, 9, and 11.

Figure 8

(A,B) L929 and Caco-2 cell viability after treatment with the Zn complex and Zn ions measured by the MTT method; (C,D) LDH leakage level of L929 and Caco-2 cells treated with Zn ions and the Zn complex; (E) concentration-dependent ROS generation by Zn ions and the Zn complex in L929 cells; (F) morphology of L929 cells treated with the Zn complex; and (G) % of silver taken up by L929 cells from the Zn complex and zinc nitrate. For (A–E), * indicates statistically significant differences (p ≤ 0.001) between the sample and control according to one-way ANOVA and the Tukey post hoc test. For (G), * indicates statistically significant differences (p ≤ 0.001) between the uptake of zinc from the Zn complex and zinc nitrate according to one-way ANOVA and the Tukey post hoc test.