Metallomics Study of Lead-Protein Interactions in Albumen by Electrochemical and Electrophoretic Methods

Lead(II) ions represent still threat to living organisms due to high burden of these ions in environment. The aim of this study was to design and perform experiments studying the interaction of lead(II) ions and proteins contained in egg albumen. For this purpose, we used electrochemical and electrophoretic techniques. We focused on a very detailed study of the interaction of lead(II) ions with the egg albumen proteins. Firstly, egg albumen was diluted with acetate buffer in the following ratios buffer: albumen - 1:0.5; 1: 1; 1: 2 and 1: 4. Additions of different concentrations of lead(II) ions into the diluted egg albumen were 0; 1; 5; 10; 25; 50; 100; 150; 200; 250; 500; 750 and 1,000 nM. Mixtures were then placed on thermoblock at 37 degrees C for 15, 30, 45 and 60 min. Primarily, we used differential pulse anodic stripping voltammetry to analyse above prepared mixtures. It clearly follows from the obtained experimental data that there is a very strong interactions of lead(II) ions and biomolecules contained in egg albumen. Rapid increase in the concentration of lead(II) ions with a linear trend was detected in the mostly diluted egg albumen (lower concentration of total protein). At higher concentrations and longer time of interactions, metal ions bind tightly or intercalate into biomolecules presented in egg albumen. Besides the effect of lead(II) ions and albumen concentrations, we aimed at the effect of various times of incubation. The obtained results were linearly plotted and the slopes of these straight lines were compared. All slopes steep decreased with the increasing time of interaction and content of egg albumen proteins (15 min interaction, the ratio of 1: 4 and the slope 0.06; the ratio of 1: 2 and the slope 0.038; the ratio of 1: 1 and the slope 0.009; the ratio of 1: 0.5 and the slope 0.0085). Moreover, it is evident a clear time-dependence of binding lead(II) ions to biomolecules. At the ratio of 1: 0.5 there was observed the bond of app. 0.06 ng Pb per min. into biomolecules of egg albumen, at the ratio of 1: 1 there was observed the bond of app. 0.08 ng Pb per min. into biomolecules of egg albumen, at the ratio of 1: 2 there was observed the bond of 0.3 ng Pb per min. into biomolecules of egg albumen and at the ratio of 1: 4 there was observed the bond of 0.55 ng Pb per min. to biomolecules of egg albumen. Probably, in the case of lower concentrations of albumen proteins, there is a change in the structure of these proteins. The samples obtained by incubation of various concentrations of lead(II) ions with egg albumen protein were also analysed by chip capillary electrophoresis under non-reducing conditions to study the influence of lead(II) ions on the protein profiles of egg albumen. We determined changes in the content of the selected proteins as lysozyme, flavoprotein, ovalbumin, ovomucoid, avidin and ovotransferrin and some unidentified as marked according to their molecular masses 55, 95, 110, 117, 123, 132 and 170 kDa. Based on determined protein profiles (as slopes of signal changes depending on the applied lead(II) concentration) at various dilutions it is clearly evident that the most significant changes are detected at ovotransferrin, avidin, lysozyme and ovomucoid. In addition, we employed optimized methods for studying of lead(II) induced complexes in died embryo of vulture. Contents of lead(II) ions, reduced and oxidised glutathione, metallothionein and zinc(II) ions were determined in albumen, yolk, liver, kidney, brain and bone obtained from the embryo. The highest lead contents were observed in liver and kidney app. 5 mu g/g tissues. The highest contents of GSH and GSSH were found in brain tissue and in albumen, which can be associated with the needs to protect neural system against reactive oxygen radicals. The highest content of MT was determined in albumen (more than 12 mu g/g) followed by yolk and kidney, and the lowest in liver. This is very interesting result, which shows a highly active defence of the embryo against lead(II) ions. The level of zinc(II) ions was highest in organs with maximum biochemical activity (liver and kidney). The results bring new and unique point of view in metallomics research and may provide new insights into mechanisms during embryonic development.