Lysozyme binding with amikacin and levofloxacin studied by tritium probe, fluorescence spectroscopy and molecular docking.

Lysozyme complexes with amikacin and levofloxacin were studied by spectroscopy approaches as well as using a tritium probe. Tritium was used as a labeling agent to trace labeled compound concentration in a system of two immiscible liquids and in the atomic form to determine the possible position of the binding site. Co-adsorption of protein and drug at the liquid-liquid interface was analyzed by scintillation phase method that allowed us to directly determine the amount of protein and drug in the mixed adsorption layer. Also, tensiometric measuring of the interfacial tension was used for calculation of binding parameters accordingly to Fainerman model. The treatment of complexes with atomic tritium followed by trypsinolysis and analysis of tritium distribution in the lysozyme peptides reveals the binding sites, binding energies in which were analyzed using molecular docking. Formation of complexes with amikacin and levofloxacin preserves secondar structure of protein. However, the formation of complex with amikacin leads to the almost total loss of the enzymatic activity of lysozyme and the redshift of the maximum on the lysozyme fluorescence band. A slight decrease in the distribution coefficient of lysozyme in the presence of amikacin assumes that the complex has higher hydrophilicity in comparison to lysozyme without additives. The most favorable for binding were the positions of the active centers that included amino acids Asp52 and Glu35, as well as in the vicinity of peptide His15-Arg21, with the participation of amino acids Tyr20, Arg14. In the case of levofloxacin, the formation of lysozyme-ligand complex in aqueous solution is possible without changing the microenvironment of the active center of the protein. Binding of levofloxacin to the active center of the enzyme was the most favorable, but Asp52 and Glu35 that are responsible for the enzymatic activity of lysozyme, were not affected.

Lysozyme-dalargin self-organization at the aqueous-air and liquid-liquid interfaces.

An experimental study of protein-peptide binding was performed by means of radiochemical and spectroscopic methods. Lysozyme and dalargin were chosen due to their biological and physiological importance. By means of tensiometry and radiochemical assays, it was found that dalargin possesses rather high surface activity at the aqueous-air and aqueous-p-xylene interfaces to be substituted by protein. Dalargin forms a hydrophobic complex with lysozyme in which the secondary structure of lysozyme is preserved. When lysozyme forms a mixed adsorption layer with dalargin at the aqueous-air surface, the peptide prevents protein from concentrating in the subsurface monolayer. In the presence of p-xylene protein in the interface, reorganization occurs quickly, so there is no lag in the interfacial tension time dependence. The interfacial tension in this case is controlled by protein and/or protein-peptide complexes. An increase in the enzymatic activity of lysozyme in the presence of dalargin was confirmed by a docking model that suggests the formation of hydrogen bonds between dalargin and amino acid residues in the active site.