An approach to apply surface hydrophobicity changes as an indicator of lysozyme modification

Lysozyme is an enzyme, which is found widespread in nature. Lysozyme occurs mostly in the natural environment as a monomer, which breaks down cell walls of Gram-positive bacteria. Formation of polymeric forms resulting from the modification of the enzyme increases its action against Gramnegative bacteria without any loss of its antibacterial activity. Since modifications lead to changes of enzyme properties, the objective evaluation of modified lysozyme is indeed a difficult task. Currently, methods basing on principles of spectrophotometry, electrophoresis and differential scanning calorimetry (DSC) are used for this purpose. None of these methods makes it possible to determine changes in the hydrophobic surface of the enzyme resulting from its modifications. The aim of this study was to characterize changes of hydrophobic surface properties of modified lysozyme in comparison to its unmodified monomer form. To determine their surface hydrophobicity, the interference of dye binding in a protein assay (BIO-RAD Dye Reagent) due to the covering of hydrophobic sites of the protein with Tween 80 (polyoxyethylene sodium monooleate) was measured. The effect of both the dye dilution, and detergent and lysozyme concentrations on the surface hydrophobicity of examined proteins was taken into consideration. As indicated by the performed experiments, apart from the factors mentioned above, the procedure for the preparation of lysozyme was essential for the obtained results. The following analytical parameters will be recommended for the determination of surface hydrophobicity of lysozyme with the examined method: dye dilution of 1 : 50, detergent concentration of 0.25 % and protein concentration of 0.25 %. Data collected in this study have shown that in comparison to the monomer form the hydrophobic surface of modified lysozyme changed markedly with the increase in the number of polymeric forms in the examined preparations. Further investigations in this respect will be recommended before the formulation of final conclusions.