Abstract
Total internal reflection fluorescence (TIRF) spectroscopy has been used to study conformational changes of hen egg white lysozyme induced by interaction with the water/quartz interface. TIRF spectra have been measured over a large temperature range and are compared to the corresponding solution spectra. It has been found that the wavelength of maximum fluorescence intensity of adsorbed lysozyme is red-shifted by about 7 nm relative to that of dissolved lysozyme in the temperature range of about 20–60°C. This observation indicates that lysozyme is partially unfolding when it is adsorbing on quartz. Using optical reflectometry a drastic temperature-induced increase of the degree of adsorption of lysozyme and staphylococcal nuclease (SNase) on silicon wafers has been measured which suggests that the corresponding adsorption processes are endothermic and thus entropically driven. The major contribution to this entropy gain will probably originate from conformational changes at lower temperatures. The experimental results indicate that proteins with a smaller Gibbs energy of unfolding have a higher tendency for adsorption. Above the temperatures of unfolding of the proteins in solution, the dehydration of hydrophobic residues, which are exposed to water in the thermally unfolded state, are the most likely driving force for the adsorption of lysozyme and SNase on silicon oxide.
Highlights
The adsorption of protein molecules at aqueous/solid interfaces is an interesting phenomenon that is relevant for medical diagnostics or protein analysis and purification [1,2]
This conclusion is supported by the Total internal reflection fluorescence (TIRF) study of lysozyme described above, in which a partial unfolding of lysozyme has been found upon adsorption at the silica/water interface in the temperature range of 20–60◦C
The drastic increase of lysozyme adsorption at the silica/water interface with increasing temperature, as found with optical reflectometry (Fig. 4), can explain the increase of the total fluorescence intensity emitted by adsorbed lysozyme molecules at elevated temperatures (Fig. 3)
Summary
The adsorption of protein molecules at aqueous/solid interfaces is an interesting phenomenon that is relevant for medical diagnostics or protein analysis and purification [1,2]. The adsorption of enzymes on a plane silica surface is investigated as a function of temperature in order to analyze the contribution of protein conformational changes to the driving forces for adsorption. The Gibbs energy of unfolding of a protein can be decreased continuously, thereby promoting adsorption-induced conformational changes which will contribute to the driving forces. Lysozyme is often classified as a “hard” protein which largely preserves its native structure at an interface, we will show in this study that there are conformational changes when lysozyme adsorbs on silica and that the associated entropy increase is probably the major driving force for adsorption on a hydrophilic surface like silicon oxide
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