Preferential binding to zinc oxide nanoparticle interface inhibits lysozyme fibrillation and cytotoxicity.

The aim of present investigation is to explore the effect of zinc oxide nanoparticles (ZnONP, 30 nm) interface on conformational dynamics and stability of lysozyme, at pH 7.4 and pH 9.0. Lysozyme adopts partially disordered conformation at pH 9.0, which adopts fibril morphology in presence of sodium dodecyl sulfate (SDS), compared to the conformation adopted at pH 7.4. However, the presence of ZnONP interface renders partially disordered lysozyme relatively regular and non-amyloidogenic conformation, and enhances the functional efficacy of lysozyme at pH 9.0. Additionally, the thermograms reveal a non-cooperative unfolding of the pH 9.0 lysozyme conformation, which accompanied with intermediate conformations that increased with increase in the interface concentration. The binding thermodynamics indicate that at pH 9.0, lysozyme conformation preferentially binds to ZnONP interface than SDS interface. The preferential binding is attributed for the resulting anti-fibrillation propensity of ZnONP interface. The data, altogether, suggest that the presence of ZnONP interface resulted in conformational rearrangements in the partially disordered lysozyme at pH 9.0 causing accumulation of non-amyloidogenic and functionally active intermediates, thus shielding the lysozyme from SDS induced fibrillation and cytotoxicity.

Unfolding and Refolding Study of a Large Dimeric Protein ß-Glucosidase from Almond Monitored by Fluorescence Spectroscopy.

In our present investigation, the unfolding and refolding of ß-glucosidase (BGL-Al) from sweet almond was investigated using tryptophan (Trp) fluorescence spectroscopy. When the unfolding of BGL-Al was induced by guanidium chloride (GdnHCl) and monitored using biological activity as well as Trp fluorescence spectroscopic measurement, we observed that the denaturation of BGL-Al could be easily induced by low concentration of GdnHCl and the enzyme was completely inactivated at 1.0 M GdnHCl. Higher unfolding in the presence of reducing agent revealed that the protein perhaps containing multiple di-sulfide bonds indicating a reason of high stability against unfolding by GdnHCl. Refolding results suggested that the protein refolded with high yield from 1 M GdnHCl denatured state, however, refolded with negligible yield from completely unfolded state. The kinetic studies of BGL-Al refolding unravel a two phase refolding process with calculated t1/2 (refolding half time) of 1.8 and 33 min, respectively. When 8-Anilino-1-naphthalenesulfonic acid (ANS) was used as extrinsic fluorophore, we found that the surface hydrophobicity of BGL-Al was continuously decreased during GdnHCl-mediated unfolding. The surface hydrophobicity of the protein was calculated to be as high as 128.32. Acrylamide quenching study demonstrated that Trp residues of BGL-Al are mostly and hence they must be located either on the surface or in the crevices accessible by quenchers.