Enzymes are very sensitive and highly complex systems, exhibiting a substantial degree of structural variability in their folded state. In the presence of cosolvents, the fluctuations among vast numbers of folded and unfolded conformations occur via many different pathways, and alternatively, enzymes can be stabilized or destabilized. To understand the contribution of osmolytes and denaturants on the stabilization, related to the associated structural changes and enzyme activity of alpha-chymotrypsin (CT), we have monitored differential scanning calorimeter (DSC), circular dichroism (CD), enzyme activity, and gel electrophoresis as a function of osmolyte or denaturant concentration. The present investigation compares the compatibility of osmolytes and deleterious effects of denaturants on the structure, function, and enzyme activity of CT. This comparison has provided new important insight on the contribution of cosolvent effects on protein folding/unfolding, enzyme activity, and understanding of protein-solvent interactions. Our DSC results reveal that the enthalpy change (DeltaH) and Gibbs free energy of change (DeltaG(u)) of CT in osmolyte (trimethylamine N-oxide (TMAO), betaine, sarcosine, proline, and sucrose) increase linearly as osmolyte concentration increases, while those values decrease sharply in the presence of denaturants (urea and guanidine hydrochloride (GdnHCl)). The modifications in the secondary structure of this beta/beta protein, as quantified by the CD spectra, showed reasonable enhancement for beta-strands in the presence of the osmolytes as compared to buffer, which contributes to its stabilization power. Evidently, we observed that naturally occurring osmolytes have a dominant contribution to the stabilization of CT while not enhancing its enzyme activity. In contrast, our results revealed that the denaturants enhanced the surface of the enzyme by binding to the surface of CT, which leads to zero enzyme activity.

The properties of confined liquid water, or liquid water in contact with hydrophobic surfaces, are significantly different from those of bulk liquid water. However, all of water's commonly described properties are related to inert "bulk liquid water" which comprises a tetrahedral hydrogen-bonded network. In this work, we report an innovative and facile method for preparing small water clusters (SWCs) with reduced affinity hydrogen bonds by letting bulk water flow through supported Au nanoparticles (NPs) under resonant illumination to give NP-treated (AuNT) water at constant temperature. Utilizing localized surface plasmon resonance on illuminated Au NPs, the strong hydrogen bonds of bulk water can be disordered when water is located at the illuminated Au/water interface. The prepared SWCs are free of Au NPs. The energy efficiency for creating SWCs is ∼17%. The resulting stable AuNT water exhibits distinct properties at room temperature, which are significantly different from the properties of untreated bulk water, examples being their ability to scavenge free hydroxyl and 2,2-diphenyl-1-picrylhydrazyl radicals and to effectively reduce NO release from lipopolysaccharide-induced inflammatory cells.

The strength of hydrogen bond (HB) decides water’s property and activity. Here we propose the mechanisms on creation and persistence of innovatively prepared liquid water, which is treated by Au nanoparticles (AuNPs) under resonant illumination of green-light emitting diode (LED) to create Au NP-treated (sAuNT) water, with weak HB at room temperature. Hot electron transfer on resonantly illuminated AuNPs, which is confirmed from Au LIII-edge X-ray absorption near edge structure (XANES) spectra, is responsible for the creation of negatively charged sAuNT water with the incorporated energy-reduced hot electron. This unique electronic feature makes it stable at least for one week. Compared to deionized (DI) water, the resulting sAuNT water exhibits many distinct properties at room temperature. Examples are its higher activity revealed from its higher vapor pressure and lower specific heat. Furthermore, Mpemba effect can be successfully explained by our purposed hypothesis based on sAuNT water-derived idea of water energy and HB.

No abstract