Human protein arginine methyltransferases (PRMTs) can be optimally active under nonphysiological conditions.

Protein arginine methylation is involved in many biological processes and can be enhanced in cancer. In mammals, these reactions are catalyzed on multiple substrates by a family of nine protein arginine methyltransferases (PRMTs). However, conditions that may regulate the activity of each enzyme and that may help us understand the physiological role of PRMTs have not been fully established. Previous studies had suggested unexpected effects of temperature and ionic strength on PRMT7 activity. Here we examine in detail the effects of temperature, pH, and ionic strength on recombinant human PRMT1, PRMT5, and PRMT7. We confirmed the unusual temperature dependence of PRMT7, where optimal activity was observed at 15 °C. On the other hand, we found that PRMT1 and PRMT5 are most active near physiological temperatures of 37 °C. However, we showed all three enzymes still have significant activity at 0 °C. Furthermore, we determined that PRMT1 is most active at a pH of about 7.7, while PRMT5 activity is not dependent on pH in the range of 6.5 to 8.5. Significantly, PRMT7 is most active at an alkaline pH of 8.5 but shows little activity at the physiological intracellular pH of about 7.2. We also detected decreased activity at physiological salt conditions for PRMT1, PRMT5, and PRMT7. We demonstrate that the loss of activity is due to the increasing ionic strength. Taken together, these results open the possibility that PRMTs respond in cells undergoing temperature, salt, or pH stress and demonstrate the potential for in vivo regulation of protein arginine methylation.

Role of pH in skin cleansing.

BACKGROUND: The importance of maintaining the acid-mantle of human stratum corneum to maintain its healthy barrier and skin's biological functions such as desquamation and lipid biosynthesis is well recognized in the literature. An outcome of this has been an increase in the number of skincare products formulated at or near the skin pH with an implication that a product formulated at skin pH will be good for skin. Such an assumption often does not take into account the specific interactions of ingredients in the product with the stratum corneum under skin pH conditions. OBJECTIVE: The objective of this research was to determine whether a skin cleansing product by virtue of its pH being same as "skin pH" is milder to skin. METHODS: A well established Forearm Controlled Application Test (FCAT) protocol was used in clinical studies to compare "skin pH" cleansing systems with neutral pH cleansing systems. Specifically, certain commercially available "skin pH" cleansing bars were compared with a neutral pH syndet bar in two separate FCAT studies. Since these bars differed in their surfactant composition, in a separate FCAT study, two identical prototype bar formulations differed only in their pH were compared. Additionally, two body wash liquid prototypes, identical in composition but differing only in their pH were also compared in another FCAT study. RESULTS: The results obtained showed that skin-cleansing systems formulated solely or predominantly with anionic surfactants under skin pH conditions can result in increased skin dryness and irritation compared to those under neutral pH conditions. The results are explained in terms of the increased electrostatic interaction of anionic surfactants with stratum corneum under low pH conditions compared to neutral pH conditions. CONCLUSION: Skin-cleansing systems formulated solely or predominantly with anionic surfactants under skin pH conditions can result in increased skin dryness and irritation compared to those under neutral pH conditions. Any skin cleansing product by virtue of its pH being same as that of "skin pH" does not guarantee that it will be good for skin. The mildness of a cleanser will be determined by the interactions of its surfactants and other ingredients with stratum corneum under its formulated pH conditions.

CONTEXTE: l'importance de la protection du manteau acide de la couche cornée humaine en vue de maintenir une barrière saine et les fonctions biologiques de la peau, telles que la desquamation et la biosynthèse lipidique, est bien reconnue dans la littérature médicale. Cela a eu pour résultat l'augmentation du nombre de produits cosmétiques formulés à un pH proche ou identique au pH de la peau, impliquant ainsi qu'un produit formulé au pH de la peau sera bon pour la peau. Cette hypothèse ne tient souvent pas compte des interactions spécifiques des ingrédients du produit avec la couche cornée dans des conditions de pH de la peau. OBJECTIF: l'objectif de cette recherche était de déterminer si un nettoyant pour la peau formulé à un pH identique au « pH de la peau ¼ est, pour cette raison, plus doux pour la peau. MÉTHODES: un protocole bien établi de test d'application contrôlée sur l'avant-bras (Forearm Controlled Application Test, FCAT) a été utilisé dans des études cliniques pour comparer les nettoyants à « pH de la peau ¼ et les nettoyants à pH neutre. Plus précisément, certains savons à « pH de la peau ¼ disponibles dans le commerce ont été comparés à un savon surgras à pH neutre dans deux études distinctes faisant appel au FCAT. La composition en termes d'agents de surface de ces savons étant différente, une étude distincte faisant appel au FCAT a comparé deux prototypes de savons de composition identique mais de pH différent. De plus, deux prototypes de savon liquide pour le corps, de composition identique mais de pH différent, ont également été comparés dans une autre étude faisant appel au FCAT. RÉSULTATS: les résultats obtenus ont montré que les nettoyants pour la peau formulés uniquement ou principalement avec des agents de surface anioniques dans des conditions de pH de la peau peuvent entraîner une augmentation de la sécheresse et de l'irritation cutanées, par rapport à ceux formulés dans des conditions de pH neutre. Les résultats s'expliquent par l'interaction électrostatique accrue des agents de surface anioniques avec la couche cornée dans des conditions de pH faible par rapport aux conditions de pH neutre. CONCLUSION: les nettoyants pour la peau formulés uniquement ou principalement avec des agents de surface anioniques dans des conditions de pH de la peau peuvent entraîner une augmentation de la sécheresse et de l'irritation cutanées, par rapport à ceux formulés dans des conditions de pH neutre. La formulation d'un nettoyant pour la peau à un pH identique au « pH de la peau ¼ ne garantit pas qu'il sera bon pour la peau. La douceur d'un nettoyant sera déterminée par les interactions de ses agents de surface et de ses autres ingrédients avec la couche cornée dans ses conditions de formulation en termes de pH.

Direct Evidence of Salinity and pH Effects on the Interfacial Interactions of Asphaltene-Brine-Silica Systems.

The hydraulic fracturing technique remains essential to unlock fossil fuel from shale oil reservoirs. However, water imbibed by shale during hydraulic fracturing triggers environmental and technical challenges due to the low flowback water recovery. While it appears that the imbibition of fracturing fluid is a complex function of physico-chemical processes in particular capillary force which is associated with wettability of oil-brine-shale, the controlling factor(s) to govern the wettability is incomplete and the literature data in this context is missing. We thus measured the adsorption/desorption of asphaltenes on silica surface in the presence of brines using quartz crystal microbalance with dissipation (QCM-D). We detected zeta potential of asphaltene-brine and brine-silica systems and calculated the disjoining pressures of the asphaltene-brine-silica system in the case of different salinity. Moreover, we performed a geochemical study to quantify the variation of surface chemical species at asphaltene and silica surfaces with different pH values and used the chemical force microscope (CFM) method to quantify the effect of pH on intermolecular forces. Our results show that lowering salinity or raising pH reduced the adhesion force between asphaltene and silica surface. For example, at a pH value of 6.5, when the concentration of injected water is reduced from 1000 mM to 100 mM and 10 mM, the adhesion force decreased by approximately 58% and 66%, respectively. In addition, for the 100 mM NaCl solution, when the pH value increased from 4.5 to 6.5 and 9, the adhesion force decreased by approximately 56% and 87%, respectively. Decreased adhesion forces between asphaltene and the silica surface could promote the desorption of asphaltene from the silica surface, resulting in a negative zeta potential for both asphaltene-silica and brine-silica interfaces and a shift of wettability towards water-wet characteristic. During such a process, -NH+ number at asphaltene surfaces decreases and the bonds between -NH+ and >SiO- break down, to further interpret the formation of a thinner asphaltene adlayer on the rock surface. This study proposes a reliable theoretical basis for the application of hydraulic fracturing technology, and a facile and possible manipulation strategy to increase flowback water from unconventional reservoirs.

Protein-RNA complexation driven by the charge regulation mechanism.

Electrostatic interactions play a pivotal role in many (bio)molecular association processes. The molecular organization and function in biological systems are largely determined by these interactions from pure Coulombic contributions to more peculiar mesoscopic forces due to ion-ion correlation and proton fluctuations. The latter is a general electrostatic mechanism that gives attraction particularly at low electrolyte concentrations. This charge regulation mechanism due to titrating amino acid and nucleotides residues is discussed here in a purely electrostatic framework. By means of constant-pH Monte Carlo simulations based on a fast coarse-grained titration proton scheme, a new computer molecular model was devised to study protein-RNA interactions. The complexation between the RNA silencing suppressor p19 viral protein and the 19-bp small interfering RNA was investigated at different solution pH and salt conditions. The outcomes illustrate the importance of the charge regulation mechanism that enhances the association between these macromolecules in a similar way as observed for other protein-polyelectrolyte systems typically found in colloidal science. Due to the highly negative charge of RNA, the effect is more pronounced in this system as predicted by the Kirkwood-Shumaker theory. Our results contribute to the general physico-chemical understanding of macromolecular complexation and shed light on the extensive role of RNA in the cell's life.

Fast and pH-Independent Elimination of trans-Cyclooctene by Using Aminoethyl-Functionalized Tetrazines.

The inverse-electron-demand Diels-Alder/pyridazine elimination tandem reaction, in which the allylic substituent on trans-cyclooctene is eliminated following reaction with tetrazines, is gaining interest as a versatile bioorthogonal process. One potential shortcoming of such currently used reactions is their propensity to proceed faster and more efficiently at lower pH, a feature caused by the nature of the tetrazines used. Here, we present aminoethyl-substituted tetrazines as the first pH-independent reagents showing invariably fast elimination kinetics at all biologically relevant pH values.

Mechanistic studies of formate oxidase from Aspergillus oryzae: A novel member of the glucose-Methanol-choline oxidoreductase enzyme superfamily that oxidizes carbon acids.

Formate oxidase (FOX) from Aspergillus oryzae is the only GMC member that oxidizes a carbon acid rather than alcohols; thus, its catalytic mechanism may be different from that of other GMC members. We have used pH, solvent viscosity, and deuterium kinetic isotope effects, to investigate the catalytic mechanism of FOX. The enzyme followed a Bi-Bi sequential steady-state kinetic mechanism. The kcat value was pH-independent between pH 2.8 and 6.8, suggesting a lack of ionizable groups in kinetic step(s) that limit the overall turnover of the enzyme. The kcat/Kformate value decreased from a value of 10,000 M-1s-1 at low pH with a pKa value of 4.4, consistent with the requirement of a protonated group for substrate binding. An inverse viscosity dependence on the kcat/Kformate value indicated an isomerization of the Michaelis complex. The kcat/Koxygen value was 340,000 M-1s-1 and pH independent up to pH 6.0. The Dkcat and D(kcat/Kformate) values were 2.5 and 1.9, respectively, indicating that substrate CH bond cleavage is rate-limiting for FOX catalysis. Analytical ultracentrifugation indicated a concentration dependence of the oligomeric state of FOX. The appkred,H value was ∼75% that of kcat,H, indicating that the anaerobic reduction of FOX was dependent on the oligomeric state of FOX.

DNA Hydrogel with Tunable pH-Responsive Properties Produced by Rolling Circle Amplification.

Recently, smart DNA hydrogels, which are generally formed by the self-assembly of oligonucleotides or through the cross-linking of oligonucleotide-polymer hybrids, have attracted tremendous attention. However, the difficulties of fabricating DNA hydrogels limit their practical applications. We report herein a novel method for producing pH-responsive hydrogels by rolling circle amplification (RCA). In this method, pH-sensitive cross-linking sites were introduced into the polymeric DNA chains during DNA synthesis. As the DNA sequence can be precisely defined by its template, the properties of such hydrogels can be finely tuned in a very facile way through template design. We have investigated the process of hydrogel formation and pH-responsiveness to provide rationales for functional hydrogel design based on the RCA reaction.

Differential zinc permeation and blockade of L-type Ca2+ channel isoforms Cav1.2 and Cav1.3.

Certain voltage-activated Ca2+ channels have been reported to act as potential zinc entry routes. However, it remains to be determined whether zinc can permeate individual Ca2+ channel isoforms. We expressed recombinant Ca2+ channel isoforms in Xenopus oocytes and attempted to record zinc currents from them using a two-electrode voltage clamp method. We found that, in an extracellular zinc solution, inward currents arising from zinc permeation could be recorded from Xenopus oocytes expressing L-type Cav1.2 or Cav1.3 isoforms, but not from oocytes expressing Cav2.2, Cav2.3, Cav3.1, or Cav3.2. Zinc currents through Cav1.2 and Cav1.3 were blocked by nimodipine, but enhanced by (±)Bay K8644, supporting the finding that zinc can permeate both L-type Cav1.2 and Cav1.3 channel isoforms. We also examined the blocking effects of low concentrations of zinc on Ca2+ currents through the L-type channel isoforms. Low micro-molar zinc potently blocked Ca2+ currents through Cav1.2 and Cav1.3 with different sensitivities (IC50 for Cav1.2 and Cav1.3=18.4 and 34.1 µM) and de-accelerated the activation and inactivation kinetics in a concentration-dependent manner. Notably, mild acidifications of the external zinc solution increased zinc currents through Cav1.2 and Cav1.3, with the increment level for Cav1.3 being greater than that for Cav1.2. In overall, we provide evidence that Cav1.2 and Cav1.3 isoforms are capable of potentially functioning as zinc permeation routes, through which zinc entry can be differentially augmented by mild acidifications.

Importance of glutamate 87 and the substrate α-amine for the reaction catalyzed by D-arginine dehydrogenase.

Pseudomonas aeruginosa D-arginine dehydrogenase (PaDADH) catalyzes the oxidation of D-arginine to iminoarginine, which is non-enzymatically hydrolyzed to 2-ketoarginine and ammonia. Here, site-directed mutagenesis and pH effects were used to investigate binding and catalysis of zwitterionic and cationic substrates for the enzyme. An unprotonated group with apparent pKa value ⩾7.9 is required for binding D-arginine or D-lysine, but not D-methionine or D-leucine. This group is E87, as suggested by its replacement with leucine. An unprotonated group with pKa of 9.5, which persists in the H48F and E87L variants, is required for amine oxidation with all substrates. Since Y53 and Y249 were previously ruled out, the pKa is assigned to the substrate α-NH3(+) group, which previous QM/MM and Kd pH-profile demonstrated to be protonated for preferred binding to the enzyme. Lack of pH effects on the (D)kred with D-leucine established 9.5 as the intrinsic pKa, and D-leucine as a non-sticky substrate. D-Arginine, D-lysine and D-methionine and their corresponding iminoproducts were significantly stickier than D-leucine, as indicated by apparent pKa values <9.5 in both kcat/Km and kcat. Restricted proton movements in catalysis were established from hollowed kcat pH profiles in wild-type PaDADH with D-lysine and in the H48F and E87L enzymes with D-arginine.

Study of the retention behavior of small polar molecules on different types of stationary phases used in hydrophilic interaction liquid chromatography.

The retention behavior of a large group of analytes (35) with varied properties (pKa and logP) was studied on eight hydrophilic interaction LC columns with different surfaces, stationary phase chemistries, and types of particles. The acetonitrile content (5-95%), buffer concentration (0.5-200 mM), and pH of the mobile phase (3.8 and 6.8) were evaluated for their effects on the retention behavior. The type of stationary phase had a significant impact on the selectivity and retention time of the tested analytes. Completely different selectivity was observed on the aminopropyl stationary phase. In this study, the influence of the buffer concentration was similar for all tested columns, except for the aminopropyl stationary phase. Increasing the buffer concentration led to decreased retention times for the basic compounds and increased retention times for the acidic compounds, while the inverse behavior was observed on the aminopropyl stationary phase. The selectivity of the individual stationary phases was evaluated at pH 3.8 and 6.8. Much lower selectivity differences between the stationary phases were observed at pH 6.8 than pH 3.8. Bare silica stationary phases were used in the comparison of the particles (fused-core and fully porous particles of 3 and 1.7 µm) and the columns provided by different manufacturers.

Rheological properties of Triumfetta cordifolia gum solutions in the concentrated regime.

The polyelectrolyte gum from Triumfetta cordifolia stem bark has recently come to the fore for its remarkable potential as an emulsifier and stabilizer for aqueous formulations. This paper presents the rheological study of T. cordifolia gum aqueous solutions in the concentrated regime (C > C** = 0.14 % w/w). To this end, both flow and oscillation tests were performed on T. cordifolia gum solutions at two distinct concentrations belonging to the concentrated regime: at 0.2 % w/w (close to C**) and at 0.7 % w/w (far above C**). The effect of temperature, pH and added salts (NaCl, CaCl2 and AlCl3) on gum viscoelastic parameters were investigated, revealing associative interactions. Under specific conditions, several remarkable and complex phenomena were observed, such as over-structuring induced by temperature, anti-thixotropy, gelation, syneresis and salting-out induced by salt addition. The charged structure of T. cordifolia gum (weak polyelectrolyte), the high divalent metal content and the presence of associative groups in its network were demonstrated as the major factors responsible for these phenomena. These findings form the basis for the structure-property relationships of T. cordifolia gum and may open up to further investigations for this gum of great potential in many fields of applications.

The Effect of Electrolyte pH and Impurities on the Stability of Electrolytic Bicarbonate Conversion.

Electrolytic bicarbonate conversion holds the promise to integrate carbon capture directly with electrochemical conversion. Most research has focused on improving the faradaic efficiencies of the system, however, the stability of the system has not been thoroughly addressed. Here, we find that the bulk electrolyte pH has a large effect on the selectivity, where a higher pH results in a lower selectivity. However, the bulk electrolyte pH has no effect on the stability of the system. A decrease in CO selectivity of 30% was observed within the first three hours of operation in an optimized system with 3 M KHCO3 and gap between the membrane and electrode. Single-pass electrolyte experiments at various constant pH values (8.5, 9.0, 9.5, and 10.0), show that only at a pH of 10 the CO selectivity was stable during three hours, reaching a faradaic efficiency toward CO of only 18% as compared to an initial 55% at pH 8.5. Trace metal impurities present in the electrolyte were found to be the cause of the decrease in stability as these deposit on the electrode surface. By complexing the trace metal ions with ethylenediaminetetraacetic acid (EDTA), the metal deposition was avoided and a stable CO selectivity was obtained.

Effect of buffers and pH in antenna sensitized Eu(III) luminescence.

The photophysics of a europium(III) complex of 1,4,7,10-tetraazacycododecane-1,4,7-triacetic acid-10-(2-methylene)-1-azathioxanthone was investigated in three buffer systems and at three pH values. The buffers-phosphate buffered saline (PBS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and universal buffer (UB)-had no effect on the europium luminescence, but a lower overall emission intensity was determined in HEPES. It was found that this was due to quenching of the 1-azathioxanthone first excited singlet state by HEPES. The effect of pH on the photophysics of the complex was found to be minimal, and protonation of the pyridine nitrogen was found to be irrelevant. Even so, pH was shown to change the intensity ratio between 1-azathioxanthone fluorescence and europium luminescence. It was concluded that the full photophysics of a potential molecular probe should be investigated to achieve the best possible results in any application.

The pH-sensitve oxygenation of FeS: Mineral transformation and immobilization of Cr(VI).

Iron sulfide (FeS) has been widely used to reduce toxic Cr(VI) into Cr(III) in anoxic aquatic environments, where pH could strongly influence Cr(VI) removal. However, it remains unclear how pH regulates the fate and transformation of FeS under oxic conditions and the immobilization of Cr(VI). The results of this study showed that typical pH conditions of natural aquatic environment significantly affected the mineral transformation of FeS. Under acidic conditions, FeS was principally transformed to goethite, amarantite, and elemental sulfur with minor lepidocrocite through proton-promoted dissolution and oxidation. Instead, under basic conditions, the main products were lepidocrocite and elemental sulfur via surface-mediated oxidation. In typical acidic or basic aquatic environment, the pronounced pathway for the oxygenation of FeS solids may alter their ability to remove Cr(VI). Longer oxygenation impeded Cr(VI) removal at acidic pH, and a decreasing ability to reduce Cr(VI) caused a drop in Cr(VI) removal performance. Cr(VI) removal decreased from 733.16 to 36.82 mg g-1 with the duration of FeS oxygenation increasing to 5760 min at pH 5.0. In contrast, newly generated pyrite from brief oxygenation of FeS improved Cr(VI) reduction at basic pH, followed by a drop in Cr(VI) removal performance due to the impaired reduction capacity with increasing to the complete oxygenation. Cr(VI) removal increased from 669.58 to 804.83 mg g-1 with increasing oxygenation time to 5 min and then decreased to 26.27 mg g-1 after the full oxygenation for 5760 min at pH 9.0. These findings provide insight into the dynamic transformation of FeS in oxic aquatic environments with various pHs and the impact on Cr(VI) immobilization.

Surface water H-bonding network is key controller of selenate adsorption on [012] α-alumina: An Ab-initio study.

Selenate adsorption onto metal oxide surfaces is a cost-effective method to remove the toxin from drinking water systems. However, the low selectivity of metal oxides requires frequent sorbent replacement. The design of selective adsorbents is stymied because the surface factors controlling selenate adsorption remain unknown. We calculate adsorption energies of selenate on the (012) α-Al2O3 surface using density functional theory to unravel the physics that controls adsorption. Our model is validated against experiment by correctly predicting selenate removal efficiency as a function pH. We find that the selenate adsorption energy on the anhydrous α-Al2O3 surface is surprisingly anti-correlated with the fully solvated adsorption energy; therefore, the direct interaction between adsorbate and sorbent is eliminated as the controlling mechanism. Rather, the change in number of surface hydrogen bonds after adsorption is the factor most correlated with the adsorption energy (R2 > 0.8); and is thus determined to be the factor controlling selenate adsorption. We find that pH affects adsorption by controlling the number of surface protons available for H-bonding to selenate. This work demonstrates that adsorption prediction should not be made based on gas phase sorption energies and suggests that surface engineering which increases surface protonation may be an effective strategy for increasing selenate sorption.

Rapid debromination of tetrabromobisphenol A by Cu/Fe bimetallic nanoparticles in water, its mechanisms, and genotoxicity after treatments.

Tetrabromobisphenol A (TBBPA), a widely used brominated flame retardants, has been detected in various environmental matrices and is known to cause various adverse effects on human bodies. This study examined the feasibility and effectiveness of remediating TBBPA using Cu/Fe bimetallic nanoparticles (Cu/Fe BNPs) at various environmental and operational conditions. In general, TBBPA removal rate and debromination efficiency increased with higher Cu doping, higher Cu/Fe BNPs loading, higher temperature, and lower pH. At optimal conditions, TBBPA was completed removed at a rate constant > 0.2 min-1 where over 90% TBBPA was transformed to BPA within 30 min. The activation energy was found to be 35.6 kJ/mol, indicating that TBBPA was predominantly removed via surface-controlled reactions. Under pH 3-7 and ≥ 25 °C, debromination was the dominant removal mechanism compared to adsorption. The complete debromination pathway and the time-evolution of intermediates byproducts at different pHs were also presented. Cu/Fe BNPs can be reused for more than 6 times with performance constancy. Genotoxic tests showed that the treated solution did not find a significant hazardous potential. The byproducts can be further degraded by additional H2O2 through Fenton reaction. These results demonstrated the efficacy of Cu/Fe BNPs for treating TBBPA and its potential for degrading other halogenated organic compounds.

Human Skin Permeation Enhancement Using PLGA Nanoparticles Is Mediated by Local pH Changes.

The steady improvement and optimization of transdermal permeation is a constant and challenging pharmaceutical task. In this study the influence of poly(lactide-co-glycolide) (PLGA) nanoparticles on the dermal permeation of the anti-inflammatory drug flufenamic acid (FFA) was investigated. For this aim, different vehicles under non-buffered and buffered conditions and different skin models (human heat separated epidermis and reconstructed human epidermis equivalents) were tested. Permeation experiments were performed using static Franz diffusion cells under infinite dosing conditions. Already the presence of drug-free nanoparticles increased drug permeation across the skin. Drug permeation was even enhanced when applying drug-loaded nanoparticles. In contrast, buffered vehicles with different pH values (pH 5.4-7.4) revealed the influence of the pH on the permeation of FFA. The change of the surrounding pH of the biodegradable nanoparticulate system was demonstrated and visualized using pH-sensitive fluorescent probes. While a potential contribution of hair follicles could be ruled out, our data suggest that the enhanced permeation of FFA through human skin in the presence of PLGA nanoparticles is mediated by a locally decreased pH during hydrolytic degradation of this polymer. This hypothesis is supported by the observation that skin permeation of the weak base caffeine was not affected.

On the clustering of bulk nanobubbles and their colloidal stability.

Bulk nanobubbles which are usually observed in pure water have a mean diameter typically around 100 nm. We use a combination of physical and chemical techniques to prove the hypothesis that the nanoentities observed in pure water are stable clusters of much smaller stable nanobubbles. The stability of bulk nanobubble clusters is affected by factors such as ionic strength or internal energy of the system. We show that bulk nanobubbles on the order of 100 nm exist in a stable cluster form in neutral or basic media, and dissociate into tiny primary nanobubbles on the order of 1 nm in acidic media, or in the presence of small amounts of salt. These new findings suggest that bulk nanobubbles which have a high surface energy unsurprisingly tend to behave in a similar manner to solid nanoparticles in terms of their agglomeration tendency, which is confirmed by the DLVO theory. The results will have important implications for our understanding and interpretation of the behaviour of bulk nanobubbles, in particular their interfacial and colloidal stability.

Choline oxidases.

Choline oxidase catalyzes the four-electron, two-step, flavin-mediated oxidation of choline to glycine betaine. The enzyme is important both for medical and biotechnological reasons, because glycine betaine is one among a limited number of compatible solutes used by cells to counteract osmotic pressure. From a fundamental standpoint, choline oxidase has emerged as one of the paradigm enzymes for the oxidation of alcohols catalyzed by flavoproteins. Mechanistic, structural, and computational studies have elucidated the mechanism of action of the enzyme from Arthrobacter globiformis at the molecular level. Both choline and oxygen access to the active site cavity are gated and tightly controlled. Amino acid residues involved in substrate binding, and their contribution, have been identified. The mechanism of choline oxidation, with a hydride transfer reaction, an asynchronous transition state, the formation and stabilization of an alkoxide transient species, and a quantum mechanical mode of reaction, has been elucidated. The importance of nonpolar side chains for oxygen localization and of the positive charge harbored on the substrate for activation of oxygen for reaction with the reduced flavin have been recognized. Interesting phenomena, like the formation of a metastable photoinduced flavin-protein adduct, the reversible formation of a bicovalent flavoprotein, and the trapping of the enzyme in inactive conformations, have been described. This review summarizes the current status of our understanding on the structure-function-dynamics of choline oxidase.

Oxidative Protein Folding Using trans-3,4-Dihydroxyselenolane Oxide.

trans-3,4-Dihydroxyselenolane oxide (DHSox), a water-soluble cyclic selenoxide reagent, is useful for rapid and quantitative formation of disulphide (SS) bonds in a reduced state of SS-containing proteins because the selenoxide is a strong but selective oxidant for thiol substrates (RSH) in a wide range of pH. Due to this advantage over common disulphide reagents, such as oxidized dithiothreitol (DTTox) and glutathione (GSSG), DHSox enables clear characterization of oxidative folding pathways of proteins. DHSox is also useful for facile diagnosis of weakly folded structure, or reactivity (i.e., pKa) of the thiols, present in a reduced polypeptide chain and the partially oxidized folding intermediates, identification of the key SS intermediates that can be oxidized directly to the native state, and preparation of SS-scrambled misfolded protein species. In this chapter, these diverse utilities of DHSox in protein folding study are demonstrated.