Disulphide Formation Research Articles

Cystine crystal nucleation and decay in the context of cystinuria pathogenesis and treatment.

Cystinuria is a rare disease which results in the precipitation of cystine in the renal filtrate, which may cause acute kidney injury due to mechanical trauma. In this work, we attempt to explore the origin of supersaturated cystine in this context to understand disease pathogenesis. This has enabled us to reproduce the clinical habit of cystine following a comprehensive study of cystine nucleation and growth in saline, artificial and human urine. Then, we describe the physical behaviour of these crystals in the presence of: cysteamine, sodium bicarbonate, captopril, tiopronin, penicillamine, glutathione and α-lipoic acid. Surprisingly, we observe that, in vitro, only cysteamine and saturated sodium bicarbonate dissolve crystals at a faster rate than saline, and that when solution pH is adjusted to physiological conditions, crystal dissolution for all agents is reduced to the rate of saline alone. We highlight that the conventional hypothesis of mixed disulphide formation in cysteamine is not the fastest mechanism of cystine dissolution, but rather that cystine dissolution (in the order of hours) is dominated by pH effects. This, combined with cysteamine's ability to take part in disulfide exchange reactions may explain cysteamine's effectiveness in this condition. Overall, our findings not only contribute to an understanding of cystinuria pathogenesis but also offer insights into how we should evaluate emerging treatments.

Enzymatic cross-linking of pea and whey proteins to enhance emulsifying and encapsulation properties

Improving the functionality of commercial plant proteins is essential for food application. Here an alternative strategy is provided to modify the structure of pea protein by microbial transglutaminase-induced cross-linking with whey protein for enhancing emulsifying and encapsulation properties. The protein structures and the physicochemical stability of β-carotene-loaded emulsions were investigated subject to protein ratio and cross-linking. Formation of disulphide and isopeptide bonds between heteroproteins according to cross-linking changed protein structures by exposing the hydrophobic sites of proteins with stiffer network. The cross-linked pea/whey protein complexes at protein ratio of 2:1 formed a stable emulsion with a droplet size of 0.10 µm, showing no phase separation for 30 days of storage and high encapsulation efficiency of 92%. These findings provided a novel strategy to design plant/dairy protein networks to protect lipophilic bioactive compounds by replacing half or more dairy protein with plant protein, leading to gradual change towards plant-based diets.

Effect of surfactant micellization on the oxidation of mercaptobenzothiazole by bioinorganic molybdenum complex

A spectrophotometric routine was used to study the effect of surfactant micellization on the oxidation of mercaptobenzothiazole (MBTZ) by bioinorganic molybdenum complex at 301 K and an absorption maxima of 560 nm. It follows an acid dependent oxidative pathway that is medium-sensitive. Charge distribution from the reaction species contributes to the redox retardation, resulting in a downturned primary salt effect with an unenhanced reaction rate. Alteration of the reaction medium with ethanol led to a lowering of oxidation time as the charge catalysis was promoted by a drop in the system permittivity. Likewise, the micellization of cetyltrimethylammonium bromide in the system increased the oxidation rate of the MBTZ due to the high impact of hydrophobic and ion interaction between the micelle and substrates. First order reaction kinetics was observed in the concentration of the redox partners and a 2:2 (complex: MBTZ) stoichiometry was obtained with the involvement of benzothiazyl radical, which resulted in the formation of benzothiazyl disulphide and a Mo2+ deactivated complex. The occurrence of counterion catalysis is pronounced in the reaction system. The standard enthalpy (31.52 ± 0.11 kJ mol−1) and Gibbs energy (74.54 ± 0.13) kJ mol−1 suggest that the process is endothermic dependent. The investigation of surfactant micellization effect on the reaction medium was quantitatively ascertained from the Piszkiewicz model of the complex interaction sequence.

DisCoTune: versatile auxiliary plasmids for the production of disulphide-containing proteins and peptides in the E.coli T7 system.

SummarySecreted proteins and peptides hold large potential both as therapeutics and as enzyme catalysts in biotechnology. The high stability of many secreted proteins helps maintain functional integrity in changing chemical environments and is a contributing factor to their commercial potential. Disulphide bonds constitute an important post‐translational modification that stabilizes many of these proteins and thus preserves the active state under chemically stressful conditions. Despite their importance, the discovery and applications within this group of proteins and peptides are limited by the availability of synthetic biology tools and heterologous production systems that allow for efficient formation of disulphide bonds. Here, we refine the design of two DisCoTune (Disulphide bond formation in E. coli with tunable expression) plasmids that enable the formation of disulphides in the highly popular Escherichia coli T7 protein production system. We show that this new system promotes significantly higher yield and activity of an industrial protease and a conotoxin, which belongs to a group of disulphide‐rich venom peptides from cone snails with strong potential as research tools and pharmacological agents.

The association of dynamic thiol-disulphide balance with white-coat hypertension.

Dynamic thiol-disulphide homeostasis is an indicator of the antioxidant system that interacts early with the oxidative environment. This study aimed to assess the dynamic thiol/disulphide balance in individuals with white-coat hypertension (WCH). This cross-sectional study included a total of 117 individuals who presented to our outpatient cardiology clinic. Seventy patients were diagnosed with WCH. All blood samples were obtained from the patients after a minimum of 8 h of fasting, centrifuged immediately, stored in Eppendorf tubes, and protected at -80°C. The two groups were well-balanced in terms of age, gender, and BMI. Native thiol and total thiol levels were lower in the WCH group than in normotensive individuals. However, disulphide levels were higher in the former. Serum disulphide concentration positively correlated with 24-h SBP, and 24-h DBP. In backward stepwise logistic regression analysis, serum disulphide [odds ratio (OR) = 1.165 (1.089-1.245), 95% confidence interval (CI), P < 0.001] and serum total thiol levels [OR = 0.992 (0.984-1.000), 95% CI, P = 0.050] were independently associated with WCH. We demonstrated that dynamic thiol/disulphide balance shifted strongly towards disulphide formation due to thiol oxidation in patients with WCH.

Association between Malondialdehyde, GSH/GSSG Ratio and Bone Mineral Density in Postmenopausal Women

Background: Osteoporosis is one of chronic degenerative diseases especially in postmenopausal women, characterized by a decreased bone mass due to imbalance activity between osteoblasts and osteoclasts. Recently, oxidative stress is believed to play an important role in osteoporosis pathogenesis. Oxidative stress is commonly considered as the consequence of an imbalance between pro and antioxidants species, which results in damage in the affected tissue. Malondialdehyde (MDA) is frequently used as a biomarker of oxidative stress in many health problems since MDA is produced at high levels during lipid peroxidation. Meanwhile, glutathione is well known as one of antioxidant which against oxidative stress by preserving its homeostasis in the reduced form of glutathione sulfhydryl (GSH) and the oxidized form of glutathione disulphide (GSSG). This study was aimed to determine the association between MDA, GSH/GSSG ratio and bone mineral density (BMD) in postmenopausal women.Materials and method: We conducted a cross-sectional study in 40 postmenopausal women. MDA and GSH/GSSG ratio were assessed by enzyme-linked immunosorbent assay (ELISA). Bone mineral density (BMD) was obtained from secondary data. The statistical analysis was conducted using Spearman rho’s correlation test.Results: Based on the test, we didn’t found significant correlation between MDA and BMD (r=-0.054, p=0.741), but we found significant moderate correlation between GSH/GSSG ratio (r=0.436, p=0.005) and BMD in postmenopausal women. Conclusion: There was no correlation between MDA and BMD in postmenopausal women. However, there was significant moderate correlation between GSH/GSSG ratio and BMD in postmenopausal women.Keywords: MDA, GSH/GSSG ratio, BMD, osteoporosis

Chlorophyll Catalysed Ultrafast Oxidation of Thiols in Water

AbstractWe have developed an efficient and eco‐friendly method of disulfides synthesis in water using surfactant (DODAB). This reaction is catalysed by chlorophyll and fluorescent lamp is used as source of energy. This, one pot, ultra fast reaction, which involve a photo‐induced electron transfer (PET) mechanism, affords a green and efficient strategy for the conversion of commercially available aromatic thiols into diversified disulfides. This rapid synthesis allows the formation of various disulphides in the range of 80–94% yield at room temperature.

Identification, Expression, and Roles of the Cystine/Glutamate Antiporter in Ocular Tissues.

The cystine/glutamate antiporter (system xc−) is composed of a heavy chain subunit 4F2hc linked by a disulphide bond to a light chain xCT, which exchanges extracellular cystine, the disulphide form of the amino acid cysteine, for intracellular glutamate. In vitro research in the brain, kidney, and liver have shown this antiporter to play a role in minimising oxidative stress by providing a source of intracellular cysteine for the synthesis of the antioxidant glutathione. In vivo studies using the xCT knockout mouse revealed that the plasma cystine/cysteine redox couple was tilted to a more oxidative state demonstrating system xc− to also play a role in maintaining extracellular redox balance by driving a cystine/cysteine redox cycle. In addition, through import of cystine, system xc− also serves to export glutamate into the extracellular space which may influence neurotransmission and glutamate signalling in neural tissues. While changes to system xc− function has been linked to cancer and neurodegenerative disease, there is limited research on the roles of system xc− in the different tissues of the eye, and links between the antiporter, aging, and ocular disease. Hence, this review seeks to consolidate research on system xc− in the cornea, lens, retina, and ocular humours conducted across several species to shed light on the in vitro and in vivo roles of xCT in the eye and highlight the utility of the xCT knockout mouse as a tool to investigate the contribution of xCT to age-related ocular diseases.

Design and in silico screening of aryl allyl mercaptan analogs as potential histone deacetylases (HDAC) inhibitors

The Zn+2 HDACIs show promising anticancer activity. Allyl mercaptan (AM), a metastabilzed monomeric form of diallyl disulphide (DADS) shows better HDACI activity. The present work screens a dataset of aryl AM derivatives 1(a-g) for potential HDACI action viain silico models. DFT calculations predicted the geometrical parameters and frontier orbital calculations suggested better chemical reactivity. Negative chemical potential and NBO hyper conjugative interactions predicted their chemical stability. ADME study confirmed favourable drug likeliness. Molecular docked models suggested the formation of coordinate bond between sulphur of allylmercaptan and Zn2+ cofactor of HDAC8. Besides, models also predicted the dominance of hydrophobic interactions. The aryl AM analogs docked perfectly with HDAC3 as well. The glide score and S-Zn distance of compounds 1a, 1f and 1g were found to be better than allylmercaptan. Therefore, the designed aryl AM analogs filtered as better HDACIs. These could be further used for design and synthesis of new improved HDACIs.

The association of thiol/disulphide homeostasis with 6-month mortality in patients with acute st-elevation myocardial infarction

Aim: Acute ST-elevation myocardial infarction (STMI) is significant determinant of mortality and long-term mortality rates has not changed significantly over the last two decades. Thiols are an important anti-oxidant mechanism in the body, and excessive reactive oxygen species oxidize the thiol into their disulphide forms. Hence, higher level of disulphide is regarded as an indicator of oxidative stress. In the present paper, we sought to investigate any probable relationship between thiol/disulphide homeostasis and 6-month all-cause mortality among STMI survivors.Material and Methods: 238 consecutive patients with STMI undergoing percutaneous coronary intervension were included. Blood samples for thiol/disulphide homeostasis were drawn on admission. Syntax I and II scores were calculated using the cineangiographic views and relevant patient demographics. Results: 6-month mortality occurred in 25 patients (mortality group), while 213 patients remained alive (non-mortality group). Total thiol, native thiol, disulphide, disulphide/total thiol , disulphide/native thiol and native thiol/total thiol were not different between the mortality and non-mortality groups (p&amp;amp;gt;0.05). Syntax I and II scores were significantly greater in mortality group (26.11±8.06 vs 19.129.80, p=0.029 for Syntax I; 50.1314.59 vs 30.639.79, p&amp;amp;lt;0.001 for Syntax II). Conclusion: Syntax II score, glomerular filtration rate (GFR) and white blood cell count (WBC) were found to predict 6-month mortality.

Oxidation of cysteine 34 of plasma albumin as a biomarker of oxidative stress

Introduction: In order to better understand the physiological and pathophysiological roles of reactive oxygen species (ROS), multiple blood and urine biomarkers of oxidative stress have been developed. The single free thiol (Cys34) in plasma albumin is a useful biomarker of oxidative stress because thiol groups are particularly sensitive to oxidation by ROS. The primary aim of this study was to develop a gel electrophoresis-based method (mPEG assay) that would be more widely accessible than existing chromatography techniques to assay the oxidation state of albumin Cys34.Method: Blood samples were collected into a solution containing polyethylene glycol maleimide (malpeg). Plasma samples were divided into two aliquots, with a reducing agent added to one aliquot. Albumin bound to malpeg was separated from albumin by gel electrophoresis. The proportion of albumin in reduced form (–SH), disulphide form (–SSX) and irreversibly oxidised form (–SO2, –SO3) could then be calculated.Results: Data for the mPEG assay was comparable to data from chromatographic and mass spectrometric assays. The mPEG assay was more sensitive than the albumin carbonyl assay for the detection of changes in albumin oxidation level in response to exposure to hydrogen peroxide or hypochlorous acid. This assay could also be performed on small blood samples (less than 10 µL) from fingerprick, thus facilitating longitudinal tracking of changes in albumin Cys34 oxidation level.Conclusion: The mPEG assay is a user-friendly, highly sensitive, specific, cost-effective gel electrophoresis-based method for the assay of the oxidations state of albumin Cys34 as a biomarker of oxidative stress.HighlightsProtein thiol groups are sensitive to oxidation by reactive oxygen species.Plasma albumin contains a reduced cysteine residue (Cys34) sensitive to oxidation.A novel gel electrophoresis-based method (mPEG) has been developed to measure the oxidation state of Cys34.The mPEG assay can be run on a drop of blood collected by fingerprick.

THE INFLUENCE OF TEMPERATURE ON THE RHEOLOGICAL PROPERTIES OF FIBROUS LUBRICATING COMPOUNDS WITH SELECTED SOLID ADDITIVES

The study evaluated the effect of temperature on the values of yield stress and cohesion energy of lubricating compounds thickened with calcium 12-hydroxystearate, prepared on the basis of mineral oil. Lubricating compounds without additives and with selected solid additives in the form of copper particles, graphite, molybdenum disulphide, and polytetrafluoroethylene were analysed. A rotational rheometer working with a plate-plate geometry was used for rheological studies. The influence of temperature on the rheological properties of lubricating compounds with additives was observed. Solid additives caused significant changes in the structure of the soap thickener, especially at negative temperatures.

Caution for the routine use of phenol red – It is more than just a pH indicator

Phenol red (PR) is the standard pH indicator in various cell and tissue culture media, as it provides a quick check for the health of the culture. PR has also been used in multiple protocols to detect cellular hydrogen peroxide as well as peroxidase activity from human peroxidase enzymes. The majority of promyelocytic leukemia cell lines (e.g. HL-60 cells) express myeloperoxidase (MPO), which may react with PR, especially as the latter is present in cell culture media at sufficient concentrations (~15 μM) to partake in redox reactions. Moreover, phenolic molecules are often efficient donor substrates for peroxidase enzymes. In this study, we hypothesized that MPO metabolism of PR via MPO-expressing HL-60 cells could result in PR metabolite(s) that could modulate cell viability. We used purified human MPO for UV–visible spectrophotometry, electron paramagnetic resonance (EPR) and LC-MS analyses to investigate PR peroxidation. 2-chloro-5,5-dimethyl-1,3-cyclohexanedione (monochloro-dimedone, MCD) was used to assess the effect of PR on MPO-catalyzed chlorination activity, and we assessed PR uptake by HL-60 cells using LC-MS analysis. Lastly, we investigated the impact of PR metabolism by intracellular MPO on cell viability (ATP, using CellTiter-Glo®), cytotoxicity (using trypan blue), and on reduced and oxidized glutathione (using GSH/GSSG-Glo™). Our results demonstrate that PR undergoes oxidative halogenation via MPO, resulting in its UV–vis spectral changes due to the formation of mono- and di-halogenated products. Moreover, a significant increase in MPO-catalyzed chlorination of MCD and an increase in glutathionyl radical detection (using EPR) were observed in the presence of PR. Our in-vitro studies revealed that PR is readily taken up by HL-60 cells and its metabolism by intracellular MPO leads to a significant decrease in cellular glutathione as well as a significant increase in glutathione disulphide formation. In spite of the latter, PR had no considerable effect on HL-60 cell viability. These results provide evidence that while no overt decrease in cell viability may be observed, PR does impart redox activity, which investigators should be wary of in experimental protocols.

Thiol-disulphide homeostasis in noncomplicated chronic otitis media

Introduction: We hypothesized that oxidative stress plays a role in the pathogenesis of chronic otitis media, chronic mucosal inflammatory disease. We aimed to investigate a novel oxidative stress marker in this study. Materıals and methods: Thirty patient with chronic otitis media as the patient group and 30 healthy volunteer subjects as the control group were admitted to the study. Blood samples were taken when they admitted to our clinic before surgical intervention. In healthy volunteers, blood samples were taken when they were admitted to our policlinic. Thiol/disulphide levels were analyzed with a newly developed method by Erel and Neselioglu.Results: 30 subjects were included in chronic otitis media group (20 females, 10 males). 30 subjects were included in the control group (8 females and 22 males). Sex distribution within the groups was significantly different (p=0.004). There was no significant difference between groups with respect to the age distribution (p=0.072). Measured native thiol, disulphide and total thiol values of the groups were not significantly different from each other. Conclusion: This study demonstrated that although dynamic thiol/disulphide homeostasis was shifted towards disulphide formation as a result of thiol oxidation in patients with chronic otitis media. But we could not find any significant difference between groups.

An Investigation of Oxidative Stress and Thiol/Disulphide Homeostasis in Graves' Disease.

Background and objectives: The aim of this study was to research oxidative stress and thiol/disulphide homeostasis in Graves’ patients. Materials and Methods: The study included 33 Graves’ patients (research group) and 35 healthy subjects (control group). Serum oxidative stress and thiol/disulphide homeostasis (a new and automated spectrophotometric method developed by Erel and Neselioglu) parameters were studied and compared between the groups. Results: The native and total thiol levels and the native thiol/total thiol ratio were lower in patients with Graves’ disease compared to the control group (p < 0.001, p < 0.001, and p = 0.006, respectively). TOS (total antioxidant status), PC (protein carbonyl), OSI (Oxidative stress index), and disulphide/native thiol and disulphide/total thiol ratios were determined to be higher in the Graves’ disease group than in the control group (p < 0.001, p = 0.001, p = 0.001, p = 0.004, and p = 0.006, respectively). In the Graves’ disease group, the free triiodothyronine (FT3) and free thyroxine (FT4) levels were significantly positively correlated with impaired thiol/disulphide homeostasis and oxidative stress parameters (p < 0.05). Conclusion: The results of the current study demonstrated that oxidative stress and thiol/disulphide homeostasis increased towards disulphide formation due to thiol oxidation in Graves’ disease. In addition, a positive correlation of FT3 and FT4 was observed with oxidative stress parameters and impaired thiol/disulphide homeostasis.

Light-induced reactions of methionine and riboflavin in model wine: Effects of hydrolysable tannins and sulfur dioxide

The riboflavin-mediated photo-degradation of methionine in white wine has been related to onset of undesired light-struck taste. This research investigated the effects of different concentrations of riboflavin and methionine, hydrolysable tannins from various sources (nut galls, chestnut and oak woods) and sulfur dioxide on methionine degradation in a model wine exposed to light. Increased methionine concentration resulted in its increased degradation with the consequent formation of volatile sulfur compounds, namely methanethiol, dimethyl disulphide and dimethyl trisulphide. Tannins, especially nut gall tannin, were effective in limiting both methionine degradation and the production of volatile sulfur compounds. Sulfur dioxide enhanced the methionine degradation although the light-struck taste was not perceived when sulfur dioxide concentration was higher than 50 mg/L. In conclusion, the use of hydrolysable tannins can represent a promising tool for protecting white wine against the light-struck taste also limiting the use of sulfur dioxide.

Thiol-disulphide balance in infertility secondary to varicocele.

Oxidative stress plays an important role in the development of infertility secondary to varicocele. We aimed to investigate the dynamic thiol-disulphide homeostasis as an oxidative stress marker in the spermatic vein of infertility secondary to varicocele. Sixty-one patients with varicocele were included in the study. Blood was drawn from the median cubital vein and the testicular venous return side before the spermatic vein was separated during surgery. Total thiol, native thiol, disulphide, ischaemia modified albumin (IMA) and albumin values were measured from both the dilated spermatic vein and the median cubital vein. The disulphide/native thiol, disulphide/total thiol and native thiol/total thiol ratios were determined. The mean age of the patients was 27.0±6.68 (15-50) years. While the albumin, native thiol and total thiol values and the native thiol/total thiol ratio were significantly lower (p=0.004, p<0.001, p<0.001, p<0.001 respectively), the IMA value and the disulphide/native thiol and disulphide/total thiol ratios were significantly higher (p<0.001, p<0.001, p<0.001 respectively) in the samples taken from spermatic venous blood. Thiol-disulphide balance had deteriorated towards disulphide formation in the spermatic vein compared with the peripheral vein. Abnormal thiol-disulphide balance may be an independent risk factor for infertility secondary to varicocele.

Molecular recognition and maturation of SOD1 by its evolutionarily destabilised cognate chaperone hCCS.

Superoxide dismutase-1 (SOD1) maturation comprises a string of posttranslational modifications which transform the nascent peptide into a stable and active enzyme. The successive folding, metal ion binding, and disulphide acquisition steps in this pathway can be catalysed through a direct interaction with the copper chaperone for SOD1 (CCS). This process confers enzymatic activity and reduces access to noncanonical, aggregation-prone states. Here, we present the functional mechanisms of human copper chaperone for SOD1 (hCCS)–catalysed SOD1 activation based on crystal structures of reaction precursors, intermediates, and products. Molecular recognition of immature SOD1 by hCCS is driven by several interface interactions, which provide an extended surface upon which SOD1 folds. Induced-fit complexation is reliant on the structural plasticity of the immature SOD1 disulphide sub-loop, a characteristic which contributes to misfolding and aggregation in neurodegenerative disease. Complexation specifically stabilises the SOD1 disulphide sub-loop, priming it and the active site for copper transfer, while delaying disulphide formation and complex dissociation. Critically, a single destabilising amino acid substitution within the hCCS interface reduces hCCS homodimer affinity, creating a pool of hCCS available to interact with immature SOD1. hCCS substrate specificity, segregation between solvent and biological membranes, and interaction transience are direct results of this substitution. In this way, hCCS-catalysed SOD1 maturation is finessed to minimise copper wastage and reduce production of potentially toxic SOD1 species.

Dynamic Thiol/Disulphide Homeostasis in Children with Nephrotic Syndrome

Background: There is a growing body of evidence showing that there is an association between oxidative damage and kidney injury in patients with nephrotic syndrome (NS). Dynamic thiol/disulphide homeostasis (DTDH) is an important antioxidant system. The aim of this study was to determine if DTDH could be used as a biomarker of antioxidant status in pediatric NS patients using a novel automated method. Methods: The study included 39 patients with NS and 40 healthy controls. The DTDH level was measured using a calorimetric and automated method developed by Erel and Neselioglu. In the NS group, DTDH was measured during first-attack episode, relapse, and remission. Results: The native thiol, total thiol, and disulphide levels were significantly lower, the native thiol/total thiol ratio was significantly lower, and the disulphide/native thiol and disulphide/total thiol ratios were significantly higher in the NS patients during relapse and first-attack episode than during remission and in the controls. In addition, the native thiol and total thiol levels, and the native thiol/total thiol ratio were significantly lower, and the disulphide/native thiol and disulphide/total thiol ratios were significantly higher in the NS group during remission than those in the control group. Conclusions: The present findings show that DTDH shifted toward disulphide formation in the NS patients, even during remission. A clearer understanding of the mechanism of this disruption to homeostasis in NS patients might lead to the development of novel therapeutic strategies.

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.