Thiol dioxygenases: from structures to functions.

Thiol oxidation to dioxygenated sulfinic acid is catalyzed by an enzyme family characterized by a cupin fold. These proteins act on free thiol-containing molecules to generate central metabolism precursors and signaling compounds in bacteria, fungi, and animal cells. In plants and animals, they also oxidize exposed N-cysteinyl residues, directing proteins to proteolysis. Enzyme kinetics, X-ray crystallography, and spectroscopy studies prompted the formulation and testing of hypotheses about the mechanism of action and the different substrate specificity of these enzymes. Concomitantly, the physiological role of thiol dioxygenation in prokaryotes and eukaryotes has been studied through genetic and physiological approaches. Further structural characterization is necessary to enable precise and safe manipulation of thiol dioxygenases (TDOs) for therapeutic, industrial, and agricultural applications.

The Michael addition of thiols to 13-oxo-octadecadienoate (13-oxo-ODE) with implications for LC-MS analysis of glutathione conjugation.

Unsaturated fatty acid ketones with αß,γδ conjugation are susceptible to Michael addition of thiols, with unresolved issues on the site of adduction and precise structures of the conjugates. Herein we reacted 13-keto-octadecadienoic acid (13-oxo-ODE or 13-KODE) with glutathione (GSH), N-acetyl-cysteine, and ß-mercaptoethanol and identified the adducts. HPLC-UV analyses indicated none of the products exhibit a conjugated enone UV chromophore, a result that conflicts with the literature and is relevant to the mass spectral interpretation of 1,4 versus 1,6 thiol adduction. Aided by the development of an HPLC solvent system that separates the GSH diastereomers and thus avoids overlap of signals in proton NMR experiments, we established the two major conjugates are formed by 1,6 addition of GSH at the 9-carbon of 13-oxo-ODE with the remaining double bond α to the thiol in the 10,11 position. N-acetyl cysteine reacts similarly, while ß-mercaptoethanol gives equal amounts of 1,4 and 1,6 addition products. Equine glutathione transferase catalyzed 1,6 addition of GSH to the two major diastereomers in 44:56 proportions. LC-MS in positive ion mode gives a product ion interpreted before as evidence of 1,4-thiol adduction, whereas here we find this ion using the authentic 1,6 adduct. LC-MS with negative ion APCI gave a fragment selective for 1,4 adduction. These results clarify the structures of thiol conjugates of a prototypical unsaturated keto-fatty acid and have relevance to the application of LC-MS for the structural analysis of keto-fatty acid glutathione conjugation.

Acidity of persulfides and its modulation by the protein environments in sulfide quinone oxidoreductase and thiosulfate sulfurtransferase.

Persulfides (RSSH/RSS-) participate in sulfur metabolism and are proposed to transduce hydrogen sulfide (H2S) signaling. Their biochemical properties are poorly understood. Herein, we studied the acidity and nucleophilicity of several low molecular weight persulfides using the alkylating agent, monobromobimane. The different persulfides presented similar pKa values (4.6-6.3) and pH-independent rate constants (3.2-9.0 × 103 M-1 s-1), indicating that the substituents in persulfides affect properties to a lesser extent than in thiols because of the larger distance to the outer sulfur. The persulfides had higher reactivity with monobromobimane than analogous thiols and putative thiols with the same pKa, providing evidence for the alpha effect (enhanced nucleophilicity by the presence of a contiguous atom with high electron density). Additionally, we investigated two enzymes from the human mitochondrial H2S oxidation pathway that form catalytic persulfide intermediates, sulfide quinone oxidoreductase and thiosulfate sulfurtransferase (TST, rhodanese). The pH dependence of the activities of both enzymes was measured using sulfite and/or cyanide as sulfur acceptors. The TST half-reactions were also studied by stopped-flow fluorescence spectroscopy. Both persulfidated enzymes relied on protonated groups for reaction with the acceptors. Persulfidated sulfide quinone oxidoreductase appeared to have a pKa of 7.8 ± 0.2. Persulfidated TST presented a pKa of 9.38 ± 0.04, probably due to a critical active site residue rather than the persulfide itself. The TST thiol reacted in the anionic state with thiosulfate, with an apparent pKa of 6.5 ± 0.1. Overall, our study contributes to a fundamental understanding of persulfide properties and their modulation by protein environments.

Caveat fluorophore: an insiders' guide to small-molecule fluorescent labels.

The last three decades have brought a revolution in fluorescence microscopy. The development of new microscopes, fluorescent labels and analysis techniques has pushed the frontiers of biological imaging forward, moving from fixed to live cells, from diffraction-limited to super-resolution imaging and from simple cell culture systems to experiments in vivo. The large and ever-evolving collection of tools can be daunting for biologists, who must invest substantial time and effort in adopting new technologies to answer their specific questions. This is particularly relevant when working with small-molecule fluorescent labels, where users must navigate the jargon, idiosyncrasies and caveats of chemistry. Here, we present an overview of chemical dyes used in biology and provide frank advice from a chemist's perspective.

Peptide ligation by chemoselective aminonitrile coupling in water.

Amide bond formation is one of the most important reactions in both chemistry and biology1-4, but there is currently no chemical method of achieving α-peptide ligation in water that tolerates all of the 20 proteinogenic amino acids at the peptide ligation site. The universal genetic code establishes that the biological role of peptides predates life's last universal common ancestor and that peptides played an essential part in the origins of life5-9. The essential role of sulfur in the citric acid cycle, non-ribosomal peptide synthesis and polyketide biosynthesis point towards thioester-dependent peptide ligations preceding RNA-dependent protein synthesis during the evolution of life5,9-13. However, a robust mechanism for aminoacyl thioester formation has not been demonstrated13. Here we report a chemoselective, high-yielding α-aminonitrile ligation that exploits only prebiotically plausible molecules-hydrogen sulfide, thioacetate12,14 and ferricyanide12,14-17 or cyanoacetylene8,14-to yield α-peptides in water. The ligation is extremely selective for α-aminonitrile coupling and tolerates all of the 20 proteinogenic amino acid residues. Two essential features enable peptide ligation in water: the reactivity and pKaH of α-aminonitriles makes them compatible with ligation at neutral pH and N-acylation stabilizes the peptide product and activates the peptide precursor to (biomimetic) N-to-C peptide ligation. Our model unites prebiotic aminonitrile synthesis and biological α-peptides, suggesting that short N-acyl peptide nitriles were plausible substrates during early evolution.

scSLAM-seq reveals core features of transcription dynamics in single cells.

Single-cell RNA sequencing (scRNA-seq) has highlighted the important role of intercellular heterogeneity in phenotype variability in both health and disease1. However, current scRNA-seq approaches provide only a snapshot of gene expression and convey little information on the true temporal dynamics and stochastic nature of transcription. A further key limitation of scRNA-seq analysis is that the RNA profile of each individual cell can be analysed only once. Here we introduce single-cell, thiol-(SH)-linked alkylation of RNA for metabolic labelling sequencing (scSLAM-seq), which integrates metabolic RNA labelling2, biochemical nucleoside conversion3 and scRNA-seq to record transcriptional activity directly by differentiating between new and old RNA for thousands of genes per single cell. We use scSLAM-seq to study the onset of infection with lytic cytomegalovirus in single mouse fibroblasts. The cell-cycle state and dose of infection deduced from old RNA enable dose-response analysis based on new RNA. scSLAM-seq thereby both visualizes and explains differences in transcriptional activity at the single-cell level. Furthermore, it depicts 'on-off' switches and transcriptional burst kinetics in host gene expression with extensive gene-specific differences that correlate with promoter-intrinsic features (TBP-TATA-box interactions and DNA methylation). Thus, gene-specific, and not cell-specific, features explain the heterogeneity in transcriptomes between individual cells and the transcriptional response to perturbations.

Thiol-based chemical probes exhibit antiviral activity against SARS-CoV-2 via allosteric disulfide disruption in the spike glycoprotein.

The development of small-molecules targeting different components of SARS-CoV-2 is a key strategy to complement antibody-based treatments and vaccination campaigns in managing the COVID-19 pandemic. Here, we show that two thiol-based chemical probes that act as reducing agents, P2119 and P2165, inhibit infection by human coronaviruses, including SARS-CoV-2, and decrease the binding of spike glycoprotein to its receptor, the angiotensin-converting enzyme 2 (ACE2). Proteomics and reactive cysteine profiling link the antiviral activity to the reduction of key disulfides, specifically by disruption of the Cys379-Cys432 and Cys391-Cys525 pairs distal to the receptor binding motif in the receptor binding domain (RBD) of the spike glycoprotein. Computational analyses provide insight into conformation changes that occur when these disulfides break or form, consistent with an allosteric role, and indicate that P2119/P2165 target a conserved hydrophobic binding pocket in the RBD with the benzyl thiol-reducing moiety pointed directly toward Cys432. These collective findings establish the vulnerability of human coronaviruses to thiol-based chemical probes and lay the groundwork for developing compounds of this class, as a strategy to inhibit the SARS-CoV-2 infection by shifting the spike glycoprotein redox scaffold.

Silicon-Containing Thiol-Specific Bioconjugating Reagent.

A new bioconjugation reagent containing silicon has been developed for the selective reaction with thiols. The inclusion of silicon significantly improves chemoselectivity and suppresses retro processes, thereby exceeding the capabilities of traditional reagents. The method is versatile and compatible with a broad range of thiols and unsaturated carbonyl compounds and yields moderate to high results. These reactions can be conducted under biocompatible conditions, thereby making them suitable for protein bioconjugation. The resulting conjugates display good stability in the presence of various biomolecules, which suggests their potential application for the synthesis of antibody-drug conjugates. Furthermore, the presence of a silicon moiety within the conjugated products opens up new avenues for drug release and bridging inorganics with other disciplines. This new class of silicon-containing thiol-specific bioconjugation reagents has significant implications for researchers working in bioanalytical science and medicinal chemistry and leads to innovative opportunities for advancing the field of bioconjugation research and medicinal chemistry.

Site-Selective Polyfluoroaryl Modification and Unsymmetric Stapling of Unprotected Peptides.

Peptide stapling is recognized as an effective strategy for improving the proteolytic stability and cell permeability of peptides. In this study, we present a novel approach for the site-selective unsymmetric perfluoroaryl stapling of Ser and Cys residues in unprotected peptides. The stapling reaction proceeds smoothly under very mild conditions, exhibiting a remarkably rapid reaction rate. It can furnish stapled products in both liquid and solid phases, and the presence of nucleophilic groups other than Cys thiol within the peptide does not impede the reaction, resulting in uniformly high yields. Importantly, the chemoselective activation of Ser ß-C(sp3)-H enables the unreacted -OH to serve as a reactive handle for subsequent divergent modification of the staple moiety with various therapeutic functionalities, including a clickable azido group, a polar moiety, a lipid tag, and a fluorescent dye. In our study, we have also developed a visible-light-induced chemoselective C(sp3)-H polyfluoroarylation of the Ser ß-position. This reaction avoids interference with the competitive reaction of Ser -OH, enabling the precise late-stage polyfluoroarylative modification of Ser residues in various unprotected peptides containing other highly reactive amino acid residues. The biological assay suggested that our peptide stapling strategy would potentially enhance the proteolytic stability and cellular permeability of peptides.

The Nitrile Bis-Thiol Bioconjugation Reaction.

Electron-poor aryl nitriles are promising reagents for bioconjugation due to their high electrophilicity and selectivity for reaction with thiols, albeit generally in a reversible manner. A transient species has previously been observed in such reactions, involving the addition of two thiols to the nitrile functional group, forming a tetrahedral amino dithioacetal (ADTA). In this work, the reaction of heteroaryl nitriles with bis-thiols is explored in an attempt to generate stable ADTAs, which could facilitate new bioconjugation protocols. By use of a 1,2-dithiol, or the incorporation of an electrophilic trap into the aryl nitrile design, the formation of stable products is achieved. The resultant "nitrile bis-thiol" (NBT) reaction is then explored in the context of protein modification, specifically to carry out antibody conjugation. By addition of these nitriles to the reduced disulfide bond of an antibody fragment, it is shown that, depending on the reagent design, cysteine-to-lysine transfer or disulfide bridged NBT products can be generated. Both represent site-selective conjugates and are shown to be stable when challenged with glutathione under physiological conditions and upon incubation in serum. Furthermore, the NBT reaction is tested in the more challenging context of a full antibody, and all four disulfide bonds are effectively modified by these new one-carbon bridging reagents. Overall, this reaction of heteroaryl-nitriles with bis-thiols is shown to be highly efficient and versatile, of tunable reversibility, and offers enticing prospects as a new addition to the toolbox of biocompatible "click"-type reactions.

Transpersulfidation or H2S Release? Understanding the Landscape of Persulfide Chemical Biology.

Persulfides (RSSH) are biologically important reactive sulfur species that are endogenously produced, protect key cysteine residues from irreversible oxidation, and are important intermediates during different enzymatic processes. Although persulfides are stronger nucleophiles than their thiol counterparts, persulfides can also act as electrophiles in their neutral, protonated form in specific environments. Moreover, persulfides are electrophilic at both sulfur atoms, and the reaction with a thiolate can lead to either H2S release with disulfide formation or alternatively result in transpersulfidation. Despite the broad acceptance of these reaction pathways, the specific properties that control whether persulfides react through the H2S-releasing or transpersulfidation pathway remain elusive. Herein, we use a combined computational and experimental approach to directly investigate the reactivity between persulfides and thiols to answer these questions. Using density functional theory (DFT) calculations, we demonstrate that increasing steric bulk or electron withdrawal near the persulfide can shunt persulfide reactivity through the transpersulfidation pathway. Building from these insights, we use a synthetic persulfide donor and an N-iodoacetyl l-tyrosine methyl ester (TME-IAM) trapping agent to experimentally monitor and measure transpersulfidation from a bulky penicillamine-based persulfide to a cysteine-based thiol, which, to the best of our knowledge, is the first direct observation of transpersulfidation between low-molecular-weight species. Taken together, these combined approaches highlight how the properties of persulfides are directly impacted by local environments, which has significant impacts in understanding the complex chemical biology of these reactive species.

Piperazine-Fused Cyclic Disulfides Unlock High-Performance Bioreductive Probes of Thioredoxins and Bifunctional Reagents for Thiol Redox Biology.

We report piperazine-fused six-membered-cyclic disulfides as redox substrates that unlock best-in-class bioreduction probes for live cell biology, since their self-immolation after reduction is unprecedentedly rapid. We develop scalable, diastereomerically pure, six-step syntheses that access four key cis- and trans-piperazine-fused cyclic dichalcogenides without chromatography. Fluorogenic redox probes using the disulfide piperazines are activated >100-fold faster than the prior art monoamines, allowing us to deconvolute reduction and cyclization rates during activation. The cis- and trans-fused diastereomers have remarkably different reductant specificities, which we trace back to piperazine boat/chair conformation effects: the cis-fused disulfide C-DiThia is activated only by strong vicinal dithiol reductants, but the trans-disulfide T-DiThia is activated even by moderate concentrations of monothiols such as GSH. Thus, in cellular applications, cis-disulfide probes selectively report on the reductive activity of the powerful thioredoxin proteins, while trans-disulfides are rapidly but promiscuously reactive. Finally, we showcase late-stage diversifications of the piperazine-disulfides, promising their broad applicability as redox-cleavable cores for probes and prodrugs that interface powerfully with cellular thiol/disulfide redox biology, for solid phase synthesis and purification, and for stimulus-responsive linkers in bifunctional reagents and antibody-drug conjugates - in addition to their dithiols' potential as high-performance reducing agents.

Identification of Serine-Containing Microcystins by UHPLC-MS/MS Using Thiol and Sulfoxide Derivatizations and Detection of Novel Neutral Losses.

Microcystins (MCs) are hepatotoxic cyclic heptapeptides produced by cyanobacteria, and their structural diversity has led to the discovery of more than 300 congeners to date. However, with known amino acid combinations, many more MC congeners are theoretically possible, suggesting many remain unidentified. Herein, two novel serine (Ser)-containing MCs were putatively identified in a Lake Erie cyanobacterial harmful algal bloom (cyanoHAB), using high-resolution UHPLC-MS as well as thiol and sulfoxide derivatization procedures. These MCs contain an α,ß-unsaturated carbonyl on methyl dehydroalanine (Mdha) residue that undergoes Michael addition to produce a thiol-derivatized MC. Derivatization reactions using various thiolation reagents were followed by MS/MS, and two Python codes were used for data analysis and structural elucidation of MCs. Two novel MCs containing Ser at position 1 (i.e., next to Mdha) were putatively identified as [Ser1]MC-RR and [Ser1]MC-YR. Using thiol- and sulfoxide-modified [Ser1]MCs, identifications were confirmed by the observation of specific neutral losses of the oxidized thiols or sulfoxides in CID-MS/MS spectra in both positive and negative electrospray ionization (ESI) modes. These novel neutral losses are unique for MCs with Mdha and an adjacent Ser residue. Data suggest that a gas-phase reaction occurs between oxygen from adjacent Ser residue and sulfur of the Mdha-bonded thiol or sulfoxide, which leads to the formation and detection of stable cyclic MC ions in MS/MS spectra at m/z values corresponding to the loss of oxidized thiols or oxidized sulfoxides from Ser1-containing MCs.

On-Chip Capture, Raman-Silent Polymer Labeling, and Digital Mapping Analysis of Escherichia coli O157:H7 in Beverages All-in-One.

Escherichia coli O157:H7 is one of the most susceptible foodborne pathogens, easily causing food poisoning and other health risks. It is of great significance to establish a quantitative method with higher sensitivity and less time consumption for foodborne pathogens analysis. The Raman-silent signal has a good performance for avoiding interference from the food matrix so as to achieve accurate signal differentiation. In this work, we presented a preparation-mapping all-in-one method for digital mapping analysis. We prepared a functionalized Raman-silent polymer label of Escherichia coli O157:H7, which was captured on a porous 4-mercaptophenylboric acid@Ag foam chip. To improve accuracy and widen the detection range, a digital mapping quantitative strategy was employed in data extraction and processing. By transfer mapping information into digitized statistical results, the limitation of obtaining reproducible intensity values just by randomly selected spots on the substrate can be addressed. With a wide linear range of 1.0 × 101-1.0 × 105 CFU mL-1 and a limit of detection of 4.4 CFU mL-1, this all-in-one method had good sensitivity performance. Also, this method achieved good precision and selectivity in a series of experiments and was successfully applied to the analysis of beverage samples.

Nanozyme-Inhibited SERS Multichannel Paper-Based Sensor Array for the Quantification and Identification of Biothiols and Cancer Cells Based on Three Ag-Based Nanomaterials.

Biothiols play essential roles in maintaining normal physiological functions, resisting oxidative stress, and protecting cell health. Establishing an effective and reliable sensor array for the accurate quantification and discrimination of diverse biothiols is extremely meaningful. In this work, Ag/Mn3O4, Ag3PO4, and Ag3Cit with excellent oxidase-mimetic activity and surface-enhanced Raman scattering (SERS)-enhanced features have been prepared and loaded onto Whatman filter paper (WFP) to build SERS paper chips as three sensing channels, which can induce 3,3',5,5'-tetramethylbenzidine (TMB) oxidation to SERS-active reporters (TMBox) and concurrently generate prominent SERS signals. Nevertheless, the addition of biothiols can suppress conversion from TMB to TMBox, which can cause the reduction of the SERS signal from TMBox. Interestingly, each SERS sensing channel can generate different TMBox signals' variations due to differences in the oxidative inhibition abilities of diverse biothiols and exclusive properties of each paper chip, which can be plotted as specific fingerprint patterns of each biothiol and further translated into intuitive two-dimensional (2D) clustering profiles through linear discriminant analysis (LDA) and hierarchical cluster analysis (HCA) techniques for precise identification of these six biothiols with the minimum concentration of 1 µM. More importantly, this SERS sensor array is exploited for the precise quantification of intracellular glutathione (GSH), and can differentiate between normal and cancer cells based on different intracellular GSH contents and even identify different types of tumor cells, demonstrating its powerful application prospects in disease diagnosis.

Acidic Derivatization of Thiols Using Diethyl 2-Methylenemalonate: Thiol-Michael Addition Click Reaction for Simultaneous Analysis of Cysteine and Cystine in Culture Media Using LC-MS/MS.

Cysteine (Cys) and its oxidized form, cystine (Cys2), play crucial roles in biological systems and have considerable applications in cell culture. However, Cys in cell culture media is easily oxidized to Cys2, leading to solubility issues. Traditional analytical methods struggle to maintain the oxidation states of Cys and Cys2 during analysis, posing a significant challenge to accurately measuring and controlling these compounds. To effectively control the Cys and Cys2 levels, a rapid and accurate analytical method is required. Here, we screened derivatizing reagents that can react with Cys even under acidic conditions to realize a novel analytical method for simultaneously determining Cys and Cys2 levels. Diethyl 2-methylenemalonate (EMM) was found to possess the desired traits. EMM, characterized by its dual electron-withdrawing attributes, allowed for a rapid reaction with Cys under acidic conditions, preserving intact information for understanding the functions of target compounds. Combined with LC-MS/MS and an internal standard, this method provided high analytical accuracy in a short analytical time of 9 min. Using the developed method, the rapid oxidation of Cys in cell culture media was observed with the headspace of the storage container considerably influencing Cys oxidation and Cys2 precipitation rates. The developed method enabled the direct and simplified analysis of Cys behavior in practical media samples and could be used in formulating new media compositions, ensuring quality assurance, and real-time analysis of Cys and Cys2 in cell culture supernatants. This novel approach holds the potential to further enhance the media performance by enabling the timely optimal addition of Cys.

NIR Fluorescent/Photoacoustic Bimodal Imaging of Ferroptosis in Pancreatic Cancer Using Biothiols-Activable Probes.

Ferroptosis modulation is a powerful therapeutic option for pancreatic ductal adenocarcinoma (PDAC) with a low 5-year survival rate and lack of effective treatment methods. However, due to the dual role of ferroptosis in promoting and inhibiting pancreatic tumorigenesis, regulating the degree of ferroptosis is very important to obtain the best therapeutic effect of PDAC. Biothiols are suitable as biomarkers of imaging ferroptosis due to the dramatic decreases of biothiol levels in ferroptosis caused by the inhibited synthesis pathway of glutathione (GSH) and the depletion of biothiol by reactive oxygen species. Moreover, a very recent study reported that cysteine (Cys) depletion can lead to pancreatic tumor ferroptosis in mice and may be employed as an effective therapeutic strategy for PDAC. Therefore, visualization of biothiols in ferroptosis of PDAC will be helpful for regulating the degree of ferroptosis, understanding the mechanism of Cys depletion-induced pancreatic tumor ferroptosis, and further promoting the study and treatment of PDAC. Herein, two biothiol-activable near-infrared (NIR) fluorescent/photoacoustic bimodal imaging probes (HYD-BX and HYD-DX) for imaging of pancreatic tumor ferroptosis were reported. These two probes show excellent bimodal response performances for biothiols in solution, cells, and tumors. Subsequently, they have been employed successfully for real-time visualization of changes in concentration levels of biothiols during the ferroptosis process in PDAC cells and HepG2 cells. Most importantly, they have been further applied for bimodal imaging of ferroptosis in pancreatic cancer in mice, with satisfactory results. The development of these two probes provides new tools for monitoring changes in concentration levels of biothiols in ferroptosis and will have a positive impact on understanding the mechanism of Cys depletion-induced pancreatic tumor ferroptosis and further promoting the study and treatment of PDAC.

Bioconjugation and Reaction-Induced Tumor Therapy via Alkynamide-Based Thiol-Yne Click Reaction.

Ferroptosis is associated with the occurrence and development of many diseases, which is the result of an imbalance in cellular metabolism and oxidation-reduction balance. Therefore, it is an effective therapeutic strategy that simultaneously regulating the intracellular oxidation-reduction system. Herein, a click reaction of alkynylamide with thiol groups in the presence of amine or in PBS (pH = 7.4) is developed, which can react efficiently with thiol substances, such as cysteine (Cys), glutathione (GSH), and bovine serum albumin (BSA). Notably, MBTB-PA, an aggregation-induced emission (AIE) photosensitizer with an alkynylamide unit, is synthesized and its intracellular behavior is visualized in situ by fluorescence imaging, demonstrating its excellent ability to target the endoplasmic reticulum. Furthermore, MBTB-PA reacted with proteins in tumor cells, consumed reducing substances, and triggered intracellular oxidative stress, resulting in cell death. Based on this reaction therapy strategy, click reaction is combined with photodynamic therapy to achieve effective killing of tumor cells by simultaneously raising the intracellular oxidative state and reducing the reductive state. This work not only develops an application of click reaction of alkynamide with thiol in bioconjugation and anti-tumor therapy, but also provides feasible ideas for organic reactions in the exploration of organisms.

Spectroscopic, electrochemical, and kinetic trends in Fe(III)-thiolate disproportionation near physiologic pH.

In addition to its primary oxygen-atom-transfer function, cysteamine dioxygenase (ADO) exhibits a relatively understudied anaerobic disproportionation reaction (ADO-Fe(III)-SR → ADO-Fe(II) + ½ RSSR) with its native substrates. Inspired by ADO disproportionation reactivity, we employ [Fe(tacn)Cl3] (tacn = 1,4,7-triazacyclononane) as a precursor for generating Fe(III)-thiolate model complexes in buffered aqueous media. A series of Fe(III)-thiolate model complexes are generated in situ using aqueous [Fe(tacn)Cl3] and thiol-containing ligands cysteamine, penicillamine, mercaptopropionate, cysteine, cysteine methyl ester, N-acetylcysteine, and N-acetylcysteine methyl ester. We observe trends in UV-Vis and electron paramagnetic resonance (EPR) spectra, disproportionation rate constants, and cathodic peak potentials as a function of thiol ligand. These trends will be useful in rationalizing substrate-dependent Fe(III)-thiolate disproportionation reactions in metalloenzymes.

Redox proteomics in melanoma cells: An optimized protocol.

Cancer development and progression are intimately related with post-translational protein modifications, e.g., highly reactive thiol moiety of cysteines enables structural rearrangements resulting in redox biological switches. In this context, redox proteomics techniques, such as 2D redox DIGE, biotin switch assay and OxIcat are fundamental tools to identify and quantify redox-sensitive proteins and to understand redox mechanisms behind thiol modifications. Given the great variability in redox proteomics protocols, problems including decreased resolution of peptides and low protein amounts even after enrichment steps may occur. Considering the biological importance of thiol's oxidation in melanoma, we adapted the biotin-switch assay technique for melanoma cells in order to overcome the limitations and improve coverage of detected proteins.