RESUMO
Thiol compounds present in human malignant prostate cells (LNCaP) were investigated after reaction with a mercurial blocking reagent. After extracting the cellular glutathione and some other low molecular weight (LMW) thiols using trichloroacetic acid the resulting the protein precipitate was extracted with buffered 8 M urea containing 2-chloromercuri-4-nitrophenol in an equimolar amount to that of the thiol present. After removing the insoluble chromatin fraction the urea soluble labeled adducts formed were chromatographed on G15 Sephadex. Three yellow coloured (A410 nm) fractions were obtained; first, the excluded protein fraction containing 16.0 ± 4.1% of the applied label followed by an intermediate fraction containing 5.9 ± 1.2%. Finally a LMW fraction emerged which contained 77.2 ± 3.7% of the total label applied and this was further analyzed by column chromatography, first on an anion exchange column and then on a PhenylSepharose 6 column to give what appeared to be a single component. LC-MS analysis of this component gave a pattern of mercuri-clusters, formed on MS ionization showing possible parent ions at 704 or 588 m/z, the former indicating that a thiol fragment of molecular weight approximately 467 could be present. No fragments with a single sulfur adduct (a 369 m/z fragment) were observed The adduct was analyzed for cysteine and other amino acids, nucleic acid bases, ribose and deoxyribose sugars, selenium and phosphorus; all were negative leading to the conclusion that a new class of unknown LMW thiol is present concealed in the protein matrices of these cells.
Assuntos
Cloromercuronitrofenóis/química , Linfonodos/química , Neoplasias da Próstata/química , Compostos de Sulfidrila/isolamento & purificação , Reagentes de Sulfidrila/química , Resinas de Troca Aniônica/química , Linhagem Celular Tumoral , Fracionamento Químico , Cromatografia Líquida , Humanos , Linfonodos/patologia , Metástase Linfática , Masculino , Peso Molecular , Neoplasias da Próstata/patologia , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Chymotrypsinogen, when reduced and taken to its molten globule-like conformation, displays a single cysteine with an unusual kinetic propensity toward oxidized glutathione (GSSG) and other organic thiol reagents. A single residue, identified by mass spectrometry like Cys1, reacts with GSSG about 1400 times faster than an unperturbed protein cysteine. A reversible protein-GSSG complex and a low pKa (8.1 ± 0.1) make possible such astonishing kinetic property which is absent toward other natural disulfides like cystine, homocystine and cystamine. An evident hyper-reactivity toward 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and 1-chloro-2,4-dinitrobenzene (CDNB) was also found for this specific residue. The extraordinary reactivity toward GSSG is absent in two proteins of the thermophilic archaeon Sulfolobus solfataricus, an organism lacking glutathione: the Protein Disulphide Oxidoreductase (SsPDO) and the Bacterioferritin Comigratory Protein 1 (Bcp1) that displays Cys residues with an even lower pKa value (7.5 ± 0.1) compared to chymotrypsinogen. This study, which also uses single mutants in Cys residues for Bcp1, proposes that this hyper-reactivity of a single cysteine, similar to that found in serum albumin, lysozyme, ribonuclease, may have relevance to drive the "incipit" of the oxidative folding of proteins from organisms where the glutathione/oxidized glutathione (GSH/GSSG) system is present.
Assuntos
Proteínas Arqueais/metabolismo , Quimotripsinogênio/metabolismo , Glutationa/metabolismo , Sequência de Aminoácidos , Archaea/metabolismo , Quimotripsinogênio/fisiologia , Cisteína/metabolismo , Dissulfetos/química , Glutationa/fisiologia , Dissulfeto de Glutationa/metabolismo , Oxirredução , Oxirredutases/metabolismo , Dobramento de Proteína , Compostos de Sulfidrila/química , Reagentes de Sulfidrila/química , Sulfolobus solfataricus/metabolismoRESUMO
Abnormal protein-protein interactions (PPIs) are the basis of multiple diseases, and the large and shallow PPI interfaces make the target "undruggable" for traditional small molecules. Peptides, emerging as a new therapeutic modality, can efficiently mimic PPIs with their large scaffolds. Natural peptides are flexible and usually have poor serum stability and cell permeability, features that limit their further biological applications. To satisfy the clinical application of peptide inhibitors, many strategies have been developed to constrain peptides in their bioactive conformation. In this report, we describe several classic methods used to constrain peptides into a fixed secondary structure which could significantly improve their biophysical properties.
Assuntos
Peptídeos/química , Amidas/química , Fenômenos Biofísicos , Dicroísmo Circular , Cristalografia por Raios X , Hidrocarbonetos/síntese química , Hidrocarbonetos/química , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Peptídeos/síntese química , Conformação Proteica em alfa-Hélice , Estabilidade Proteica , Estrutura Secundária de Proteína , Técnicas de Síntese em Fase Sólida , Reagentes de Sulfidrila/químicaRESUMO
Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. Yet maleimide-based conjugates display limited stability in vivo because the succinimidyl thioether linkage can undergo a retro-Michael reaction. This, of course, can lead to the release of the radioactive payload or its exchange with thiol-bearing biomolecules in circulation. Both of these processes can produce elevated activity concentrations in healthy organs as well as decreased activity concentrations in target tissues, resulting in reduced imaging contrast and lower therapeutic ratios. In 2018, we reported the creation of a modular, stable, and easily accessible phenyloxadiazolyl methyl sulfone reagent - dubbed 'PODS' - as a platform for thiol-based bioconjugations. We have clearly demonstrated that PODS-based site-selective bioconjugations reproducibly and robustly create homogenous, well-defined, highly immunoreactive, and highly stable radioimmunoconjugates. Furthermore, preclinical experiments in murine models of colorectal cancer have shown that these site-selectively labeled radioimmunoconjugates exhibit far superior in vivo performance compared to radiolabeled antibodies synthesized via maleimide-based conjugations. In this protocol, we will describe the four-step synthesis of PODS, the creation of a bifunctional PODS-bearing variant of the ubiquitous chelator DOTA (PODS-DOTA), and the conjugation of PODS-DOTA to the HER2-targeting antibody trastuzumab.
Assuntos
Imunoconjugados/metabolismo , Reagentes de Sulfidrila/síntese química , Animais , Humanos , Maleimidas/química , Camundongos , Reagentes de Sulfidrila/química , Trastuzumab/farmacologiaRESUMO
This paper introduces three ways to determine host-guest complexation of cucurbit[7]uril (CB[7]) with homocysteine (Hcy). After preincubating Hcy and cysteine (Cys) with CB[7], Ellman's reagent (DTNB) was used to detect Hcy and Cys. Only Cys reacted with DTNB and Hcy gave a retarded color change. This suggests that the -SH group of Hcy is buried inside CB[7]. Human cystathionine γ-lyase (hCGL) decreased the level of Hcy degradation after preincubating Hcy and CB[7]. These results suggest that the amount of free Hcy available was decreased by the formation of a Hcy-CB[7] complex. The immunological signal of anti-Hcy monoclonal antibody was decreased significantly by preincubating CB[7] with Hcy. The ELISA results also show that ethanethiol group (-CH2CH2SH) of Hcy, which is an epitope of anti-Hcy monoclonal antibody, was blocked by the cavity in CB[7]. Overall, CB[7] can act as a host by binding selectively with Hcy, but not Cys. The calculated half-complexation formation concentration of CB[7] was 58.2 nmol using Ellman's protocol, 97.9 nmol using hCGL assay and 87.7 nmol using monoclonal antibody. The differing binding abilities of Hcy and Cys towards the CB[7] host may offer a simple and useful method for determining the Hcy concentration in plasma or serum.
Assuntos
Bioensaio/métodos , Hidrocarbonetos Aromáticos com Pontes/química , Homocisteína/análise , Homocisteína/química , Imidazóis/química , Anticorpos Monoclonais/imunologia , Cistationina gama-Liase/química , Cisteína/química , Ácido Ditionitrobenzoico/química , Epitopos/imunologia , Homocisteína/imunologia , Humanos , Modelos Moleculares , Estrutura Molecular , Reagentes de Sulfidrila/químicaRESUMO
To determine the binding-site of a combinatorially-selected peptide possessing a fluoroprobe, a novel cysteine reactive small photo-crosslinker that can be excited by a conventional long-wavelength ultraviolet handlamp (365 nm) was synthesized via Suzuki coupling with three steps. The crosslinker is rationally designed, not only as a bioisostere of the fluoroprobe, but as a caged-fluorophore, and the photo-crosslinked target protein became fluorescent with a large Stokes-shift. By introducing the crosslinker to a designated sulfhydryl (SH) group of a combinatorially-selected peptide, the protein-binding site of the targeted peptide was deduced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/fluorescence imaging followed by matrix-assisted laser desorption ionization-time of flight tandem mass spectrometry (MALDI-TOF-MS/MS) analysis.
Assuntos
Reagentes de Ligações Cruzadas/química , Corantes Fluorescentes/química , Peptídeos/química , Reagentes de Sulfidrila/química , Sítios de Ligação , Técnicas de Química Combinatória , Cisteína/química , Peptídeos/metabolismo , Ligação Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
There is a dire need for new treatments for Alzheimer's disease (AD). Principal drugs have reached maturity, and the number of people affected by AD is growing at a rapid rate. After years of research and many clinical trials, only symptomatic treatments are available. An effective disease-modifying drug for AD needs to be discovered. The research presented in this paper aims to facilitate in the discovery of new potential targets that could help in the ongoing AD research. Aryl methanesulfonate derivatives were screened for their acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitory activities. IC50 values between 0.660 and 3.397 µM against AChE and 0.885 and 2.596 µM against BuChE were obtained.
Assuntos
Inibidores da Colinesterase/farmacologia , Descoberta de Drogas , Mesilatos/farmacologia , Nootrópicos/farmacologia , Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Animais , Butirilcolinesterase/química , Butirilcolinesterase/metabolismo , Ácido Ditionitrobenzoico/química , Electrophorus , Proteínas de Peixes/antagonistas & inibidores , Proteínas de Peixes/metabolismo , Cavalos , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Concentração Osmolar , Espectrofotometria , Reagentes de Sulfidrila/químicaRESUMO
In the process of developing [18F]FBEM coupled target peptide, we have instituted a robust automated synthesis of [18F]FBEM, a sulfhydryl (-SH) site specific agent for radiolabeling of peptides and proteins. The radiosynthesis generated 1.67-3.89 GBq (45.1-105.1â¯mCi, 7.5-18.8% non-decay corrected yield) of [18F]FBEM from 22.2 GBq (600â¯mCi) of starting [18F]fluoride with molar activity of 31.8⯱â¯5.3 GBq/µmol (0.86⯱â¯0.14â¯mCi/nmol) (nâ¯=â¯3) at the end of synthesis. Radiochemical purity was greater than 98%, and total synthesis time was ~90â¯min.
Assuntos
Radioisótopos de Flúor/química , Peptídeo 1 Semelhante ao Glucagon/análogos & derivados , Maleimidas/química , Maleimidas/síntese química , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/síntese química , Animais , Cromatografia Líquida de Alta Pressão , Peptídeo 1 Semelhante ao Glucagon/síntese química , Peptídeo 1 Semelhante ao Glucagon/química , Peptídeo 1 Semelhante ao Glucagon/normas , Maleimidas/normas , Peptídeos/química , Proteínas/química , Controle de Qualidade , Radioquímica/instrumentação , Radioquímica/métodos , Compostos Radiofarmacêuticos/normas , Reagentes de Sulfidrila/síntese química , Reagentes de Sulfidrila/químicaRESUMO
Hydroxycinnamoyl-Coenzyme A (CoA) hydroxycinnamoyl transferases are BAHD family acyltransferases that transfer hydroxycinnamoyl moieties from a CoA-thioester to an acceptor amine or alcohol to form an N-hydroxycinnamoyl amide or O-hydroxycinnamoyl ester, respectively, with the concomitant release of free CoA. One approach to measure reaction rates for these enzymes is to quantify the hydroxycinnamoyl amide or ester reaction product following chromatographic separation of reaction components. This approach can be labor-intensive and time-consuming. As an alternative, we examined the use of 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB, Ellman's reagent) to spectrophotometrically quantify, in real time, the release of free CoA during the transferase reaction. Using a hydroxycinnamoyl-CoA:l-DOPA hydroxycinnamoyl transferase as a model, we show that DTNB has little to no effect on the transferase reaction and can be used to provide a good estimate of hydroxycinnamoyl amide formation, thus allowing for the quick and easy collection of reaction rate data and determination of transferase kinetic parameters. This approach should be applicable to a wide range of hydroxycinnamoyl-CoA and other BAHD acyltransferases.
Assuntos
Aciltransferases/metabolismo , Coenzima A/metabolismo , Ácido Ditionitrobenzoico/química , Proteínas de Plantas/metabolismo , Espectrofotometria/métodos , Cinética , Reagentes de Sulfidrila/químicaRESUMO
Proteins with great sequence similarity usually have similar structure, function and other physicochemical properties. But in many cases, one or more of the physicochemical or functional characteristics differ, sometimes very considerably, among these homologous proteins. To better understand how critical amino acids determine quantitative properties of function in proteins, the responsible residues must be located and identified. This can be difficult to achieve, particularly in cases where multiple amino acids are involved. In this work, two triosephosphate isomerases with very high similarity from two related human parasites were used to address one such problem. We demonstrate that a seventy-fold difference in the reactivity of an interface cysteine to the sulfhydryl reagent methylmethane sulfonate in these two enzymes depends on three amino acids located far away from this critical residue and which could not have been predicted using other current methods. Starting from previous observations with chimeric proteins involving these two triosephosphate isomerases, we developed a strategy involving additive mutant enzymes and selected site directed mutants to locate and identify the three amino acids. These three residues seem to induce changes in the interface cysteine in reactivity by increasing (or decreasing) its apparent pKa. Some enzymes with four to seven mutations also exhibited altered reactivity. This study completes a strategy for identifying key residues in the sequences of proteins that can have applications in future protein structure-function studies.
Assuntos
Aminoácidos/química , Cisteína/química , Reagentes de Sulfidrila/química , Triose-Fosfato Isomerase/química , Trypanosoma/enzimologia , Sequência de Aminoácidos , Aminoácidos/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Homologia de Sequência de Aminoácidos , Triose-Fosfato Isomerase/genéticaRESUMO
High-efficient enrichment of glycopeptides prior to mass spectrometry is essential for glycoproteomics analysis. Hydrophilic interaction chromatography (HILIC) approach is a prominent strategy for glycopeptides identification. In this work, glucose functionalized magnetic graphene hydrophilic nanocomposite (MagG/Au/Glu) was synthesized as a novel HILIC material via a facile surface modification strategy. Different from previous click synthesis of saccharides-functionalized materials, glucose was easily thiolated via Traut's reagent and then immobilized on the materials via efficient Au-S coupling, greatly simplifying the synthesis process. Combining the rapid magnetic response, huge surface area from graphene and excellent hydrophilicity from glucose, MagG/Au/Glu nanocomposites afforded convenience of the operation and affinity for glycopeptides. Thus, the nanocomposites exhibited superior performance of high sensitivity, selectivity and reusability in glycopeptide enrichment from tryptic digests of standard glycoprotein HRP. Encouragingly, with the usage of MagG/Au/Glu nanocomposites, a total of 305 glycopeptides assigned to 108 glycoproteins were identified from complex real sample human serum, indicating a great potential for the application of glycoproteomics research.
Assuntos
Proteínas Sanguíneas/química , Glucose/química , Glicopeptídeos/isolamento & purificação , Grafite/química , Nanopartículas de Magnetita/química , Nanocompostos/química , Proteínas Sanguíneas/isolamento & purificação , Cromatografia/métodos , Glicopeptídeos/química , Ouro/química , Peroxidase do Rábano Silvestre/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Reagentes de Sulfidrila/químicaAssuntos
Anticorpos Monoclonais/análise , Imunoglobulina M/análise , Cadeias kappa de Imunoglobulina/análise , Paraproteinemias/diagnóstico , Paraproteínas/análise , Idoso , Anticorpos Monoclonais/química , Anticorpos Monoclonais/urina , Eletroforese das Proteínas Sanguíneas , Humanos , Imunoeletroforese , Imunoglobulina M/química , Imunoglobulina M/urina , Cadeias kappa de Imunoglobulina/química , Cadeias kappa de Imunoglobulina/urina , Masculino , Mercaptoetanol/química , Peso Molecular , Oxirredução , Paraproteinemias/sangue , Paraproteinemias/urina , Paraproteínas/química , Paraproteínas/urina , Substâncias Redutoras/química , Reprodutibilidade dos Testes , Reagentes de Sulfidrila/químicaRESUMO
Previous studies indicated that bound sulfur species (BSS), including hydrogen polysulfide (H2Sn), have various physiological functions in mammalian cells. Although H2Sn molecules have been considered as secondary metabolites derived from hydrogen sulfide (H2S) based on in vitro studies or predetermined reaction formula, the physiological form of BSS and their endogenous concentration remain unclear. In the present study, we aimed to improve the usual method using monobromobimane (mBB) followed by high performance liquid chromatographic (HPLC) analysis for HSï¼ for simultaneous determination of H2S, H2S2, H2S3 and cysteine persulfide in biological samples. We demonstrated that mBB derivatization of H2S and H2Sn standards under alkaline conditions (pH 9.5) induced significant decreases in H2S2 and H2S3 levels and a significant increase in the H2S level in an incubation time-dependent manner. Conversely, the derivatization of mBB adducts of H2S2 and H2S3 were stable under neutral conditions (pH 7.0), which is physiologically relevant. Therefore, we re-examined the method using mBB and applied an improved method for the evaluation of H2S, H2S2, and H2S3 in mouse brain under physiological pH conditions. The concentrations of H2S and H2S2 were 0.030 ± 0.004µmol/g protein and 0.026 ± 0.002µmol/g protein, respectively. Although the level of H2S3 was below the quantification limit of this method, H2S3 was detected in mouse brain. Using the method established here, we reveal for the first time the existence of endogenous H2S2 and H2S3 in mammalian brain tissues. H2S2 and H2S3 exert anti-oxidant activity and anti-carbonyl stress effects through the regulation of redox balance in neuronal cells. Thus, our observations provide novel insights into the physiological functions of BSS in the brain and into neuronal diseases involved in redox imbalance.
Assuntos
Encéfalo/metabolismo , Cisteína/análogos & derivados , Dissulfetos/isolamento & purificação , Sulfeto de Hidrogênio/isolamento & purificação , Sulfetos/isolamento & purificação , Animais , Química Encefálica , Compostos Bicíclicos com Pontes/química , Cromatografia Líquida de Alta Pressão , Cisteína/isolamento & purificação , Cisteína/metabolismo , Dissulfetos/metabolismo , Sulfeto de Hidrogênio/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reagentes de Sulfidrila/química , Sulfetos/metabolismo , Espectrometria de Massas em TandemRESUMO
The selective reaction of chemical reagents with reduced protein thiols is critical to biological research. This reaction is utilized to prevent cross-linking of cysteine-containing peptides in common proteomics workflows and is applied widely in discovery and targeted redox investigations of the mechanisms underlying physiological and pathological processes. However, known and commonly used thiol blocking reagents like iodoacetamide, N-ethylmaleimide, and others were found to cross-react with oxidized protein sulfenic acids (-SOH) introducing significant errors in studies employing these reagents. We have investigated and are reporting here a new heteroaromatic alkylsulfone, 4-(5-methanesulfonyl-[1,2,3,4]tetrazol-1-yl)-phenol (MSTP), as a selective and highly reactive -SH blocking reagent compatible with biological applications.
Assuntos
Descoberta de Drogas , Fenóis/química , Sulfonas/química , Tetrazóis/química , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Humanos , Espectrometria de Massas , Modelos Biológicos , Estrutura Molecular , Reagentes de Sulfidrila/química , Reagentes de Sulfidrila/farmacocinética , Reagentes de Sulfidrila/farmacologia , Sulfonas/farmacocinética , Sulfonas/farmacologiaRESUMO
Recently, there have been a limited number of new, validated targets for small-molecule drug discovery in the pharmaceutical industry. Although there are approximately 30â¯000 genes in the human genome, only 2% are targeted by currently approved small-molecule drugs. One reason that many targets remain neglected by drug discovery programs is the absence of biochemical assays enabling evaluation of the potency of inhibitors in a quantitative and high-throughput manner. To overcome this issue, we developed a biochemical assay to evaluate the potency of both reversible and irreversible inhibitors using a nonspecific thiol-labeling fluorescent probe. The assay can be applied to any targets with a cysteine residue in a pocket that can accommodate small-molecule ligands. By constructing a mathematical model, we showed that the potency of compounds can be quantitatively evaluated by performing an activity-based protein profiling assay. In addition, the validity of the theory was confirmed experimentally using epidermal growth factor receptor kinase as a model target. This approach provides an assay system for targets for which biochemical assays cannot be developed. Our approach can potentially not only expand the number of exploitable targets but also accelerate the lead optimization process by providing quantitative structure-activity relationship information.
Assuntos
Compostos de Boro/metabolismo , Descoberta de Drogas/métodos , Receptores ErbB/antagonistas & inibidores , Corantes Fluorescentes/metabolismo , Maleimidas/metabolismo , Modelos Moleculares , Inibidores de Proteínas Quinases/farmacologia , Reagentes de Sulfidrila/metabolismo , Animais , Sítios de Ligação , Ligação Competitiva , Biocatálise , Compostos de Boro/química , Domínio Catalítico , Cisteína/química , Receptores ErbB/química , Receptores ErbB/genética , Receptores ErbB/metabolismo , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Ensaios de Triagem em Larga Escala , Humanos , Cinética , Ligantes , Maleimidas/química , Conformação Molecular , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Relação Quantitativa Estrutura-Atividade , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Células Sf9 , Spodoptera , Reagentes de Sulfidrila/químicaRESUMO
BACKGROUND: Apolipoprotein E (apoE) is closely involved in the pathogenesis of apoE-related diseases, such as Alzheimer's disease and cardiovascular disease. The redox modulation of cysteine-thiols in a protein is involved in various pathophysiological regulations; however, that of apoE has not been studied in detail. Herein, we devised an analytical method to determine the redox status of serum apoE and assessed its relation to serum cholesterol levels and apoE phenotype. METHODS: The present method was based on a band shift assay, using a photocleavable maleimide-conjugated polyethylene glycol. RESULTS: The basic characteristics of the present method were found to be satisfactory to determine the redox status of serum apoE quantitatively. Serum apoE was separated into its reduced-form (r-), non-reduced-form (nr-), apoE-AII complex, and homodimer using this method. R-apoE could be detected as a 40-kDa band, whereas nr-apoE remained as monomeric apoE. R-apoE displayed a preference for VLDL; however, the levels showed the correlation with HDL-cholesterol levels (p<0.005). Redox status of serum apoE was significantly different among apoE phenotypes. The quantitative ratios of nr-apoE to total apoE in serum from subjects with apoE4/E3 were higher than in serum from subjects with apoE3/E3 (p<0.0001) and apoE3/E2 (p<0.001). CONCLUSION: The redox status of serum apoE might be related to the synthesis of HDL. The information concerning the redox status of serum apoE provided by the present method may be a potent indicator to evaluate various apoE-related diseases.
Assuntos
Apolipoproteínas E/sangue , HDL-Colesterol/sangue , Apolipoproteína A-II/sangue , Apolipoproteína A-II/química , Apolipoproteína A-II/isolamento & purificação , Apolipoproteína E2/sangue , Apolipoproteína E2/química , Apolipoproteína E2/isolamento & purificação , Apolipoproteína E3/sangue , Apolipoproteína E3/química , Apolipoproteína E3/isolamento & purificação , Apolipoproteína E4/sangue , Apolipoproteína E4/química , Apolipoproteína E4/isolamento & purificação , Apolipoproteínas E/química , Apolipoproteínas E/isolamento & purificação , HDL-Colesterol/química , Cisteína/química , Diamida/química , Dimerização , Ditiotreitol/química , Ensaio de Desvio de Mobilidade Eletroforética , Células HEK293 , Humanos , Indicadores e Reagentes/química , Peso Molecular , Oxirredução , Processos Fotoquímicos , Polietilenoglicóis/química , Solubilidade , Reagentes de Sulfidrila/química , Raios UltravioletaRESUMO
After mild reduction of serum albumin, seven among the 34 cysteines forming the disulfide network displayed a surprising hyper-reactivity. Compared to the thiol group of glutathione, the average reactivity of these cysteines towards disulfides and thiol reagents was more than 100 times higher. Using mass spectrometry and kinetic data, we identified all these unusual residues, with Cys75, Cys123 and Cys264 showing the highest reactivity. This effect was mainly due to a low pKa of the sulfhydryl groups and may explain the very fast formation of early disulfides in the nascent protein suggesting the existence of a hierarchical propensity to form such covalent links in selected regions during oxidative folding. An identical pattern of hyper-reactive cysteines was found in albumins from six different mammals. This hyper-reactivity is much higher than the one found in other proteins containing multiple cysteines only devoted to structural disulfide bonds. It is possible that such hyper-reactive cysteines could also be present in other proteins, although their existence has been completely ignored so far.
Assuntos
Cisteína/química , Dissulfetos/química , Albumina Sérica/química , Reagentes de Sulfidrila/química , Animais , Sítios de Ligação , Bovinos , Cisteína/metabolismo , Dissulfetos/metabolismo , Cães , Glutationa/química , Glutationa/metabolismo , Cabras , Cavalos , Humanos , Cinética , Modelos Moleculares , Domínios Proteicos , Dobramento de Proteína , Albumina Sérica/metabolismo , Ovinos , Especificidade da Espécie , Compostos de Sulfidrila/química , Compostos de Sulfidrila/metabolismo , Reagentes de Sulfidrila/metabolismoRESUMO
Water-soluble trialkylphosphines such as tris(carboxyethyl)phosphine (TCEP) and trishydroxypropyl phosphine (THPP) are effective agents for reducing disulfide bonds in proteins and are increasingly becoming the reagents of choice for bioconjugation strategies that modify cysteine (thiol containing) amino acids. These reducing agents are often considered as being chemically compatible with Michael acceptors such as maleimides and, as such, are often not removed prior to performing protein conjugation reactions. Here, we demonstrate the rapid and irreversible reaction of both TCEP and THPP with derivatives of the commonly employed thiol alkylating groups, maleimide and vinyl sulfone. Mechanistic investigations revealed distinct differences between the reactions of TCEP and THPP with maleimide, leading to the production of either nonproductive ylenes or succidimidyl derivatives, respectively. Importantly, we also demonstrate the incorporation of nonproductive ylenes formed between maleimide and TCEP into the Pneumococcal capsular polysaccharide Pn6b following strategies employed toward the production of conjugate vaccines.
Assuntos
Fosfinas/química , Proteínas/química , Alquilantes/química , Dissulfetos/química , Espectroscopia de Ressonância Magnética , Maleimidas/química , Polissacarídeos/química , Solubilidade , Reagentes de Sulfidrila/química , Sulfonas/química , ÁguaRESUMO
The reagents and methods for purification and use of the most commonly used denaturants, guanidine hydrochloride (guanidine-HCl) and urea, are described. Other protein denaturants and reagents used to fold proteins are briefly mentioned. Sulfhydryl reagents (reducing agents) and "oxido-shuffling" (or oxidative regeneration) systems are also described.
Assuntos
Bioquímica/métodos , Indicadores e Reagentes/química , Dobramento de Proteína , Proteínas/química , Guanidina/química , Cinética , Oxirredução , Desnaturação Proteica , Estabilidade Proteica , Proteínas/isolamento & purificação , Substâncias Redutoras/química , Solventes/química , Reagentes de Sulfidrila/química , Ureia/químicaRESUMO
Grape pomace is a winemaking by-product that can be used to extract oenological tannins. Recently, some grape skin tannins were shown to contain very high amounts of two polyfunctional thiol precursors (3-S-glutathionylhexan-1-ol, 3-S-cysteinylhexan-1-ol) whose free forms are responsible for appreciated tropical-like flavours. This study shows that an oxidative treatment (no SO2) of white grape pomace and the presence of grape leaves and stems can increase the content of the above mentioned precursors. Moreover, it shows significant differences between Sauvignon Blanc, Gewuerztraminer and Mueller-Thurgau grape pomace for the 3-mercaptohexan-1-ol precursors and 4-S-cysteinyl-4-methylpentan-2-one. The grape cultivar is crucial, but the technological ability of enhancing the level of the volatile thiol precursors simply by treating the grape marc in different ways is a promising and powerful tool for the production of potentially flavouring tannins intended for food and beverage industry.