RESUMO
Biomolecules play vital roles in many biological processes and diseases, making their identification crucial. Herein, we present a colorimetric sensing method for detecting biomolecules like cysteine (Cys), homocysteine (Hcy), and glutathione (GSH). This approach is based on a reaction system whereby colorless 3,3',5,5'-tetramethylbenzidine (TMB) undergoes catalytic oxidation to form blue-colored oxidized TMB (ox-TMB) in the presence of hydrogen peroxide (H2O2), utilizing the peroxidase and catalase-mimicking activities of metal-phenolic coordination frameworks (MPNs) of Cu-TA, Co-TA, and Fe-TA nanospheres. The Fe-TA nanospheres demonstrated superior activity, more active sites and enhanced electron transport. Under optimal conditions, the Fe-TA nanospheres were used for the detection of biomolecules. When present, biomolecules inhibit the reaction between TMB and H2O2, causing various colorimetric responses at low detection limits of 0.382, 0.776 and 0.750 µM for Cys, Hcy and GSH. Furthermore, it was successfully applied to real water samples with good recovery results. The developed sensor not only offers a rapid, portable, and user-friendly technique for multi-target analysis of biomolecules at low concentrations but also expands the potential uses of MPNs for other targets in the environmental field.
Assuntos
Benzidinas , Colorimetria , Cisteína , Glutationa , Peróxido de Hidrogênio , Colorimetria/métodos , Peróxido de Hidrogênio/química , Glutationa/química , Glutationa/análise , Cisteína/química , Cisteína/análise , Benzidinas/química , Homocisteína/análise , Homocisteína/química , Estruturas Metalorgânicas/química , Limite de Detecção , Fenóis/química , Fenóis/análise , Oxirredução , Catálise , Peroxidase/química , Catalase/químicaRESUMO
BACKGROUND: Cysteine (Cys), glutathione (GSH), and homocysteine (Hcy), as three major biothiols are involved in a variety of physiological processes and play a crucial role in plant growth. Abnormal levels of Cys can cause plants to fail to grow properly. To date, although a very large number of fluorescent probes have been reported for the detection of biothiols, very few of them can be used for the selective discrimination of Cys from GSH and Hcy due to their structural similarity, and only a few of them can be used for plant imaging. RESULTS: Here, three fluorescent probes (o-/m-/p-TMA) based on TMN fluorophore and the ortho-/meta-/para-substituted maleimide recognition groups were constructed to investigate the selective response effect of Cys. Compared to the o-/m-TMA, p-TMA can selectively detect Cys over GSH and Hcy with a rapid response time (10 min) and a low detection limit (0.26 µM). The theoretical calculation confirmed that the intermediate p-TMA-Cys-int has shorter interatomic reaction distances (3.827 Å) compared to o-/m-TMA-Cys (5.533/5.287 Å), making it more suitable for further transcyclization reactions. Additionally, p-TMA has been employed for selective tracking of exogenous and endogenous Cys in Arabidopsis thaliana using both single-/two-photon fluorescence imaging. Furthermore, single cell walls produced obvious two-photon fluorescence signals, indicating that p-TMA can be used for high-concentration Cys analysis in single cells. Surprisingly, p-TMA can be used as a fluorescent dye for protein staining in SDS-PAGE with higher sensitivity (7.49 µg/mL) than classical Coomassie brilliant blue (14.11 µg/mL). SIGNIFICANCE: The outstanding properties of p-TMA make it a promising multifunctional molecular tool for the highly selective detection of Cys over GSH and Hcy in various complex environments, including water solutions, zebrafish, and plants. Additionally, it has the potential to be developed as a fluorescent dye for a simple and fast SDS-PAGE fluorescence staining method.
Assuntos
Cisteína , Eletroforese em Gel de Poliacrilamida , Corantes Fluorescentes , Glutationa , Homocisteína , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Cisteína/análise , Cisteína/química , Glutationa/análise , Glutationa/química , Homocisteína/análise , Homocisteína/química , Animais , Fótons , Imagem Óptica , Arabidopsis/química , Humanos , Ciclização , Peixe-ZebraRESUMO
The escalating drug resistance among microorganisms underscores the urgent need for innovative therapeutic strategies and a comprehensive understanding of bacteria's defense mechanisms against oxidative stress and antibiotics. Among the recently discovered barriers, the endogenous production of hydrogen sulfide (H2S) via the reverse transsulfuration pathway, emerges as a noteworthy factor. In this study, we have explored the catalytic capabilities and crystal structure of cystathionine γ-lyase from Pseudomonas aeruginosa (PaCGL), a multidrug-opportunistic pathogen chiefly responsible for nosocomial infections. In addition to a canonical L-cystathionine hydrolysis, PaCGL efficiently catalyzes the production of H2S using L-cysteine and/or L-homocysteine as alternative substrates. Comparative analysis with the human enzyme and counterparts from other pathogens revealed distinct structural features within the primary enzyme cavities. Specifically, a distinctly folded entrance loop could potentially modulate the access of substrates and/or inhibitors to the catalytic site. Our findings offer significant insights into the structural evolution of CGL enzymes across different pathogens and provide novel opportunities for developing specific inhibitors targeting PaCGL.
Assuntos
Domínio Catalítico , Cistationina gama-Liase , Sulfeto de Hidrogênio , Pseudomonas aeruginosa , Pseudomonas aeruginosa/enzimologia , Cistationina gama-Liase/metabolismo , Cistationina gama-Liase/química , Cristalografia por Raios X , Especificidade por Substrato , Sulfeto de Hidrogênio/metabolismo , Sulfeto de Hidrogênio/química , Modelos Moleculares , Cisteína/metabolismo , Cisteína/química , Conformação Proteica , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Humanos , Homocisteína/metabolismo , Homocisteína/química , CatáliseRESUMO
Boron neutron capture therapy is a unique form of adjuvant cancer therapy for various malignancies including malignant gliomas. The conjugation of boron compounds and human serum albumin (HSA)-a carrier protein with a long plasma half-life-is expected to extend systemic circulation of the boron compounds and increase their accumulation in human glioma cells. We report on the synthesis of fluorophore-labeled homocystamide conjugates of human serum albumin and their use in thiol-'click' chemistry to prepare novel multimodal boronated albumin-based theranostic agents, which could be accumulated in tumor cells. The novelty of this work involves the development of the synthesis methodology of albumin conjugates for the imaging-guided boron neutron capture therapy combination. Herein, we suggest using thenoyltrifluoroacetone as a part of an anticancer theranostic construct: approximately 5.4 molecules of thenoyltrifluoroacetone were bound to each albumin. Along with its beneficial properties as a chemotherapeutic agent, thenoyltrifluoroacetone is a promising magnetic resonance imaging agent. The conjugation of bimodal HSA with undecahydro-closo-dodecaborate only slightly reduced human glioma cell line viability in the absence of irradiation (~30 µM of boronated albumin) but allowed for neutron capture and decreased tumor cell survival under epithermal neutron flux. The simultaneous presence of undecahydro-closo-dodecaborate and labeled amino acid residues (fluorophore dye and fluorine atoms) in the obtained HSA conjugate makes it a promising candidate for the combination imaging-guided boron neutron capture therapy.
Assuntos
Antineoplásicos/uso terapêutico , Compostos de Boro/uso terapêutico , Terapia por Captura de Nêutron de Boro , Sistemas de Liberação de Medicamentos , Homocisteína/química , Albumina Sérica Humana/química , Antineoplásicos/síntese química , Antineoplásicos/química , Compostos de Boro/síntese química , Compostos de Boro/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Homocisteína/análogos & derivados , Humanos , Estrutura MolecularRESUMO
A new homocysteine thiolactone derivative, thiolactomide (1), was isolated along with a known compound, N-acetyl homocysteine thiolactone (2), from a culture extract of soil-derived Streptomyces sp. RK88-1441. The structures of these compounds were elucidated by detailed NMR and MS spectroscopic analyses with literature study. In addition, biological evaluation studies revealed that compounds 1 and 2 both exert neuroprotective activity against 6-hydroxydopamine (6-OHDA)-mediated neurotoxicity by blocking the generation of hydrogen peroxide in neuroblastoma SH-SY5Y cells.
Assuntos
Homocisteína/análogos & derivados , Fármacos Neuroprotetores/farmacologia , Streptomyces/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Homocisteína/química , Homocisteína/farmacologia , Humanos , Peróxido de Hidrogênio/metabolismo , Estrutura Molecular , Fármacos Neuroprotetores/química , Oxidopamina/toxicidade , Microbiologia do SoloRESUMO
The presence of reduced aminothiols, including homocysteine (Hcy), cysteine (Cys), cysteinyl-glycine (CG), and glutathione (GSH), is significantly increased in the pathological state. However, there have been no reports on the relationship between reduced aminothiols (Hcy, Cys, CG, and GSH) and different genders, ages, and drug combinations in human blood. The accurate quantification of these reduced thiols in biological fluids is important for monitoring some special pathological conditions of humans. However, the published methods typically not only require cumbersome and technically challenging processing procedures to ensure reliable measurements, but are also laborious and time-consuming, which may disturb the initial physiological balance and lead to inaccurate results. We developed a hollow fiber centrifugal ultrafiltration (HFCF-UF) method for sample preparation coupled with a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method and used it to determine four reduced aminothiols (Hcy, Cys, CG, and GSH) in human blood for the first time. A total of 96 clinical patients were enrolled in our study. The influence of different genders, ages, and drug combinations on the levels of four reduced thiols in human blood was also discussed by SPSS 24.0. The sample preparation was simplified to a single 5 min centrifugation step in a sealed system that did not disturb the physiological environment. The validation parameters for the methodological results were excellent. The procedure was successfully applied to monitoring the concentrations of four reduced aminothiols (Hcy, Cys, CG, and GSH) in 96 clinical blood samples. There were no significant differences in Hcy, Cys, CG, or GSH for the different genders, ages, or combinations with methotrexate or vancomycin (P > 0.05). However, there was a significant increase in Hcy concentration in patients treated with valproic acid who were diagnosed with epilepsy (p=0.0007). It is advisable to measure reduced Hcy level in patients taking valproic acid. The developed HFCF-UF method was simple and accurate. It can be easily applied in clinical research to evaluate oxidative stress in further study.
Assuntos
Análise Química do Sangue/métodos , Cisteína/sangue , Dipeptídeos/sangue , Glutationa/sangue , Homocisteína/sangue , Ultrafiltração/métodos , Antibacterianos/sangue , Antibacterianos/química , Cromatografia Líquida de Alta Pressão/métodos , Cisteína/química , Dipeptídeos/química , Inibidores Enzimáticos/sangue , Inibidores Enzimáticos/química , Congelamento , Glutationa/química , Homocisteína/química , Humanos , Limite de Detecção , Metotrexato/sangue , Metotrexato/química , Estrutura Molecular , Espectrometria de Massas em Tandem/métodos , Temperatura , Ácido Valproico/sangue , Ácido Valproico/química , Vancomicina/sangue , Vancomicina/químicaRESUMO
High levels of homocysteine are reported as a risk factor for Alzheimer's disease (AD). Correspondingly, inborn hyperhomocysteinemia is associated with an increased predisposition to the development of dementia in later stages of life. Yet, the mechanistic link between homocysteine accumulation and the pathological neurodegenerative processes is still elusive. Furthermore, despite the clear association between protein aggregation and AD, attempts to develop therapy that specifically targets this process have not been successful. It is envisioned that the failure in the development of efficacious therapeutic intervention may lie in the metabolomic state of affected individuals. We recently demonstrated the ability of metabolites to self-assemble and cross-seed the aggregation of pathological proteins, suggesting a role for metabolite structures in the initiation of neurodegenerative diseases. Here, we provide a report of homocysteine crystal structure and self-assembly into amyloid-like toxic fibrils, their inhibition by polyphenols, and their ability to seed the aggregation of the AD-associated ß-amyloid polypeptide. A yeast model of hyperhomocysteinemia indicates a toxic effect, correlated with increased intracellular amyloid staining that could be rescued by polyphenol treatment. Analysis of AD mouse model brain sections indicates the presence of homocysteine assemblies and the interplay between ß-amyloid and homocysteine. This work implies a molecular basis for the association between homocysteine accumulation and AD pathology, potentially leading to a paradigm shift in the understanding of AD initial pathological processes.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Homocisteína/metabolismo , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/ultraestrutura , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Encéfalo/patologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Homocisteína/química , Humanos , Espectrometria de Mobilidade Iônica , Cinética , Camundongos Transgênicos , Modelos Biológicos , Polifenóis/farmacologia , Saccharomyces cerevisiae/metabolismoRESUMO
Homocysteine is a non-proteinogenic sulfhydryl-containing amino acid derived from methionine and is a homologue of cysteine [...].
Assuntos
Bioquímica/métodos , Doença/etiologia , Homocisteína/metabolismo , Biologia Molecular/métodos , Animais , Homocisteína/química , HumanosRESUMO
BODIPY dyes are photostable neutral derivatives of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene. These are widely used as chemosensors, laser materials, and molecular probes. At the same time, BODIPY dyes have small or moderate Stokes shifts like most other fluorophores. Large Stokes shifts are preferred for fluorophores because of higher sensitivity of such probes and sensors. The new boron containing BODIPY dye was designed and synthesized. We succeeded to perform an annulation of pyrrole ring with coumarin heterocyclic system and achieved a remarkable difference in absorption and emission maximum of obtained fluorophore up to 100 nm. This BODIPY dye was equipped with linker arm and was functionalized with a maleimide residue specifically reactive towards thiol groups of proteins. BODIPY residue equipped with a suitable targeting protein core can be used as a suitable imaging probe and agent for Boron Neutron Capture Therapy (BNCT). As the most abundant protein with a variety of physiological functions, human serum albumin (HSA) has been used extensively for the delivery and improvement of therapeutic molecules. Thiolactone chemistry provides a powerful tool to prepare albumin-based multimodal constructions. The released sulfhydryl groups of the homocysteine functional handle in thiolactone modified HSA were labeled with BODIPY dye to prepare a labeled albumin-BODIPY dye conjugate confirmed by MALDI-TOF-MS, UV-vis, and fluorescent emission spectra. Cytotoxicity of the resulting conjugate was investigated. This study is the basis for a novel BODIPY dye-albumin theranostic for BNCT. The results provide further impetus to develop derivatives of HSA for delivery of boron to cancer cells.
Assuntos
Compostos de Boro/química , Terapia por Captura de Nêutron de Boro/métodos , Neoplasias/tratamento farmacológico , Albumina Sérica Humana/química , Corantes/química , Sistemas de Liberação de Medicamentos , Corantes Fluorescentes/química , Homocisteína/química , Humanos , Lactonas/química , Maleimidas/química , Sondas Moleculares , Medicina de Precisão , Espectrometria de Fluorescência , Espectrometria de Massas por Ionização por Electrospray , Espectrofotometria UltravioletaRESUMO
Observations that copper and homocysteine levels are simultaneously elevated in patients with cardiovascular disease has generated interest in investigating the interactions between copper and homocysteine. Several prior studies have shown that complexes of copper and homocysteine are toxic, leading to cardiovascular damage in vitro. It is not clear, however, why related effects do not occur with other structurally similar, more abundant cellular thiols such as glutathione and cysteine. Herein, a mechanism for a selective redox interaction between copper and homocysteine is demonstrated. It involves a kinetically favored intramolecular hydrogen atom transfer that results in an alpha-amino carbon-centered radical known to promote biomolecular damage.
Assuntos
Doenças Cardiovasculares/metabolismo , Cobre/metabolismo , Homocisteína/metabolismo , Cobre/química , Glutationa/química , Glutationa/metabolismo , Homocisteína/química , Humanos , Hidrogênio/química , Hidrogênio/metabolismo , Oxirredução , Compostos de Sulfidrila/química , Compostos de Sulfidrila/metabolismoRESUMO
A novel fluorescent probe (RA), based on the rosamine skeleton bearing acrylate group, has been reasonably designed and prepared, which employed an addition-cyclization-elimination sequence reaction mechanism to detect cysteine. RA displayed rapid response to cysteine within 1.5 min, and exhibited satisfactory selectivity for cysteine over H2S, glutathione (Glu), and homocysteine (Hcy), due to the formation of seven-membered lactam favored kinetically. Fluorescence ratio was utilized to detect cysteine from 6.0 to 20.0 µM with a detection limit of 0.29 µM. More, RA was used to monitor cysteine in BSA, water, milk, milk powder, cabbage, radish, apple, and pear.
Assuntos
Cisteína/análise , Corantes Fluorescentes/química , Compostos Heterocíclicos com 3 Anéis/química , Leite/química , Plantas/química , Rodaminas/química , Soroalbumina Bovina/química , Espectrometria de Fluorescência/métodos , Água/química , Animais , Brassica/química , Brassica/metabolismo , Glutationa/química , Homocisteína/química , Limite de Detecção , Malus/química , Malus/metabolismo , Leite/metabolismo , Plantas/metabolismo , Pyrus/química , Pyrus/metabolismo , Raphanus/química , Raphanus/metabolismoRESUMO
A BrPAPS based Cu2+ complex has been developed as a colorimetric probe for the selective recognition of homocysteine (Hcy) over cysteine (Cys) and glutathione (GSH) in an aqueous solution via the indicator displacement assay. BrPAPS formed a complex with Cu2+ in a 1:1 ratio (BrPAPS-Cu2+) accompanied by the color change from yellow to red. Detecting Hcy is based on high affinity of Hcy for Cu2+. The addition of Hcy to BrPAPS-Cu2+ caused the complex formation of Hcy with Cu2+ in a 2:1 stoichiometry, resulting a hypsochromic shift with change back of color from red to yellow by the release of BrPAPS from BrPAPS-Cu2+. The absorption response is linear with the Hcy concentration in the range of 0-20 µM with a detection limit of 1.46 µM. Moreover, the detection of Hcy was not significantly affected by other amino acids from the competition experiments. Thus, BrPAPS-Cu2+ can be used as a simple probe for Hcy in aqueous solution.
Assuntos
Colorimetria/métodos , Complexos de Coordenação/química , Cobre/química , Homocisteína/análise , Homocisteína/química , Compostos Azo/química , Análise Química do Sangue/métodos , Compostos Cromogênicos/química , EspectrofotometriaRESUMO
As a kind of bioactive sulfur species, biothiols (Cys, Hcy, and GSH) play an irreplaceable role in regulating the redox balance of life processes. Because of their similar chemical structures and properties, a sulfydryl group, and an amino group, it is an important challenge to distinguish two or more of them at the same time. Herein, a fluorescent sensor (NTPC) based on the coumarin structure was developed to discriminate Cys/Hcy and GSH simultaneously. The sensor has no fluorescence due to the d-PET effect but displays strong fluorescence after its reaction with biothiols. There are two potential reaction sites (nitrophenyl sulfide group and aldehyde group) in the structure of NTPC, resulting in different fluorescent signal changes after reacting with biothiols (green for Cys and Hcy and red for GSH). Under double-wavelength excitation, the sensor shows low background fluorescence, high selectivity, and low detection limits toward biothiols. Moreover, the sensor can be used to discriminate different biothiols (Cys/Hcy and GSH) in cells and zebra fish by different fluorescence signals with low toxicity and might provide a promising tool for studying the roles of different biothiols in various physiological and pathological processes.
Assuntos
Cisteína , Corantes Fluorescentes , Glutationa/química , Homocisteína/química , Animais , Estrutura Molecular , Peixe-ZebraRESUMO
S-adenosyl-l-homocysteine hydrolase (SAHase) is a major regulator of cellular methylation reactions that occur in eukaryotic and prokaryotic organisms. SAHase activity is also a significant source of l-homocysteine and adenosine, two compounds involved in numerous vital, as well as pathological processes. Therefore, apart from cellular methylation, the enzyme may also influence other processes important for the physiology of particular organisms. Herein, presented is the structural characterization and comparison of SAHases of eukaryotic and prokaryotic origin, with an emphasis on the two principal domains of SAHase subunit based on the Rossmann motif. The first domain is involved in the binding of a substrate, e.g., S-adenosyl-l-homocysteine or adenosine and the second domain binds the NAD+ cofactor. Despite their structural similarity, the molecular interactions between an adenosine-based ligand molecule and macromolecular environment are different in each domain. As a consequence, significant differences in the conformation of d-ribofuranose rings of nucleoside and nucleotide ligands, especially those attached to adenosine moiety, are observed. On the other hand, the chemical nature of adenine ring recognition, as well as an orientation of the adenine ring around the N-glycosidic bond are of high similarity for the ligands bound in the substrate- and cofactor-binding domains.
Assuntos
Adenosil-Homocisteinase/metabolismo , Adenina/química , Adenosina/química , Motivos de Aminoácidos , Animais , Cristalografia por Raios X , Bases de Dados de Proteínas , Homocisteína/química , Humanos , Ligantes , Metilação , Conformação Molecular , NAD , Nucleotídeos/química , Ligação Proteica , Domínios Proteicos , Dobramento de Proteína , SoftwareRESUMO
A cyclodextrin-based fluorescence light-up and ratiometric sensor is reported for highly selective and sensitive recognition of glutathione over cystein and homocystein. The sensing scheme developed builds up on a supramolecular assembly formed between a molecular rotor dye (ThT) and a polyanionic supramolecular host (sulfated-ß-cyclodextrin, SCD). The detection scheme is accomplished as follows: firstly, the bivalent Cu2+ quenches the emission from ThT-SCD assembly by causing the dissociation of ThT molecules from SCD surface. Secondly, when GSH is added to the copper-quenched system, owing to specific interaction between Cu2+ and GSH, Cu2+ is removed from the SCD which again allows the formation of ThT-SCD assembly. Indeed, this scheme of disassembly and reassembly successively caused by Cu2+ and GSH in the aqueous solution empowers our sensor framework to work as a good ratiometric sensor for the detection of GSH. The sensor scheme shows a linear response in the range 0-250 µM with a LOD of 2.4 ± 0.2 µM in aqueous solution and 13.6 ± 0.5 µM in diluted human serum sample. The sensor system is excited at 410 nm and the emission signal is plotted as a ratio of intensity at 545 nm (aggregate band) and 490 nm (monomer band). This ratiometric sensor system is highly selective to glutathione over cystein, homocystein, and other amino acids. Additionally, response of the sensor system towards GSH in complex biological media of serum samples demonstrates its potential for practical utility. Graphical abstract.
Assuntos
Benzotiazóis/química , Técnicas Biossensoriais , Cisteína/química , Homocisteína/química , Espectrometria de Fluorescência , beta-Ciclodextrinas/química , Cobre/químicaRESUMO
Herein, gold@platinum (Au@Pt) bimetallic nanoparticles with high catalytic ability were in situ decorated onto a molybdenum disulfide (MoS2) surface to obtain nanocomposites (MoS2-Au@Pt) with high peroxidase-mimicking activity, which were used to construct a colorimetric sensor for cysteine (Cys) detection. Interestingly, this sensor can efficiently distinguish Cys from homocysteine (Hcy), glutathione (GSH) and 19 other amino acids with high sensitivity. As expected, the colorimetric sensor can determine the Cys content in Cys supplement tablets due to its high stability and repeatability. Finally, the detection mechanism was studied.
Assuntos
Colorimetria/métodos , Cisteína/análise , Dissulfetos/química , Ouro/química , Molibdênio/química , Nanocompostos/química , Platina/química , Materiais Biomiméticos/química , Materiais Biomiméticos/metabolismo , Glutationa/química , Homocisteína/química , Peróxido de Hidrogênio/química , Nanopartículas Metálicas/química , Peroxidases/química , Peroxidases/metabolismoRESUMO
Homocysteine thiolactone (HTL) is a chemically reactive thioester that has been implicated in cardiovascular disease. So far, its presence has been documented in human and mouse plasma and urine. Here, using a new method, we show that HTL is present in human saliva. The assay involves chloroform-methanol extraction of HTL, lyophilization, and derivatization with N-trimethylsilyl-N-methyl trifluoroacetamide (MSTFA) and trimethylchlorosilane (TMCS). The method is based on a gas chromatography coupled with mass spectrometry (GC-MS) and quantifies HTL in a linear range from 0.05 to 1 µmol L-1 saliva and urine. The limit of quantification (LOQ) was 0.05 µmol L-1. With respect to saliva specimen, the accuracy was 98.7-112.6%, and 90.2-100.5%, while the precision was 7.1-13.5% and 12.5-15.0% for the intra- and inter-day variation, respectively. In relation to urine samples, the accuracy was 91.9-110.9% and 91.2-103.3%, while the precision varied from 2.2% to 14.5% and 7.4% to 14.3% for intra- and inter-day measurements, respectively. Using this method, we show that in apparently healthy individuals (n = 18), HTL levels in saliva are not positively correlated with urinary HTL levels. Undoubtedly, larger population should be investigated to get more meaningful results.
Assuntos
Cromatografia Gasosa-Espectrometria de Massas/métodos , Homocisteína/análogos & derivados , Saliva/química , Adulto , Idoso , Feminino , Homocisteína/análise , Homocisteína/química , Homocisteína/urina , Humanos , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Adulto JovemRESUMO
RATIONALE: Oxidative stress is an imbalance between reactive free radical oxygen species and antioxidant defenses. Its consequences can lead to numerous pathologies. Regulating oxidative stress is the complex interplay between antioxidant recycling and thiol-containing regulatory proteins. Understanding these regulatory mechanisms is important for preventing onset of oxidative stress. The aim of this study was to investigae S-thiol protein chemistry associated with oxidized vitamin C (dehydroascorbate, DHA), homocysteine (HcySH) and glutathione (GSH) using mass spectrometry. METHODS: Glutaredoxin-1 (Grx-1) was incubated with DHA, with and without GSH and HcySH. Disulfide formation was followed by electrospray ionization mass spectrometry (ESI-MS) of intact proteins and by LC/ESI-MS/MS of peptides from protein tryptic digestions. The mechanism of DHA-mediated S-thiolation was investigated using two synthetic peptides: AcFHACAAK and AcFHACE. Three proteins, i.e. human hemoglobin (HHb), recombinant peroxiredoxin 2 (Prdx2) and Grx-1, were S-homocysteinylated followed by S-transthiolyation with GSH and investigated by ESI-MS and ESI-MS/MS. RESULTS: ESI-MS analysis reveals that DHA mediates disulfide formation and S-thiolation by HcySH as well as GSH of Grx-1. LC/ESI-MS/MS analysis allows identification of Grx-1 S-thiolated cysteine adducts. The mechanism by which DHA mediates S-thiolation of heptapeptide AcFHACAAK is shown to be via initial formation of a thiohemiketal adduct. In addition, ESI-MS of intact proteins shows that GSH can S-transthiolate S-homocysteinylated Grx-1_ HHb and Prdx2. The GS-S-protein adducts over time dominate the ESI-MS spectrum profile. CONCLUSIONS: Mass spectrometry is a unique analytical technique for probing complex reaction mechanisms associated with oxidative stress. Using model proteins, ESI-MS reveals the mechanism of DHA-facilitated S-thiolation, which consists of thiohemiketal formation, disulfide formation or S-thiolation. Furthermore, protein S-thiolation by HcySH can be reversed by reversible GSH thiol exchange. The use of mass spectrometry with in vitro models of protein S-thiolation in oxidative stress may provide significant insight into possible mechanisms of action occurring in vivo.
Assuntos
Ácido Desidroascórbico , Glutationa , Homocisteína , Espectrometria de Massas por Ionização por Electrospray/métodos , Compostos de Sulfidrila/análise , Ácido Desidroascórbico/análise , Ácido Desidroascórbico/química , Ácido Desidroascórbico/metabolismo , Glutationa/análise , Glutationa/química , Glutationa/metabolismo , Homocisteína/análise , Homocisteína/química , Homocisteína/metabolismo , Humanos , Estresse Oxidativo/fisiologia , Proteínas/análise , Proteínas/química , Proteínas/metabolismo , Compostos de Sulfidrila/química , Compostos de Sulfidrila/metabolismo , Espectrometria de Massas em Tandem/métodosRESUMO
A near-infrared (NIR) and water-soluble probe was synthesized and studied for the detection of Cys/Hcy in aqueous solution, living cells and mice. The probe was composed of cyanine derivative as the NIR fluorescent reporting unit and pyrimidiny-thioether moiety as the Cys/Hcy responsive unit. Treatment with Cys/Hcy induced the formation of sulfur-substituted products, then intramolecular rearrangement reaction would occur to produce amino-substituted products and resulting in enhanced red fluorescence emissions. It could be applied to sense Cys/Hcy both in solution with the detection limit of 0.17 µM (or 0.32 µM) and in living cells. Cell imaging experiments proved that such a probe exhibited good cell penetration. In addition, the probe could detect Cys/Hcy in live mice with strong turn-on fluorescent response.
Assuntos
Cisteína/química , Corantes Fluorescentes/química , Homocisteína/química , Raios Infravermelhos , Água/química , Animais , Linhagem Celular , Sobrevivência Celular , Cisteína/metabolismo , Corantes Fluorescentes/metabolismo , Homocisteína/metabolismo , Camundongos , Imagem Óptica , SolubilidadeRESUMO
Zwitterionic metal-organic frameworks (MOFs) of {[Cu(Cbdcp)(Dps)(H2O)3]·6H2O}n (MOF 1) and [Cu4(Dcbb)4(Dps)2(H2O)2]n (MOF 2) (H3CbdcpBr = N-(4-carboxybenzyl)-(3,5-dicarboxyl)pyridinium bromide; H2DcbbBr = 1-(3,5-dicarboxybenzyl)-4,4'-bipyridinium bromide; Dps = 4,4'-dipyridyl sulfide) quench the fluorescence of cytosine-rich DNA tagged with 5-carboxytetramethylrhodamine (TAMRA, emission at 582 nm, denoted as C-rich P-DNA-1) and yield the corresponding P-DNA-1@MOF hybrids. Exposure of these hybrids to Ag+ results in the release of the P-DNA-1 strands from the MOF surfaces as double-stranded, hairpin-like C-AgI-C (ds-DNA-1@Ag+) with the restoration of TAMRA fluorescence. The ds-DNA-1@Ag+ formed on the surface of 1 can subsequently sense biothiols cysteine (Cys), glutathione (GSH), and homocysteine (Hcy) due to the stronger affinity of mercapto groups for Ag+ that serves to unfold the ds-DNA-1@Ag+ duplex, reforming P-DNA-1, which is re-adsorbed by MOF 1 accompanied by quenching of TAMRA emission. Meanwhile, MOF 2 is also capable of co-loading a thymine-rich probe DNA tagged with 5-carboxyfluorescein (FAM, emission at 518 nm, denoted as T-rich P-DNA-2) to achieve synchronous sensing of Ag+ and Hg2+, resulting from the simultaneous yet specific ds-DNA-1@Ag+ and T-HgII-T duplex (ds-DNA-2@Hg2+) formation, as well as the distinctive emission wavelengths of TAMRA and FAM. Detection limits are as low as 5.3 nM (Ag+), 14.2 nM (Cys), 13.5 nM (GSH), and 9.1 nM (Hcy) for MOF 1, and 7.5 nM (Ag+) and 2.6 nM (Hg2+) for MOF 2, respectively. The sequential sensing of Ag+ and biothiols by MOF 1, and the synchronous sensing of Ag+ and Hg2+ by MOF 2 are rapid and specific, even in the presence of other mono- and divalent metal cations or other biothiols at much higher concentrations. Molecular simulation studies provide insights regarding the molecular interactions that underpin these sensing processes.