Determination of acidity and nucleophilicity in thiols by reaction with monobromobimane and fluorescence detection (original) (raw)
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Talanta, 2004
Hydrophobic interaction chromatography coupled online with chemical vapour atomic fluorescence spectrometry (HIC-CVGAFS) has been optimized for the analysis of thiolic proteins in denaturing conditions. Proteins are pre-column simultaneously denatured and derivatized in phosphate buffer solution containing 8.0moldm(-3) urea and p-hydroxymercurybenzoate (PHMB) and the derivatized denatured proteins are separated on a silica HIC Eichrom Propyl column in the presence of 8.0M urea in the mobile phase. Post-column online reaction of derivatized denatured proteins with bromine, generated in situ by KBr/KBrO(3) in HCl medium, allowed the fast conversion of the uncomplexed PHMB and of the PHMB bound to proteins to inorganic mercury also in presence of urea. Hg(2+), present in solution as Hg(2+)-urea complex, is selectively detected by AFS in a Ar/H(2) miniaturized flame after sodium borohydride reduction to Hg. Under optimized conditions, online bromine treatment gives a 100+/-2% recovery of both free and protein-complexed PHMB. Denatured glyceraldehyde-3-phosphate dehydrogenase, aldolase, lactate dehydrogenase, trioso phosphate isomerase and beta-lactoglobulin have been examined. As the sensitivity and limit of detection of proteins in the HIC-CVGAFS apparatus depends on number of SH groups reacting with PHMB, the denaturation process, which increases the number of PHMB-reactive thiolic groups in proteins, improves the analytical performances of the described system in protein analysis. The detection limit for the denatured proteins examined was found in the range of 10(-10)-10(-12)moldm(-3), depending on the considered protein, with linear calibration curves spanning over four decades of concentration.
Organic & Biomolecular Chemistry, 2013
A new and simple chemodosimetric probe L 1 is utilized for the selective detection of biothiols in the presence of other relevant amino acids under physiological conditions (pH = 7.4). This eventually led to a turn-off luminescence response due to an effective photoinduced electron transfer based signaling mechanism. A comparison of the results of the fluorescence kinetic analysis and 1 H NMR studies of the reaction between thiol and L 1 or the analogous compound L 2 revealed the role of intramolecular hydrogen bonding in activating the imine functionality towards nucleophilic addition. Such an example is not common in contemporary literature. Conventional MTT assay studies revealed that this probe (L 1) has low cytotoxicity. Results of the cell imaging studies revealed that this probe was cell membrane permeable and could detect the intracellular distribution of biothiols within living HeLa cells. Furthermore, our studies with human blood plasma demonstrated the possibility of using this reagent for the quantitative optical detection of total biothiols in biological fluid. Such an example for the detection of biothiols in real biological samples is rare in the contemporary literature. These results clearly demonstrate the possibility of using this reagent in medicinal biology and diagnostic applications. † Electronic supplementary information (ESI) available: Characterization data for L 1 and L 2 , hydrolytic stability test, determination of detection limit, scanning of L 1 and L 2 with different amino acids. See
Thiol Reactive Probes and Chemosensors
2012
Thiols are important molecules in the environment and in biological processes. Cysteine (Cys), homocysteine (Hcy), glutathione (GSH) and hydrogen sulfide (H 2 S) play critical roles in a variety of physiological and pathological processes. The selective detection of thiols using reaction-based probes and sensors is very important in basic research and in disease diagnosis. This review focuses on the design of fluorescent and colorimetric probes and sensors for thiol detection. Thiol detection methods include probes and labeling agents based on nucleophilic addition and substitution, Michael addition, disulfide bond or Se-N bond cleavage, metal-sulfur interactions and more. Probes for H 2 S are based on nucleophilic cyclization, reduction and metal sulfide formation. Thiol probe and chemosensor design strategies and mechanism of action are discussed in this review.
Fluorescence-based detection of thiols in vitro and in vivo using dithiol probes
Analytical Biochemistry, 2006
Thiols play a central role in maintaining biological homeostasis. Their levels can change dramatically in response to oxidative stress associated with toxic insults, bacterial infection, and disease. Therefore, a reagent that can monitor thiol levels both in vitro and in vivo would be useful for assays and as a biomarker. Such a reagent should (i) be selective for thiols, (ii) be able to penetrate cell walls, and (iii) have a low reduction potential so as not to create oxidative stress in a cell. We have developed such a fluorescent reagent (DSSA) based on a dithiol linker: (i) the use of a dithiol linker makes it selective for thiols; (ii) the use of fluorophores that populate neutral states at physiological pH improves cell wall penetration; and (iii) because of the reagent's low reduction potential (À0.60 V), it will not stress cells oxidatively. For example, 5 lM of reagent is responsive to changes in glutathione levels in the physiologically relevant range of 1 to 10 mM, yet this would oxidize less than 1% of cellular glutathione. In Escherichia coli, decreased thiol levels were detected in cells deficient in glutathione synthesis. In zebrafish embryos, the DSSA reagent permitted detection of unusually high thiol levels in the zebrafish chorion.
ELECTROPHORESIS, 2005
Highly sensitive simultaneous detection of cultured cellular thiols by laser induced fluorescencecapillary electrophoresis We have recently described a new method to determine physiological thiols, in which the quantification of plasma homocysteine, cysteine, cysteinylglycine, glutathione, and glutamylcysteine was achieved after derivatization with 5-iodoacetamidofluorescein. Samples were separated and measured by capillary electrophoresis with laserinduced fluorescence in an uncoated fused-silica capillary, using a phosphate/borate run buffer and the organic base N-Methyl-D-glucamine as effective electrolyte addictive to obtain a baseline peak separation. In this paper, we propose an improvement of our method useful for the analysis of the intracellular thiols in different cultured cells. In particular, we studied run buffer and injection conditions in order to increase the sensitivity of the assay and we found that, by incrementing two times the injected volume and using the water plug before the sample injection, the sensitivity of our previous method was increased by about ten times. To maintain a good resolution between peaks, particularly between homocysteine and the internal standard d-penicillamine, we lengthened the run time by incrementing the concentration of the electrolyte buffer and the organic base d-glucamine and by decreasing the cartridge temperature from 40 to 257C. After these changes in electrophoretical parameters, cellular thiols were baseline-resolved in less than 14 min instead of 9 min as in our previous method, but the limit of quantification is increased from 50 to 1 nmol/L. This new procedure allows also to measure the intracellular thiols commonly found at low concentration, such as cysteinylglycine, glutamylcysteine, and homocysteine. The new analytical method performance was assessed by measuring the intracellular thiols in three different cell lines, i.e., HUVEC, ECV304, and R1 stem cells.
Journal of Chromatography B, 2016
Biothiols such as homocysteine, cysteine, and glutathione play many biologically important roles, especially in reduction-oxidation homeostasisand resistance to oxidative stress, and the measurement of their concentrations in model animal fluids is important in clarifying the pathology of thiol-related diseases. In this study, an analytical method for total biothiols in mouse serum using hydrophilic interaction liquid chromatography (HILIC) with fluorescence detection was developed. Mouse serum samples were derivatized with ammonium7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F), after reduction by tris(2-carboxyethyl)phosphine. Five biothiols (homocysteine, cysteine, cysteinylglycine, glutathione, and γ-glutamylcysteine) in the mouse sera were separated within 16 min on an amide-type HILIC column. The method possessed good linearity, good reproducibility with an intra-day variance of less than 3%, and low detection limits of 0.2-4 nM. Concentrations of homocysteine, cysteine, cysteinylglycine, glutathione, and γ-glutamylcysteine in the mouse serum samples were calculated as 6.7 ± 0.3, 227.7 ± 16.9, 1.2 ± 0.4, 77.5 ± 29.2, and 8.2 ± 0.9 μM, respectively (mean ± S.D., n = 4). Furthermore, HILIC-negative electrospray ionization-mass spectrometry(MS) analysis using a high-resolution mass spectrometer was conducted to determine the exact masses of two unknown peaks, which were found in the mouse serum samples with high signal intensity and were not detected in human plasma samples. The exact masses of the unknown compounds were determined as 1184.519 and 800.281 (as SBD-derivatized negative ions),which possessed a product ion common to SBD-thiols (m/z 230.954, as[SBD-SH]-) upon tandem MS spectrometric analysis.
Analytica Chimica Acta, 2010
An analytical method was developed for the determination of thiols in biological samples. Reverse phase chromatography coupled to ICP quaduropole MS or Orbitrap MS was employed for the separation and detection of thiols. For the determination of total thiols, oxidized thiols were reduced using dithiothreitol (DTT). Reduction efficiencies for species of interest were found to be close to 100%. Reduced thiols were derivatized by p-hydroxymercuribenzoate (PHMB) and then separated on a C8 column. Optimization of the extraction, separation and detection steps of the HPLC-ICP-MS and HPLC-Orbitrap MS methods was carried out. Detection limits for cysteine, homocysteine, selenocysteine, glutathione, selenomethionine and cysteinyl-glycine were found to be 18, 34, 39, 12, 128 and 103 fmol, respectively, using HPLC-Orbitrap MS and 730, 1110, 440, 1110 and 580 fmol for cysteine, homocysteine, selenocysteine, glutathione, and cysteinyl-glycine using HPLC-ICP-MS. Contrary to expectation, the LODs and RSDs are higher for the HPLC-ICP-MS instrument, therefore HPLC-Orbitrap MS was used for the determination of thiols in yeast samples. Three different brands of baker's yeast and a selenized yeast were analyzed. The GSH and cysteine levels found in these samples ranged from 4.45 to 17.87 mol g −1 and 0.61 to 1.32 mol g −1 , respectively.