The Experts below are selected from a list of 39 Experts worldwide ranked by ideXlab platform
Robert C Fahey - One of the best experts on this subject based on the ideXlab platform.
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determination of biothiols by Bromobimane labeling and high performance liquid chromatography
Methods in Enzymology, 1995Co-Authors: Gerald L Newton, Robert C FaheyAbstract:Publisher Summary This chapter discusses the determination of biothiols by Bromobimane labeling and high-performance liquid chromatography (HPLC). Most thiols undergo rapid autoxidation when exposed to air at neutral or basic pH, a process that is catalyzed by heavy metals of the type found in most cells. A primary consideration in preparing and analyzing samples for thiols is to prevent their oxidative loss during the sample preparation and analysis. This can be accomplished by alkylation of the thiol group near neutral pH with the Bromobimanes. The chapter focuses on monoBromobimane (mBBr), an uncharged reagent that readily penetrates cells, 1-4 and 4- p -sulfobenzoyloxymethyl-6-bromomethy-1-3,7-dimethyl-1,5-diazabicyclo [3.3.0] octa-3,6-diene-2,8-dione (SBBr), an anionic reagent that does not enter cells. Many biological thiols are relatively small and ionic, which makes them easy to separate from more hydrophobic thiols but difficult to separate from each other using reversed-phase HPLC. The bimane derivatives of these ionic thiols are more hydrophobic than the thiol itself and are readily resolved by HPLC analysis on silica-based reversed-phase columns. The highly selective, rapid reactivity of Bromobimanes toward thiols, the stability and fluorescence yield of the thiol derivatives, the ease of separation of the derivatives by reversed-phase HPLC, and the availability of both cell-penetrating and nonpenetrating forms make the use Bromobimanes an extremely powerful approach to the analysis of low-molecular-weight biothiols.
J.-e. Hällgren - One of the best experts on this subject based on the ideXlab platform.
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Determination of glutathione in Scots pine needles by high-performance liquid chromatography as its mono-Bromobimane derivative
Journal of Chromatography A, 2001Co-Authors: Gunnar Wingsle, Göran Sandberg, J.-e. HällgrenAbstract:A method for determination of glutathione in its reduced (GSH) and oxidized (GSSG) forms in Scots pine extracts by reversed-phase high-performance liquid chromatography utilizing monoBromobimane as the derivatization reagent was developed. The recovery and the precision for GSH in pine needles was high, ca. 99 and 3.6%, respectively. The determination of GSSG showed lower recovery (ca. 80%) and poorer precision (13.3%). The identity of the putative GSH-bimane derivative was confirmed indirectly by gas chromatography-mass spectrometry. GSH comprised 77% of water-extractable thiols in pine needles, and the ratio GSSG/GSH was low (0.024).
Michael A. Baldwin - One of the best experts on this subject based on the ideXlab platform.
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Reactivity of zinc finger cysteines: chemical modifications within labile zinc fingers in estrogen receptor.
Journal of the American Society for Mass Spectrometry, 2005Co-Authors: Christian Atsriku, Gary K. Scott, Christopher C. Benz, Michael A. BaldwinAbstract:Estrogen receptor (ER, alpha isoform) is a 67 kDa zinc finger transcription factor that plays a fundamental role in both normal reproductive gland development and breast carcinogenesis, and also represents a critical molecular target for breast cancer therapy. We are investigating the structural consequences of chemical exposures thought to modify essential zinc finger cysteine residues in human ER. The current study employs mass spectrometry to probe ER zinc finger structural changes induced by a redox-reactive vitamin K3 analog, menadione; a commonly used cysteine alkylator, iodoacetic acid; and a thiol alkylating fluorophore, monoBromobimane. Although they are slower to react, the sterically bulkier reagents, monoBromobimane and menadione, effectively alkylate the most susceptible ER zinc finger cysteine sulfhydryl groups. Menadione arylation results first in Michael addition of the hydroquinone followed by rapid oxidation to the corresponding quinone, evidenced by a 2 Da mass loss per cysteine residue. Mass spectrometric analysis performed under MALDI conditions reveals both hydroquinone and quinone forms of arylated menadione, whereas only the quinone product is detectable under ESI conditions. Tandem mass spectrometry of a synthetic peptide encompassing the C-terminal half of the structurally more labile second zinc finger of ER (ZnF2B) demonstrates that the two nucleophilic thiols in ZnF2B (Cys-237, Cys-240) are not chemically equivalent in their reactivity to Bromobimane or menadione, consistent with their unequal positioning near basic amino acids that affect thiol pKa, thereby rendering Cys-240 more reactive than Cys-237. These findings demonstrate important differential susceptibility of ER zinc finger cysteine residues to thiol reactions.
Nechama S. Kosower - One of the best experts on this subject based on the ideXlab platform.
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Bromobimane probes for thiols.
Methods in Enzymology, 2004Co-Authors: Edward M. Kosower, Nechama S. KosowerAbstract:Publisher Summary This chapter discusses Bromobimane probes for thiols and describes the use of four Bromobimanes for fluorescent labeling of biochemical and biological systems. The four Bromobimanes are (1) mBBr, (2) bBBr, (3) qBBr, and (4) SBBr. The Bromobimanes are essentially nonfluorescent and are relatively stable in the dry state when stored in the dark. Chromatography on thin-layer silica yields a yellow nonfluorescent spot that develops a blue fluorescence after several minutes of exposure to 360-nm light. The change is a convenient characteristic for the identification of the Bromobimanes and suggests sensitivity to light (photolysis). Bromobimanes in solution react with small thiols (e.g., the tripeptide thiol glutathione [GSH]), and with reactive protein thiol groups (e.g., hemoglobin). The reactions of Bromobimanes with thiols are second order and dependent on pH, the active nucleophile being the thiolate anion, such as GS − . The reaction of Bromobimane with a thiolate converts the nonfluorescent agent into water-soluble fluorescent products. The chapter discusses the chemical and photophysical properties of Bromobimanes, labeling procedures, labeling of thiols in tissues, and other applications of thiol labeling.
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fluorescent localization of thiols and disulfides in marsupial spermatozoa by Bromobimane labelling
Molecular Reproduction and Development, 1994Co-Authors: K E Mate, Nechama S. Kosower, I G White, J C RodgerAbstract:The acrosome of marsupial spermatozoa is a robust structure which, unlike its placental counterpart, resists disruption by detergent or freeze/thawing and does not undergo a calcium ionophore induced acrosome reaction. In this study specific fluorescent thiol labels, Bromobimanes, were used to detect reactive thiols in the intact marsupial spermatozoon and examine whether disulfides play a role in the stability of the acrosome. Ejaculated brushtail possum (Trichosurus vulpecula) and tammar wallaby (Macropus eugenii) spermatozoa were washed by swim up and incubated with or without dithiothreitol (DTT) in order to reduce disulfides to reactive thiols. Spermatozoa were then washed by centrifugation and treated with monoBromobimane (mBBr), a membranepermeable Bromobimane, or with monobromotrimethylammoniobimane (qBBr), a membrane-impermeable Bromobimane. Labelled spermatozoa were examined by fluorescence microscopy and sperm proteins (whole sperm proteins and basic nuclear proteins) were analysed by gel electrophoresis. The membrane-permeable agent mBBr lightly labelled the perimeter of the acrosome of non-DTT-treated possum and wallaby spermatozoa, indicating the presence of peri-acrosomal thiol groups. After reduction of sperm disulfides by DTT, mBBr labelled the entire acrosome of both species. The membrane-impermeable agent qBBr did not label any part of the acrosome in non-DTT or DTT-treated wallaby or possum spermatozoa. Thiols and disulfides are thus associated with the marsupial acrosome. They are not found on the overlying plasma membrane but are either in the acrosomal membranes and/or matrix. The sperm midpiece and tail were labelled by mBBr, with increased fluorescence observed in DTT-treated spermatozoa. The nucleus was not labelled in non-DTT or DTT-treated spermatozoa. Electrophoretic analysis confirmed the microscopic observations: Basic nuclear protein (protamines) lacked thiols or disulfide groups. Based on these findings, the stability of the marsupial acrosome may be due in part to disulfide stabilization of the acrosomal membranes and/or acrosomal matrix. In common with placental mammals, thiol and disulfide containing proteins appear to play a role in the stability of sperm tail structures. It was confirmed that the fragile marsupial sperm nucleus lacked thiols and disulfides. © 1994 Wiley-Liss, Inc.
Gerald L Newton - One of the best experts on this subject based on the ideXlab platform.
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determination of biothiols by Bromobimane labeling and high performance liquid chromatography
Methods in Enzymology, 1995Co-Authors: Gerald L Newton, Robert C FaheyAbstract:Publisher Summary This chapter discusses the determination of biothiols by Bromobimane labeling and high-performance liquid chromatography (HPLC). Most thiols undergo rapid autoxidation when exposed to air at neutral or basic pH, a process that is catalyzed by heavy metals of the type found in most cells. A primary consideration in preparing and analyzing samples for thiols is to prevent their oxidative loss during the sample preparation and analysis. This can be accomplished by alkylation of the thiol group near neutral pH with the Bromobimanes. The chapter focuses on monoBromobimane (mBBr), an uncharged reagent that readily penetrates cells, 1-4 and 4- p -sulfobenzoyloxymethyl-6-bromomethy-1-3,7-dimethyl-1,5-diazabicyclo [3.3.0] octa-3,6-diene-2,8-dione (SBBr), an anionic reagent that does not enter cells. Many biological thiols are relatively small and ionic, which makes them easy to separate from more hydrophobic thiols but difficult to separate from each other using reversed-phase HPLC. The bimane derivatives of these ionic thiols are more hydrophobic than the thiol itself and are readily resolved by HPLC analysis on silica-based reversed-phase columns. The highly selective, rapid reactivity of Bromobimanes toward thiols, the stability and fluorescence yield of the thiol derivatives, the ease of separation of the derivatives by reversed-phase HPLC, and the availability of both cell-penetrating and nonpenetrating forms make the use Bromobimanes an extremely powerful approach to the analysis of low-molecular-weight biothiols.
