Determination of low-molecular-weight thiols using monobromobimane fluorescent labeling and high-performance liquid chromatography

Fahey, Robert C.; Newton, Gerald L.

doi:10.1016/0076-6879(87)43016-4

Cited by 350 publications

(223 citation statements)

References 14 publications

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“…Four pellets (∼0.5 mL each) were extracted with 2.5 mL of 50% acetonitrile in 20 mM N- [2-hydroxyethyl]piperazine-N' -[2-ethanesulfonic acid], pH 8, containing 2 mM mBBr (Calbiochem), and incubated at 60°C for 15 min (26). The extract was cooled on ice and acidified with 5 μL of 5 M methanesulfonic acid.…”

Section: Analysis Of Thiols From B Anthracismentioning

confidence: 99%

Structure of the Type III Pantothenate Kinase from Bacillus anthracis at 2.0 Å Resolution: Implications for Coenzyme A-Dependent Redox Biology^,

Parsonage

Paige

et al. 2007

Biochemistry

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Coenzyme A (CoASH) is the major low-molecular weight thiol in Staphylococcus aureus and a number of other bacteria; the crystal structure of the S. aureus coenzyme A-disulfide reductase (CoADR), which maintains the reduced intracellular state of CoASH, has recently been reported [Mallett, T.C., Wallen, J.R., Karplus, P.A., Sakai, H., Tsukihara, T., and Claiborne, A. (2006) Biochemistry 45, 11278-11289]. In this report we demonstrate that CoASH is the major thiol in Bacillus anthracis; a bioinformatics analysis indicates that three of the four proteins responsible for † This work was supported by National Institutes of Health (NIH) Grants GM-35394 (A.C), AI-49174 (R.C.F.), and GM-62896 (S.J.), by a grant from the Southeast Regional Center of Excellence for Biodefense and Emerging Infections (SERCEB, to A.C.), and by Cancer Center (CORE) Support Grant CA21765 and ALSAC (S.J.). C.P. was the recipient of a Graduate Fellowship from the U.S. Department of Homeland Security (DHS). SERCEB is supported by an award from the NIH (National Institute of Allergy and Infectious Diseases; NIAID). The DHS Scholarship and Fellowship Program is administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement with the U.S. Department of Energy (DOE). ORISE is managed by Oak Ridge Associated Universities under DOE contract number DE-AC05-06OR23100. The findings, opinions, and recommendations expressed in this paper are those of the authors and are not necessarily those of NIAID, SERCEB, NIH, DHS, DOE, or ORISE. Data for this study were measured at beamline X12C of the National Synchrotron Light Source. With the identification of two distinct new bacterial pantothenate kinase classes in 2005, different investigators have used either type I, -II, and -III CoaAs (e.g., type II Staphylococcus aureus CoaA) or PanK-I, -II, and -III nomenclature to identify the three bacterial enzyme classes. In this report we refer to the three bacterial enzyme classes as bacterial type I, type II, and type III PanKs; CoaA is synonymous with the type I PanK. 5 J. Ravel, personal communication. Supporting Information Available A figure depicting a structure-based alignment of 13 type III PanK sequences ( Figure S1) is included as Supporting Information (one page). This material is available free of charge via the Internet at http://pubs.acs.org NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2009 January 5. In contrast, a novel type III pantothenate kinase (PanK) catalyzes the first committed step in the biosynthetic pathway in B. anthracis; unlike the E. coli type I PanK, this enzyme is not subject to feedback inhibition by CoASH. The crystal structure of B. anthracis PanK (BaPanK), solved using multiwavelength anomalous dispersion data and refined at a resolution of 2.0 Å, demonstrates that BaPanK is a new member of the Acetate and Sugar Kinase/Hsc70/Actin (ASKHA) superfamily. The Pan and ATP substrates have been modeled into the active-site cleft; in addition to prov...

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Section: Analysis Of Thiols From B Anthracismentioning

confidence: 99%

Structure of the Type III Pantothenate Kinase from Bacillus anthracis at 2.0 Å Resolution: Implications for Coenzyme A-Dependent Redox Biology^,

Parsonage

Paige

et al. 2007

Biochemistry

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“…Thiol assays mBBr fluorescent labelling and HPLC methods were used to determine the intracellular thiol (cysteine, γ-GC and glutathione) contents according to methods in the literature (Fahey and Newton 1987). A 90 μl aliquot of a cell suspension was added to 200 μl 50 mM HEPPSO (pH 7.5) containing 50% aqueous acetonitrile, 1 mM dithiothreitol, and 5 mM DTPA, following which 5 μl mBBr (50 mM in acetonitrile) was added.…”

Section: Construction Of Strains and Plasmidsmentioning

confidence: 99%

Using Lactococcus lactis for glutathione overproduction

Hugenholtz

Sybesma

et al. 2004

Appl Microbiol Biotechnol

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Glutathione and γ-glutamylcysteine were produced in Lactococcus lactis using a controlled expression system and the genes gshA and gshB from Escherichia coli encoding the enzymes γ-glutamylcysteine synthetase and glutathione synthetase. High levels of γ-glutamylcysteine were found in strains growing on chemically defined medium and expressing either gshA alone or both gshA and gshB. As anticipated, glutathione was found in a strain expressing gshA and gshB. The level of glutathione production could be increased by addition of the precursor amino acid cysteine to the medium. The addition of cysteine led to an increased activity of glutathione synthetase, which is remarkable because the amino acid is not a substrate of this enzyme. The final intracellular glutathione concentration attained was 358 nmol mg −1 protein, which is the highest concentration reported for a bacterium, demonstrating the suitability of engineered L. lactis for fine-chemical production and as a model for studies of the impact of glutathione on flavour formation and other properties of food.

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“…However, cysteine and thiosulfate, which elute near 6 min, are poorly separated, and Coenzyme A elutes at 1*5 min as a peak too broad to detect at low CoA levels. Coenzyme A elutes as a sharp peak at 26 min and is easily quantitated using a tetrabutylammonium ion pairing system (Method 2, Fahey and Newton, 1986), and cysteine and thiosulfate vere also well resolved in this protocol, eluting at 6 and 21 min, respectively. The use of these two different protocols for analysis of all samples provided reasonable assurance as to the correctness of the peak assignments.…”

Section: Methodsmentioning

confidence: 99%

“…A combination of two HPLC protocols was used to achieve full resolution of the low molecular weight thiols. The reverse phase protocol (Method l) described by Fahey and Newton (1986) resolved most of the compounds listed in Tables 1-3. However, cysteine and thiosulfate, which elute near 6 min, are poorly separated, and Coenzyme A elutes at 1*5 min as a peak too broad to detect at low CoA levels.…”

Section: Methodsmentioning

confidence: 99%

Evolution of glutathione metabolism in phototrophic microorganisms

Fahey

Buschbacher

Newton

1986

Origins Life Evol Biosphere

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Determination of low-molecular-weight thiols using monobromobimane fluorescent labeling and high-performance liquid chromatography

Cited by 350 publications

References 14 publications

Structure of the Type III Pantothenate Kinase from Bacillus anthracis at 2.0 Å Resolution: Implications for Coenzyme A-Dependent Redox Biology^,

Structure of the Type III Pantothenate Kinase from Bacillus anthracis at 2.0 Å Resolution: Implications for Coenzyme A-Dependent Redox Biology^,

Using Lactococcus lactis for glutathione overproduction

Evolution of glutathione metabolism in phototrophic microorganisms

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Determination of low-molecular-weight thiols using monobromobimane fluorescent labeling and high-performance liquid chromatography

Cited by 350 publications

References 14 publications

Structure of the Type III Pantothenate Kinase from Bacillus anthracis at 2.0 Å Resolution: Implications for Coenzyme A-Dependent Redox Biology,

Structure of the Type III Pantothenate Kinase from Bacillus anthracis at 2.0 Å Resolution: Implications for Coenzyme A-Dependent Redox Biology,

Using Lactococcus lactis for glutathione overproduction

Evolution of glutathione metabolism in phototrophic microorganisms

Contact Info

Product

Resources

About

Structure of the Type III Pantothenate Kinase from Bacillus anthracis at 2.0 Å Resolution: Implications for Coenzyme A-Dependent Redox Biology^,

Structure of the Type III Pantothenate Kinase from Bacillus anthracis at 2.0 Å Resolution: Implications for Coenzyme A-Dependent Redox Biology^,