Spectrophotometric Determination of Hydrogen Sulfide

Fogo, James K.; Popowsky, Milton

doi:10.1021/ac60030a028

Cited by 568 publications

(189 citation statements)

References 7 publications

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“…3-Mercaptopyruvate:Dithiothreitol Sulfurtransferase Activity of TUM1-3-Mercaptopyruvate:dithiothreitol sulfurtransferase activities of TUM1 were quantified using the methylene blue method (32). The reaction mixtures were incubated for 3 min at 37°C in a final volume of 400 l of 50 mM Tris-HCl, 200 mM NaCl, pH 10.5.…”

Section: Methodsmentioning

confidence: 99%

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Characterization and Interaction Studies of Two Isoforms of the Dual Localized 3-Mercaptopyruvate Sulfurtransferase TUM1 from Humans

Fräsdorf

Radon

Leimkühler

2014

Journal of Biological Chemistry

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Background: Localization and identification of interaction partners of two splice variants of the human 3-mercaptopyruvate sulfurtransferase TUM1. Results: We show that TUM1 interacts with proteins involved in Moco and FeS cluster biosynthesis. Conclusion: Human TUM1 is a dual localized protein in the cytosol and mitochondria with distinct roles in sulfur transfer and interaction partners. Significance: The study contributes to the sulfur transfer pathway for the biosynthesis of sulfur-containing biofactors.

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Section: Methodsmentioning

confidence: 99%

“…-To analyze whether TUM1-Iso1 or TUM1-Iso2 stimulated L-cysteine desulfurase activity of human NFS1, each isoform was incubated with the stable NFS1⌬1-55⅐ISD11 complex and sulfide production was determined by methylene blue quantification (23,32). The results in Fig.…”

Section: Analysis Of the L-cysteine Desulfurase Activity Of Nfs1 In Tmentioning

confidence: 99%

Characterization and Interaction Studies of Two Isoforms of the Dual Localized 3-Mercaptopyruvate Sulfurtransferase TUM1 from Humans

Fräsdorf

Radon

Leimkühler

2014

Journal of Biological Chemistry

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“…Sulfite reductase activity was assayed as sulfide production, using an adaptation of the previously described method (7,12) for the colorimetric determination of sulfide through the formation of methylene blue. The complete assay procedure is given below.…”

Section: Methodsmentioning

confidence: 99%

Characterization of anaerobic sulfite reduction by Salmonella typhimurium and purification of the anaerobically induced sulfite reductase

1989

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Mutants of Salmonella typhimurium that lack the biosynthetic sulfite reductase (cysI and cysJ mutants) retain the ability to reduce sulfite for growth under anaerobic conditions (E. L. Barrett and G. W. Chang, J. Gen. Microbiol., 115:513-516, 1979). Here we report studies of sulfite reduction by a cysI mutant of S. typhimurium and purification of the associated anaerobic sulfite reductase. Sulfite reduction for anaerobic growth did not require a reducing atmosphere but was prevented by an argon atmosphere contaminated with air (<0.33%). It was also prevented by the presence of 0.1 mM nitrate, which argues against a strictly biosynthetic role for anaerobic sulfite reduction. Anaerobic growth in liquid minimal medium, but not on agar, was found to require additions of trace amounts (lo-7 M) of cysteine. Spontaneous mutants that grew under the argon contaminated with air also lost the requirement for 10-7 M cysteine for anaerobic growth in liquid. A role for sulfite reduction in anaerobic energy generation was contraindicated by the findings that sulfite reduction did not improve cell yields, and anaerobic sulfite reductase activity was greatest during the stationary phase of growth. Sulfite reductase was purified from the cytoplasmic fraction of the anaerobically grown cysI mutant and was purified 190-fold. The most effective donor in crude extracts was NADH. NADPH and methyl viologen were, respectively, 40 and 30% as effective as NADH. Oxygen reversibly inhibited the enzyme. Two highmolecular-weight proteins separated by gel filtration (Mr 360,000 and 490,000, respectively) were required for maximal activity with NADH. Indirect evidence, including in vitro complementation experiments with a cysG mutant extract, suggested that the 3609000-Mr component contains siroheme and is the terminal reductase.This component was further purified to near homogeneity and was found to consist of a single subunit of molecular weight 67,500. The anaerobic sulfite reductase showed some resemblance to the biosynthetic sulfite reductase, but apparently it has a unique, as yet unidentified function.Although much is known about sulfite reduction by the sulfate-reducing bacteria (1, 23, 28), very little is known about the anaerobic production of H2S from sulfite by members of the family Enterobacteriaceae. The ability to perform this reaction is routinely used in diagnostic laboratories to differentiate Salmonella species (H2S positive) from Escherichia coli (H2S negative). We have hypothesized that the enzymes involved may explain the observation that cysI and cysJ (biosynthetic sulfite reductase) mutants of S. typhimurium behave as prototrophs under anaerobic conditions (2), whereas E. coli cysI and cysJ mutants are auxotrophs under both aerobic and anaerobic conditions (E. L. Barrett, unpublished findings). Like E. coli, S. typhimurium mutants defective in cysG, which is required for synthesis of the siroheme prosthetic group (25), do not produce H2S from sulfite and do not behave as prototrophs under anaerobic growth conditions. The...

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“…The pH optimum of Nfs1-⌬1-55/ Isd11 was determined to be at 8.0 and the temperature optimum was at 46°C (data not shown). Enzyme assays were performed at 37°C by varying the concentrations of L-cysteine and enzyme activity was determined using the methylene blue method, detecting the release of H 2 S in the assay (28). Enzyme activity was only detectable for the Nfs1⌬1-55/Isd11 complex.…”

Section: Functional Complementation Of the E Coli Cl100(iscs ϫ ) Strmentioning

confidence: 99%

A Novel Role for Human Nfs1 in the Cytoplasm

Marelja

Stöcklein

Nimtz

et al. 2008

Journal of Biological Chemistry

108

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The human MOCS3 gene encodes a protein involved in activation and sulfuration of the C terminus of MOCS2A, the smaller subunit of the molybdopterin (MPT) synthase. MPT synthase catalyzes the formation of the dithiolene group of MPT that is required for the coordination of the molybdenum atom in the last step of molybdenum cofactor (Moco) biosynthesis. The two-domain protein MOCS3 catalyzes both the adenylation and the subsequent generation of a thiocarboxylate group at the C terminus of MOCS2A by its C-terminal rhodanese-like domain (RLD). The low activity of MOCS3-RLD with thiosulfate as sulfur donor and detailed mutagenesis studies showed that thiosulfate is most likely not the physiological sulfur source for Moco biosynthesis in eukaryotes. It was suggested that an L-cysteine desulfurase might be involved in the sulfuration of MOCS3 in vivo. In this report, we investigated the involvement of the human L-cysteine desulfurase Nfs1 in sulfur transfer to MOCS3-RLD. A variant of Nfs1 was purified in conjunction with Isd11 in a heterologous expression system in Escherichia coli, and the kinetic parameters of the purified protein were determined. By studying direct protein-protein interactions, we were able to show that Nfs1 interacted specifically with MOCS3-RLD and that sulfur is transferred from L-cysteine to MOCS3-RLD via an Nfs1-bound persulfide intermediate. Because MOCS3 was shown to be located in the cytosol, our results suggest that cytosolic Nfs1 has an important role in sulfur transfer for the biosynthesis of Moco.

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Spectrophotometric Determination of Hydrogen Sulfide

Cited by 568 publications

References 7 publications

Characterization and Interaction Studies of Two Isoforms of the Dual Localized 3-Mercaptopyruvate Sulfurtransferase TUM1 from Humans

Characterization and Interaction Studies of Two Isoforms of the Dual Localized 3-Mercaptopyruvate Sulfurtransferase TUM1 from Humans

Characterization of anaerobic sulfite reduction by Salmonella typhimurium and purification of the anaerobically induced sulfite reductase

A Novel Role for Human Nfs1 in the Cytoplasm

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