Determination of sulfhydryl groups with 2,2′- or 4,4′-dithiodipyridine

Grassetti, D.R.; Murray, John F.

doi:10.1016/0003-9861(67)90426-2

Cited by 980 publications

(443 citation statements)

References 13 publications

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“…To speed up the oxidation reaction as well as to allow direct monitoring of the reaction, the thiol groups were activated with 2,2′-dithiodipyridine (DTDP) (Grassetti and Murray 1967;Pedersen and Jacobsen 1980;Riener et al 2002) (Fig. 1a).…”

Section: Resultsmentioning

confidence: 99%

Protein-DNA chimeras for single molecule mechanical folding studies with the optical tweezers

et al. 2008

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Here we report on a method that extends the study of the mechanical behavior of single proteins to the low force regime of optical tweezers. This experimental approach relies on the use of DNA handles to specifically attach the protein to polystyrene beads and minimize the non-specific interactions between the tethering surfaces. The handles can be attached to any exposed pair of cysteine residues. Handles of different lengths were employed to mechanically manipulate both monomeric and polymeric proteins. The low spring constant of the optical tweezers enabled us to monitor directly refolding events and fluctuations between different molecular structures in quasiequilibrium conditions. This approach, which has already yielded important results on the refolding process of the protein RNase H (Cecconi et al. in Science 309: 2057-2060-2005, appears robust and widely applicable to any protein engineered to contain a pair of reactive cysteine residues. It represents a new strategy to study protein folding at the single molecule level, and should be applicable to a range of problems requiring tethering of protein molecules.

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Section: Resultsmentioning

confidence: 99%

Protein-DNA chimeras for single molecule mechanical folding studies with the optical tweezers

et al. 2008

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“…Total thiol concentration in red blood cells was obtained by the stoichiometric reaction of 4,4Ј-dithiodipyride (4-PDS) with thiols, which generates 4-thipyridone (4-TP) (Grassetti and Murray, 1967;Bonaventura et al, 1998). Washed red blood cells were lysed on ice for 30min with a 10-fold excess volume of 10mmoll -1 phosphate buffer, pH 7.6, and centrifuged at high speed to remove membrane debris prior to 4-PDS addition.…”

Section: Sulfhydryl Measurementsmentioning

confidence: 99%

“…A 4-molar excess of 4-PDS over haem was used and the reaction was allowed to proceed for 1h at room temperature. The reaction product 4-TP was quantified spectrophotometrically at 324nm using the extinction coefficient 19.8mmoll -1 cm -1 (Grassetti and Murray, 1967).…”

Section: Sulfhydryl Measurementsmentioning

confidence: 99%

Circulating nitric oxide metabolites and cardiovascular changes in the turtleTrachemys scriptaduring normoxia, anoxia and reoxygenation

Jacobsen

Hansen

Frank

et al. 2012

Journal of Experimental Biology

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SUMMARYTurtles of the genus Trachemys show a remarkable ability to survive prolonged anoxia. This is achieved by a strong metabolic depression, redistribution of blood flow and high levels of antioxidant defence. To understand whether nitric oxide (NO), a major regulator of vasodilatation and oxygen consumption, may be involved in the adaptive response of Trachemys to anoxia, we measured NO metabolites (nitrite, S-nitroso, Fe-nitrosyl and N-nitroso compounds) in the plasma and red blood cells of venous and arterial blood of Trachemys scripta turtles during normoxia and after anoxia (3h) and reoxygenation (30min) at 21°C, while monitoring blood oxygen content and circulatory parameters. Anoxia caused complete blood oxygen depletion, decrease in heart rate and arterial pressure, and increase in venous pressure, which may enhance heart filling and improve cardiac contractility. Nitrite was present at high, micromolar levels in normoxic blood, as in some other anoxia-tolerant species, without significant arterial-venous differences. Normoxic levels of erythrocyte S-nitroso compounds were within the range found for other vertebrates, despite very high measured thiol content. Fe-nitrosyl and N-nitroso compounds were present at high micromolar levels under normoxia and increased further after anoxia and reoxygenation, suggesting NO generation from nitrite catalysed by deoxygenated haemoglobin, which in turtle had a higher nitrite reductase activity than in hypoxia-intolerant species. Taken together, these data indicate constitutively high circulating levels of NO metabolites and significant increases in blood NO after anoxia and reoxygenation that may contribute to the complex physiological response in the extreme anoxia tolerance of Trachemys turtles.

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“…Therefore, the method was modified so that enzyme was the mixed disulfide, and substrate was the attacking thiolate. To compensate for the relatively slow reaction of CysI3' with DTNB, the thiol reagent 4,4'-dithiodipyridine (DPDS) (Grassetti & Murray, 1967) was substituted. DPDS was found to react rapidly with Cys'".…”

Section: Preparation and Purification Of C138s-c35smentioning

confidence: 99%

Formation and properties of mixed disulfides between thioredoxin reductase from Escherichia coli and thioredoxin: Evidence that cysteine‐138 functions to initiate dithiol‐disulfide interchange and to accept the reducing equivalent from reduced flavin

et al. 1998

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Mutation of one of the cysteine residues in the redox active disulfide of thioredoxin reductase from Escherichia coli results in C135S with CysI3' remaining or C138S with CysI3' remaining. The expression system for the genes encoding thioredoxin reductase, wild-type enzyme, C135S, and C138S has been re-engineered to allow for greater yields of protein. Wild-type enzyme and C135S were found to be as previously reported, whereas discrepancies were detected in the characteristics of C138S. It was shown that the original C138S was a heterogeneous mixture containing C138S and wild-type enzyme and that enzyme obtained from the new expression system is the correct species. C138S obtained from the new expression system having 0.1 % activity and 7% flavin fluorescence of wild-type enzyme was used in this study. Reductive titrations show that, as expected, only 1 mol of sodium dithionite/mol of FAD is required to reduce C138S. The remaining thiol in C135S and C138S has been reacted with 5,5'-dithiobis-(2-nitrobenzoic acid) to form mixed disulfides. The half time of the reaction was <5 s for in C135S and approximately 300 s for C Y S '~~ in (2138s showing that CysI3' is much more reactive. The resulting mixed disulfides have been reacted with Cys32 in C35S mutant thioredoxin to form stable, covalent adducts C138S-C35S and C135S-C35S. The half times show that Cys"' is approximately fourfold more susceptible to attack by the nucleophile. These results suggest that Cys 13' may be the thiol initiating dithiol-disulfide interchange between thioredoxin reductase and thioredoxin.Keywords: dithiol-disulfide interchange; flavoprotein; thioredoxin reductase Thioredoxin reductase from Escherichia coli is a pyridine nucleotidedisulfide oxidoreductase containing FAD and a disulfide in each active site. The redox active disulfide is composed of C Y S "~ and Cys'". Reducing equivalents move from NADPH to FAD, from Abbreviations: DTNB, 5,5'-dithiobis-(2-nitrobenzoic acid); TNB anion, 5-thio-2-nitrobenzoate anion: DTT, 1,4-dithiothreitol; DPDS, 4,4'-dithio-C138S and C135S, thioredoxin reductase in which C Y S '~~ or C Y S '~~ has dipyridine: PDS, 4-thiopyridone; IPTG, isopropyl P-D-thiogalactoside; been changed to Ser, respectively; C32S and C35S, thioredoxin in which Cys32 or has been changed to Ser, respectively: C138S-C35S, a covalent adduct where the remaining active site thiol of C138S is linked via a disulfide to the remaining active site thiol of C35S, analogous designations are used for the three other possible combinations of mutated thioredoxin reductases and thioredoxins. reduced flavin to the active site disulfide; dithiol-disulfide interchange effects reduction of the substrate thioredoxin. The redox active disulfide of thioredoxin is composed of Cys3' and Cys3'. Modification by site-directed mutagenesis of the active site in the enzyme has produced the single thiol mutants C138S (with CysI3' remaining from the active site disulfide) and C135S (with CysI3' remaining) (Prongay et al., 1989). The spectral characteristics...

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Determination of sulfhydryl groups with 2,2′- or 4,4′-dithiodipyridine

Cited by 980 publications

References 13 publications

Protein-DNA chimeras for single molecule mechanical folding studies with the optical tweezers

Protein-DNA chimeras for single molecule mechanical folding studies with the optical tweezers

Circulating nitric oxide metabolites and cardiovascular changes in the turtleTrachemys scriptaduring normoxia, anoxia and reoxygenation

Formation and properties of mixed disulfides between thioredoxin reductase from Escherichia coli and thioredoxin: Evidence that cysteine‐138 functions to initiate dithiol‐disulfide interchange and to accept the reducing equivalent from reduced flavin

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