Assay of thiols and disulfides based on the reversibility of N-ethylmaleimide alkylation of thiols combined with electrolysis

Nishiyama, Junko; Kuninori, Toyo

doi:10.1016/0003-2697(92)90457-i

Cited by 20 publications

(17 citation statements)

References 8 publications

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“…Maleimides on the other hand are more reactive towards amine residues, and at higher pH's this reaction is favored [22,23]. However, hydrolysis of maleimides to a mixture of isomeric maleamic acids can compete significantly with thiol modification particularly above pH 8 [24,25]. Thus careful attention must be given to the use of maleimide dyes in protein labeling, both with respect to the dye:thiol ratio and the pH of the labeling reaction, in order to specifically label Cys residues.…”

Section: Discussionmentioning

confidence: 96%

Thiol‐reactive dyes for fluorescence labeling of proteomic samples

2003

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Covalent derivatization of proteins with fluorescent dyes prior to separation is increasingly used in proteomic research. This paper examines the properties of several commercially available iodoacetamide and maleimide dyes and discusses the conditions and caveats for their use in labeling of proteomic samples. The iodoacetamide dyes BODIPY TMR cadaverine IA and BODIPY Fl C(1)-IA were highly specific for cysteine residues and showed little or no nonspecific labeling even at very high dye:thiol ratios. These dyes also showed minimal effects on pI's of standard proteins. Some iodoacetamide dyes, (5-TMRIA and eosin-5-iodoacetamide) and some maleimide dyes (ThioGlo I and Rhodamine Red C(2) maleimide) exhibited nonspecific labeling at high dye:thiol ratios. Labeling by both iodoacetamide and maleimide dyes was inhibited by tris(2-carboxyethyl)phosphine (TCEP); interactions between TCEP and dye were also observed. Thiourea, an important component of sample solubilization cocktails, inhibited labeling of proteins with iodoacetamide dyes but not with maleimide dyes. Maleimide dyes may serve as an alternative for labeling proteins where it is essential to have thiourea in the solubilization buffer. Covalent derivatization by BODIPY TMR cadaverine IA, BODIPY Fl C(1)-IA or Rhodamine Red C(2) maleimide was also demonstrated to be compatible with in-gel digestion and peptide mass fingerprinting by matrix assisted laser desorption/ionization-mass spectrometry and allowed successful protein identification.

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Section: Discussionmentioning

confidence: 96%

Thiol‐reactive dyes for fluorescence labeling of proteomic samples

2003

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“…For a more direct assessment of the selectivity of the method, it has been tried to block all the thiolic groups of native proteins by derivatization with N-ethylmaleimide (NEM) (33), but the results were not conclusive. Indeed, although the NEM treatment decreased the number of mercury-accessible ϪSH groups, it was not clear if it could be addressed to the nonselectivity of the binding of mercury or to the circumstance that NEM failed to block all the mercury-accessible ϪSH groups of native proteins.…”

Section: Consideration About Selectivity Of Cvafs Methodsmentioning

confidence: 99%

Application of Mercury Cold Vapor Atomic Fluorescence Spectrometry to the Characterization of Mercury-Accessible −SH Groups in Native Proteins

Bramanti¹,

D’Ulivo²,

Lampugnani³

et al. 1999

Analytical Biochemistry

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“…The solution was found to contain 45 µM of papain according to absorbance at 278 nm (ε 278 = 58,500 M -1 cm -1 , [4]). Cysteine hydrochloride (60 mM, 47.3 mg in 25 mL) was dissolved in argon-bubbled buffer 6.7 and stored under argon (the tube was stoppered with a rubber septum and the gas phase was perfused for 5 min with argon via two hypodermic needles while the solution was stirred to exchange dissolved air for argon).…”

Section: Reactivation Of Papain With Cysteinementioning

confidence: 99%

“…As a consequence, the quantitative measurement of SH groups is a routine task in many applied disciplines where a quick and easy method is much preferred. Electrochemical [3,4] and fluorimetric assays [5,6,7,8] of protein sulfhydryls are very sensitive and accurate but they involve lengthy procedures (complete proteolysis, electrolysis, HPLC separation). Sensitivity and reliability are also good in an enzymatic method where protein sulfhydryls are first reacted with cystamine and the released cysteamine cleaves papain-S-S-CH 3 , yielding active papain-SH which is then assayed with a fluorigenic substrate [9].…”

Section: Introductionmentioning

confidence: 99%

Quick measurement of protein sulfhydryls with Ellman's reagent and with 4,4′-dithiodipyridine

2002

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Since its introduction in 1959, Ellman's reagent (5,5'-dithio-bis(2-nitrobenzoic acid)) has been the favorite reagent for spectrophotometric measurement of protein sulfhydryls. Meanwhile however, evidence has accumulated that many protein sulfhydryls give an incomplete reaction with Ellman's reagent, even during prolonged assay times. In the present study, the kinetic problem was solved by including cystamine as a "mediator" between the protein sulfhydryl and Ellman's reagent, as previously applied in an enzymatic thiol assay [9]. As an alternative, 4,4'-dithiodipyridine (DTDP) was used in place of Ellman's reagent. Due to its small size, amphiphilic nature, and lack of charge, DTDP quickly reacts with poorly accessible protein sulfhydryls, without any catalysis by cystamine. The DTDP method and the Ellman/cystamine method were both optimized for maximal sensitivity, minimal sample consumption (detection limit 0.2 nmol mL(-1), determination limit 0.6 nmol mL(-1)), and minimal assay time (5 min). In validation experiments, both methods gave identical results and the measured sulfhydryls/protein matched the expected values. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00216-002-1347-2.

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Assay of thiols and disulfides based on the reversibility of N-ethylmaleimide alkylation of thiols combined with electrolysis

Cited by 20 publications

References 8 publications

Thiol‐reactive dyes for fluorescence labeling of proteomic samples

Thiol‐reactive dyes for fluorescence labeling of proteomic samples

Application of Mercury Cold Vapor Atomic Fluorescence Spectrometry to the Characterization of Mercury-Accessible −SH Groups in Native Proteins

Quick measurement of protein sulfhydryls with Ellman's reagent and with 4,4′-dithiodipyridine

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