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

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...

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Published inAnalytical and bioanalytical chemistry Vol. 373; no. 4-5; pp. 266 - 276
Main Authors Riener, Christian K, Kada, Gerald, Gruber, Hermann J
Format Journal Article
LanguageEnglish
Published Germany 01.07.2002
Subjects
Accessibility
Animals
Assaying
Calibration
Consumption
Disulfides
Dithionitrobenzoic Acid
Electronics
Humans
Mathematical analysis
Muramidase - analysis
Papain - analysis
Proteins
Proteins - analysis
Proteins - standards
Pyridines
Reagents
Reproducibility of Results
Sensitivity and Specificity
Serum Albumin, Bovine - analysis
Servers
Spectrophotometry, Ultraviolet
Sulfhydryl Compounds - analysis
Sulfhydryl Compounds - standards
Sulfhydryl Reagents
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Summary: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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ISSN:1618-2642
1618-2650
DOI:10.1007/s00216-002-1347-2