Analytical and Bioanalytical Chemistry

, Volume 373, Issue 4–5, pp 266–276

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

  • Christian K. Riener
  • Gerald Kada
  • Hermann J. Gruber
Original Paper

Abstract

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.

Thiol Mercaptan Assay Ellman's reagent 4,4′-Dithiodipyridine 

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Supplementary material

216_2002_1347_MOESM1_ESM.pdf (20 kb)
Determination of sulfhydryl group concentration in a BSA stock solution (PDF 21 KB)

Copyright information

© Springer-Verlag 2002

Authors and Affiliations

  • Christian K. Riener
    • 1
  • Gerald Kada
    • 1
  • Hermann J. Gruber
    • 1
  1. 1.Institute of Biophysics, J. Kepler University, Altenberger Str. 69, A-4040 Linz, AustriaAustria

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