Analytical and Bioanalytical Chemistry

, Volume 405, Issue 19, pp 6379–6384 | Cite as

Using UV-absorbance of intrinsic dithiothreitol (DTT) during RP-HPLC as a measure of experimental redox potential in vitro

  • Angie Seo
  • Janelle L. Jackson
  • Jolene V. Schuster
  • Didem Vardar-Ulu


Many in-vitro experiments performed to study the response of thiol-containing proteins to changes in environmental redox potentials use dithiothreitol (DTT) to maintain a preset redox environment throughout the experiments. However, the gradual oxidation of DTT during the course of the experiments, and the interaction between DTT and other components in the system, can significantly alter the initial redox potential and complicate data interpretation. Having an internal reporter of the actual redox potential of the assayed sample facilitates direct correlation of biochemical findings with experimental redox status. Reversed-phase high-performance liquid chromatography (RP-HPLC) is a widely used, well-established tool for analysis and purification of biomolecules, including proteins and peptides. Here, we describe a simple, robust, and quantitative RP-HPLC method we developed and tested for determination of the experimental redox potential of an in-vitro sample at the time of the experiment. It exploits the specific UV-absorbance of the oxidized intrinsic DTT in the samples and retains the high resolving power and high sensitivity of RP-HPLC with UV detection.


Reversed-phase high-performance liquid chromatography (RP-HPLC) Dithiothreitol (DTT) Redox potential Nernst equation Disulfide bond UV-absorbance 



Partial financial support for this work was provided by the Wellesley College Sophomore Early Research Program to AS. JLJ was funded by a Howard Hughes Medical Institute IV Award DVU and JVS was funded by Camille and Henry Dreyfus Start-up Award and NIH/NCI grant number R15CA143892-01A1.

Supplementary material

216_2013_7063_MOESM1_ESM.pdf (69 kb)
ESM 1 (PDF 69.0 KB)


  1. 1.
    Herrmann JM, Jakob U (2008) Special issue: redox regulation of protein folding. Preface. Biochim Biophys Acta 1783(4):519CrossRefGoogle Scholar
  2. 2.
    Narayan M (2012) Disulfide bonds: protein folding and subcellular protein trafficking. FEBS J 279(13):2272–82CrossRefGoogle Scholar
  3. 3.
    Okumura M, Shimamoto S, Hidaka Y (2012) A chemical method for investigating disulfide-coupled peptide and protein folding. FEBS J 279(13):2283–95CrossRefGoogle Scholar
  4. 4.
    Sato Y, Inaba K (2012) Disulfide bond formation network in the three biological kingdoms, bacteria, fungi and mammals. FEBS J 279(13):2262–71CrossRefGoogle Scholar
  5. 5.
    Jones DP (2008) Radical-free biology of oxidative stress. Am J Physiol Cell Physiol 295(4):C849–68CrossRefGoogle Scholar
  6. 6.
    Gilbert HF (1996) Thiol/disulfide exchange and redox potentials of proteins. In: Lenaz G, Milazzo G (eds) Bioelectrochemistry of Biomacromolecules. Birkhäuser, Basel, pp 256–324Google Scholar
  7. 7.
    Cleland WW (1964) Dithiothreitol, a New Protective Reagent for Sh Groups. Biochemistry 3:480–2CrossRefGoogle Scholar
  8. 8.
    Getz EB et al (1999) A comparison between the sulfhydryl reductants tris(2-carboxyethyl)phosphine and dithiothreitol for use in protein biochemistry. Anal Biochem 273(1):73–80CrossRefGoogle Scholar
  9. 9.
    (2011)The Grace Davison Discovery Sciences, Vydac Reference Handbook: Protein & Peptide Analysis and Purification, G.D.D. Sciences, Editor W. R. Grace & Co.-ConnGoogle Scholar
  10. 10.
    Jones DP (2002) Redox potential of GSH/GSSG couple: assay and biological significance. Methods Enzymol 348:93–112CrossRefGoogle Scholar
  11. 11.
    Vardar D et al (2003) Nuclear magnetic resonance structure of a prototype Lin12-Notch repeat module from human Notch1. Biochemistry 42(23):7061–7CrossRefGoogle Scholar
  12. 12.
    Krezel A et al (2001) Coordination of heavy metals by dithiothreitol, a commonly used thiol group protectant. J Inorg Biochem 84:77–88CrossRefGoogle Scholar
  13. 13.
    Charrier JG, Anastasio C (2012) On dithiothreitol (DTT) as a measure of oxidative potential for ambient particles: evidence for the importance of soluble transition metals. Atmos Chem Phys 12(5):11317–11350CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Angie Seo
    • 1
  • Janelle L. Jackson
    • 1
  • Jolene V. Schuster
    • 2
  • Didem Vardar-Ulu
    • 1
  1. 1.Department of ChemistryWellesley CollegeWellesleyUSA
  2. 2.Science DepartmentBlair AcademyBlairstownUSA

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