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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
Note

Abstract

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.

Keywords

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

Notes

Acknowledgments

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)

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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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