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

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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.

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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.

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Authors and Affiliations

  1. Department of Chemistry, Wellesley College, 106 Central Street, Wellesley, MA, 02481, USA

    Angie Seo, Janelle L. Jackson & Didem Vardar-Ulu

  2. Science Department, Blair Academy, 2 Park Street, P.O. Box 600, Blairstown, NJ, 07825, USA

    Jolene V. Schuster

Authors
  1. Angie Seo
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  2. Janelle L. Jackson
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  3. Jolene V. Schuster
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  4. Didem Vardar-Ulu
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Corresponding author

Correspondence to Didem Vardar-Ulu.

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Seo, A., Jackson, J.L., Schuster, J.V. et al. Using UV-absorbance of intrinsic dithiothreitol (DTT) during RP-HPLC as a measure of experimental redox potential in vitro. Anal Bioanal Chem 405, 6379–6384 (2013). https://doi.org/10.1007/s00216-013-7063-2

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  • DOI: https://doi.org/10.1007/s00216-013-7063-2

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