Using UV-absorbance of intrinsic dithiothreitol (DTT) during RP-HPLC as a measure of experimental redox potential in vitro.
Anal Bioanal Chem
; 405(19): 6379-84, 2013 Jul.
Article
en En
| MEDLINE
| ID: mdl-23743664
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.
Texto completo:
1
Base de datos:
MEDLINE
Asunto principal:
Cromatografía Líquida de Alta Presión
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Ditiotreitol
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Cromatografía de Fase Inversa
Tipo de estudio:
Evaluation_studies
Idioma:
En
Revista:
Anal Bioanal Chem
Año:
2013
Tipo del documento:
Article