A fluorometric method to quantify protein glutathionylation using glutathione derivatization with 2,3-naphthalenedicarboxaldehyde

Deepthi Menon, Philip G. Board*

*Corresponding author for this work

    Research output: Contribution to journalArticlepeer-review

    21 Citations (Scopus)

    Abstract

    This study reports the development of a new assay for the rapid determination of protein glutathionylation in tissues and cell lines using commercially available reagents and standard instrumentation. In this method cells are homogenized in the presence of N-ethylmaleimide to eliminate free thiols and the proteins are precipitated with acetone. Subsequently, the disulfide-bound glutathione is eluted from the protein by the addition of tris(2-carboxyethyl)phosphine and reacted with 2,3-napthalenedicarboxaldehyde to generate a highly fluorescent product. Lymphoblastoid cell lines were found to have glutathionylation levels in the range of 0.3-3 nmol/mg protein, which were significantly elevated after treatment of the cells with S-nitrosoglutathione. Mouse tissues including liver, kidney, lung, heart, brain, spleen, and testes were found to have glutathionylation levels between 1 and 2.5 nmol/mg protein and the levels tended to increase after treatment of mice with doxorubicin. In contrast, mouse skeletal muscle glutathionylation was significantly higher (4.2 ± 0.33 nmol/mg, p < 0.001) than in other tissues in untreated mice and decreased to 1.9 ± 0.15 nmol/mg after doxorubicin treatment. This new method allows rapid measurement of cellular glutathionylation in a high-throughput 96-well plate format.

    Original languageEnglish
    Pages (from-to)132-136
    Number of pages5
    JournalAnalytical Biochemistry
    Volume433
    Issue number2
    DOIs
    Publication statusPublished - 15 Feb 2013

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