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ARS Home » Midwest Area » Urbana, Illinois » Global Change and Photosynthesis Research » Research » Publications at this Location » Publication #208808
Title: Measurement of reduced, oxidized and total ascorbate content in plants

Author
item GILLESPIE, KELLY - UNIVERSITY OF ILLINOIS
item Ainsworth, Elizabeth - Lisa

Submitted to: Nature Protocols
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/9/2007
Publication Date: 4/12/2007
Citation: Gillespie, K.M., Ainsworth, E.A. 2007. Measurement of reduced, oxidized and total ascorbate content in plants. Nature Protocols. 2:871-874.

Interpretive Summary: Ascorbate is one of the major antioxidant metabolites in plant tissues. This protocol describes a microplate-adapted colorimetric ascorbate assay, in which ferric ion is reduced by ascorbate to the ferrous ion. The ferrous ion reacts with alpha-bipyridl to form a complex with characteristic absorbance at 525 nm. By chemically reducing any dehydroascorbate in a sample, total ascorbate can be assayed by the alpha-bipyridl method, and dehydroascorbate estimated by subtracting the reduced portion from the total ascorbate pool. The assay is performed in microcentrifuge tubes and assessed in a 96-well plate reader. This protocol is provided along with rapid, microplate-based protocols for measuring oxygen radical absorbance capacity and total phenolic content in plant tissues. Collectively, these assays provide scientists and researchers with general diagnostic tools for assessing the antioxidant capacity of leaf tissue extracts.

Technical Abstract: Ascorbate is one of the major antioxidant metabolites in plant tissues. This protocol describes a microplate-adapted colorimetric ascorbate assay, in which ferric ion is reduced by ascorbate to the ferrous ion. The ferrous ion reacts with alpha-bipyridl to form a complex with characteristic absorbance at 525 nm. By chemically reducing any dehydroascorbate in a sample, total ascorbate can be assayed by the alpha-bipyridl method, and dehydroascorbate estimated by subtracting the reduced portion from the total ascorbate pool. The assay is performed in microcentrifuge tubes and assessed in a 96-well plate reader. With the described protocol, reduced ascorbate, dehydroascorbate, and total ascorbate of at least 64 experimental samples easily can be analyzed in one day.