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  An Amplex Red-based fluorometric and spectrophotometric assay for L-asparaginase using its natural substrate.

Karamitros, C. S., Lim, J., & Konrad, M. (2013). An Amplex Red-based fluorometric and spectrophotometric assay for L-asparaginase using its natural substrate. Analytical Biochemistry, 445, 20-23. doi:10.1016/j.ab.2013.09.028.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0014-CF29-6 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-0028-EB85-F
Genre: Journal Article

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 Creators:
Karamitros, C. S.1, Author              
Lim, J.1, Author              
Konrad, M.1, Author              
Affiliations:
1Research Group of Enzyme Biochemistry, MPI for biophysical chemistry, Max Planck Society, ou_578612              

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Free keywords: L-Asparaginase; Leukemia; L-Aspartate oxidase; Coupled enzyme assay; Amplex Red
 Abstract: We report on the development of a sensitive real-time assay for monitoring the activity of L-asparaginase that hydrolyzes L-asparagine to L-aspartate and ammonia. In this method, L-aspartate is oxidized by L-aspartate oxidase to iminoaspartate and hydrogen peroxide (H2O2), and in the detection step horseradish peroxidase uses H2O2 to convert the colorless, nonfluorescent reagent Amplex Red to the red-colored and highly fluorescent product resorufin. The assay was validated in both the absorbance and the fluorescence modes. We show that, due to its high sensitivity and substrate selectivity, this assay can be used to measure enzymatic activity in human serum containing L-asparaginase.

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Language(s): eng - English
 Dates: 2013-10-07
 Publication Status: Published online
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 Rev. Method: Peer
 Identifiers: DOI: 10.1016/j.ab.2013.09.028
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Title: Analytical Biochemistry
Source Genre: Journal
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Pages: - Volume / Issue: 445 Sequence Number: - Start / End Page: 20 - 23 Identifier: -