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Journal Article

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

MPS-Authors
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Karamitros,  C. S.
Research Group of Enzyme Biochemistry, MPI for biophysical chemistry, Max Planck Society;

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Lim,  J.
Research Group of Enzyme Biochemistry, MPI for biophysical chemistry, Max Planck Society;

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Konrad,  M.
Research Group of Enzyme Biochemistry, MPI for biophysical chemistry, Max Planck Society;

Fulltext (public)

1878116.pdf
(Publisher version), 545KB

Supplementary Material (public)

1878116_Suppl_1.doc
(Supplementary material), 3MB

Citation

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.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0014-CF29-6
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.