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An Amplex Red-based fluorometric and spectrophotometric assay forl-asparaginase using its natural substrateNotes & Tips

Authors
Journal
Analytical Biochemistry
0003-2697
Publisher
Elsevier
Publication Date
Volume
445
Identifiers
DOI: 10.1016/j.ab.2013.09.028
Keywords
  • L-Asparaginase
  • Leukemia
  • L-Aspartate Oxidase
  • Coupled Enzyme Assay
  • Amplex Red

Abstract

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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