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Fluorimetric Assay for Measuring Neprilysin Activity Using HPLC

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Abstract

A rapid and reproducible assay for the determination of neprilysin activity is reported. This method is based on fluorimetric detection of a dansylated dipeptide, 5-dimethylaminonaphthalene-1-sulfonyl-d-Ala-Gly, enzymatically formed from the substrate 5-dimethylaminonaphthalene-1-sulfonyl-d-Ala-Gly-Phe(pNO2)-Gly after separation by high-performance liquid chromatography using a C-18 reversed-phase column by isocratic elution. The detection limit of this method is at a level as low as 0.2 nmol mL−1, it yields highly reproducible results and the time consumed for analysis is less than 6.8 min per sample for separation and quantification. By using this assay, the stimulatory effect of (+)MK-801 (dizocilpine maleate)—a noncompetitive N-methyl-[d]-aspartate receptor antagonist—on this enzyme activity was observed in rat brain regions 3 days after the treatment. This new method would be useful for clarification of the physiological role of this enzyme.

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Authors and Affiliations

  1. Department of Analytical Chemistry of Medicines, Showa Pharmaceutical University, 3-3165 Higashi-tamagawagakuen, Machida, Tokyo, 194-8543, Japan

    Gou Takahashi, Miki Tabata & Toshiyuki Chikuma

  2. Department of Medicinal Pharmacology, Showa Pharmaceutical University, Machida, Tokyo, 194-8543, Japan

    Kyoji Taguchi

Authors
  1. Gou Takahashi
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  2. Miki Tabata
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  3. Kyoji Taguchi
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  4. Toshiyuki Chikuma
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Correspondence to Toshiyuki Chikuma.

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Takahashi, G., Tabata, M., Taguchi, K. et al. Fluorimetric Assay for Measuring Neprilysin Activity Using HPLC. Chromatographia 78, 593–597 (2015). https://doi.org/10.1007/s10337-015-2856-4

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  • DOI: https://doi.org/10.1007/s10337-015-2856-4

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