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A high-throughput fluorimetric assay for angiotensin I-converting enzyme

Nature Protocols volume 1, pages 1961–1964 (2006)Cite this article

Abstract

Angiotensin I-converting enzyme (ACE) assays are commonly used for measuring enzymatic activity in clinical and biological samples. The fluorimetric procedure described is sensitive, rapid and involves unsophisticated procedures and inexpensive reagents. It uses the substrate hippuryl-L-histidyl-L-leucine, and the fluorescent adduct of the enzyme-catalyzed product L-histidyl-L-leucine is quantified fluorimetrically. This assay has been adapted for a 96-well plate format that produces comparable data to previously described assays and has the advantage of greater efficiency with respect to both time and reagents. The protocol can be used for routine purposes or for more detailed kinetic analyses. The apparent Km and kcat values for purified testis ACE determined from a double reciprocal plot were 3.0 mM and 195.7 s−1, respectively. The protocol can be completed within 4 h.

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Figure 1: Standard curve of HL versus fluorescence intensity at excitation 360 nm and emission 485 nm.
Figure 2
Figure 3: The rate of HL formation catalyzed by purified recombinant testis ACE versus HHL concentration (mM).

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Acknowledgements

This work was supported by the South African National Research Foundation, the Wellcome Trust (UK) grant 070060.

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Authors and Affiliations

  1. Division of Medical Biochemistry, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Observatory, 7925, South Africa

    Sylva L Schwager & Edward D Sturrock

  2. Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, SP, Brazil

    Adriana K Carmona

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  1. Sylva L Schwager
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Correspondence to Edward D Sturrock.

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Schwager, S., Carmona, A. & Sturrock, E. A high-throughput fluorimetric assay for angiotensin I-converting enzyme. Nat Protoc 1, 1961–1964 (2006). https://doi.org/10.1038/nprot.2006.305

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