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A continuous fluorescence resonance energy transfer angiotensin I-converting enzyme assay

Nature Protocols volume 1, pages 1971–1976 (2006)Cite this article

Abstract

Angiotensin I-converting enzyme (ACE) is involved in various physiological and physiopathological conditions; therefore, the measurement of its catalytic activity may provide essential clinical information. This protocol describes a sensitive and rapid procedure for determination of ACE activity using fluorescence resonance energy transfer (FRET) substrates containing o-aminobenzoic acid (Abz) as the fluorescent group and 2,4-dinitrophenyl (Dnp) as the quencher acceptor. Hydrolysis of a peptide bond between the donor/acceptor pair generates fluorescence that can be detected continuously, allowing quantitative measurement of the enzyme activity. The FRET substrates provide a useful tool for kinetic studies and for ACE determination in biological fluids and crude tissue extracts. An important benefit of this method is the use of substrates selective for the two active sites of the enzyme, namely Abz-SDK(Dnp)P-OH for N-domain, Abz-LFK(Dnp)-OH for C-domain and Abz-FRK(Dnp)P-OH for somatic ACE. This methodology can be adapted for determinations using a 96-well fluorescence plate reader.

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Figure 1: Schematic representation of the FRET peptide mechanism with the Abz/Dnp donor/acceptor pair.
Figure 2
Figure 3
Figure 4: Determination of total hydrolysis of Abz-FRK(Dnp)P-OH by ACE.
Figure 5: Michaelis–Menten curve for the hydrolysis of Abz-FRK(Dnp)-P-OH by purified rabbit lung ACE.
Figure 6: Plasma ACE determination.

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Acknowledgements

This work was supported by the Brazilian agencies Fundação de Amparo à Pesquisa do Estado de São Paulo (grant no 2003/09994-7), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (grant no. 470.905/04-2).

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Authors and Affiliations

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

    Adriana K Carmona, Maria A Juliano & Luiz Juliano

  2. 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

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  1. Adriana K Carmona
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  3. Maria A Juliano
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Correspondence to Edward D Sturrock.

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Carmona, A., Schwager, S., Juliano, M. et al. A continuous fluorescence resonance energy transfer angiotensin I-converting enzyme assay. Nat Protoc 1, 1971–1976 (2006). https://doi.org/10.1038/nprot.2006.306

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