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Related Experiment Videos

Internally quenched fluorogenic substrates for angiotensin I-converting enzyme.

M C Araujo1, R I Melo, E Del Nery

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

Journal of Hypertension
|July 14, 1999
PubMed
Summary

New fluorogenic substrates enable sensitive, continuous assays for angiotensin I-converting enzyme (ACE). These internally quenched peptides allow real-time measurement of ACE activity, even in human serum, offering a valuable tool for enzyme studies.

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Area of Science:

  • Biochemistry
  • Enzymology
  • Fluorogenic Assays

Background:

  • Angiotensin-converting enzyme (ACE) plays a crucial role in the renin-angiotensin system.
  • Sensitive and continuous assay methods are needed for ACE activity measurement.
  • Existing methods may have limitations in continuous monitoring or sample types.

Purpose of the Study:

  • To develop novel internally quenched fluorogenic substrates for ACE.
  • To enable sensitive and continuous fluorimetric assays of enzymatic activity.
  • To validate these substrates for use in biological samples, including human serum.

Main Methods:

  • Synthesis of internally quenched fluorogenic bradykinin-related peptides (Abz-GFSPFRX-EDDnp and Abz-GFSPFXQ-EDDnp).
  • Assay of peptide hydrolysis by ACE using continuous fluorescence monitoring (excitation 320 nm, emission 420 nm).

Related Experiment Videos

  • Characterization of substrate performance with plasma guinea pig ACE and inhibition studies with ACE inhibitors (captopril, lisinopril).

    • Main Results:

    • Internally quenched peptides were effectively hydrolyzed by ACE, releasing fluorescence upon cleavage.
    • Optimal substrates (Abz-GFSPFRA-EDDnp, Abz-GFSPFFQ-EDDnp) showed cleavage at specific bonds (R-A, F-Q).
    • ACE activity assays demonstrated sensitivity to NaCl concentration and optimal pH > 8.0; results correlated well with a standard assay (r=0.959) and were inhibited by captopril and lisinopril.

    Conclusions:

    • Developed internally quenched fluorogenic substrates for ACE are effective tools.
    • These substrates allow continuous fluorimetric measurement of ACE activity.
    • The substrates are suitable for ACE studies in various biological matrices, including human serum.