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Related Experiment Videos

Membrane permeabilization by alpha-helical peptides: a flow cytometry study

P Midoux1, R Mayer, M Monsigny

  • 1Centre de Biophysique Moléculaire, CNRS et Université d'Orléans, France.

Biochimica Et Biophysica Acta
|November 1, 1995
PubMed
Summary
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Flow cytometry effectively monitors alpha-helical peptide-induced cell membrane permeabilization using ethidium bromide. This method enables rapid screening of peptides for membrane permeabilization applications.

Area of Science:

  • Biophysics
  • Cell Biology
  • Biochemistry

Background:

  • Alpha-helical peptides can permeabilize mammalian cell membranes.
  • Monitoring this permeabilization is crucial for understanding peptide-cell interactions and developing new applications.
  • Flow cytometry offers a sensitive platform for quantitative cellular analysis.

Purpose of the Study:

  • To establish and validate flow cytometry as a method for monitoring alpha-helical peptide-induced cell membrane permeabilization.
  • To investigate the membrane permeabilization activity of various alpha-helical peptides, including melittin and its derivatives, and influenza hemagglutinin-derived peptides.
  • To characterize the dose-dependency and pH-sensitivity of peptide-induced membrane permeabilization.

Main Methods:

  • Utilizing flow cytometry to detect ethidium bromide fluorescence, which indicates DNA accessibility following plasma membrane permeabilization.

Related Experiment Videos

  • Employing HL60 promyelocytic cells as a model system.
  • Synthesizing and testing modified peptides (e.g., succinylated melittin, E5CA peptide variants) and peptide-protein conjugates.

    • Main Results:

    • Melittin rapidly permeabilized HL60 cells in a dose-dependent manner at neutral pH (ED50 = 0.5 microM).
    • Succinylated melittin and the anionic E5CA peptide demonstrated pH-dependent permeabilization, with activity decreasing at higher pH values.
    • Peptide-protein conjugation reversibly abolished membrane permeabilization activity, and specific amino acid substitutions altered pH sensitivity.

    Conclusions:

    • Flow cytometry with ethidium bromide is a facile and effective method for quantifying alpha-helical peptide-induced membrane permeabilization.
    • The study characterizes the activity of specific peptides, highlighting the influence of charge and pH on their membrane interaction.
    • This approach facilitates rapid screening and selection of peptides for applications requiring controlled membrane permeabilization.