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A quantitative model for using acridine orange as a transmembrane pH gradient probe

S Clerc1, Y Barenholz

  • 1Department of Biochemistry, Hebrew University-Hadassah Medical School, Jerusalem, Israel.

Analytical Biochemistry
|May 30, 1998
PubMed
Summary
This summary is machine-generated.

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Optical probes like acridine orange can monitor internal vesicle acidification. This study confirms acridine orange aggregation quantifies transmembrane pH gradients in biological vesicles.

Area of Science:

  • Biochemistry
  • Biophysics
  • Membrane Biology

Background:

  • Proton pumps drive acidification within membrane vesicles.
  • Transmembrane pH gradients alter acridine orange's spectral properties.
  • Acridine orange accumulation and aggregation are hypothesized causes.

Purpose of the Study:

  • To validate the hypothesis that acridine orange aggregation causes spectral changes.
  • To establish a theoretical basis for using acridine orange to quantify pH gradients.

Main Methods:

  • Utilized liposomes with ammonium sulfate gradients as a model system.
  • Measured acridine orange fluorescence intensity under varying conditions.
  • Compared experimental data with a theoretical model of dye accumulation and aggregation.

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Main Results:

  • Acridine orange fluorescence was sensitive to gradient magnitude, vesicle volume, and temperature.
  • The theoretical model accurately predicted spectral changes based on proton concentration ratios.
  • Quantitative relationships between pH gradients and spectral shifts were established.

Conclusions:

  • Acridine orange aggregation within vesicles is confirmed as the cause of spectral changes.
  • Characterizing dye aggregation provides a theoretical foundation for using acridine orange.
  • Acridine orange is a reliable optical probe for quantifying transmembrane pH gradients.