The 'delta pH'-probe 9-aminoacridine: response time, binding behaviour and dimerization at the membrane

Area of Science:

• Biophysical Chemistry
• Membrane Biophysics
• Lipid Bilayer Dynamics

Background:

• Investigating the behavior of fluorescent probes within liposomes is crucial for understanding membrane properties.
• Transmembrane pH gradients are fundamental to cellular processes and can be mimicked in model systems.
• 9-aminoacridine (9-AA) is a pH-sensitive fluorescent dye used to probe membrane potential and gradients.

Purpose of the Study:

• To investigate the fluorescence quenching of 9-aminoacridine (9-AA) in response to transmembrane pH gradients in various lipid systems.
• To elucidate the role of lipid composition, particularly headgroup charge and phase transition, in modulating 9-AA fluorescence quenching.
• To determine the kinetics and dependencies of 9-AA fluorescence quenching and recovery.

Main Methods:

• Utilized stopped-flow mixing to rapidly induce transmembrane pH gradients in liposomes.
• Employed fluorescence spectroscopy to monitor the quenching and recovery of 9-AA.
• Investigated different lipid compositions, including phosphatidylcholine, phosphatidylglycerol, and phosphatidylserine, and varied temperatures relative to lipid phase transitions.
• Used the entrapped dye pyranine to independently monitor the pH gradient decay.

Main Results:

• Observed rapid fluorescence quenching of 9-AA upon imposition of an acidic interior pH gradient, attributed to the pH response of 9-AA.
• Fluorescence recovery, indicative of pH gradient dissipation, occurred significantly faster (10-30 times) than pyranine-monitored delta pH decay.
• Quenching was hindered below the phase transition temperature of dipalmitoylphosphatidylglycerol and absent in vesicles with only uncharged phosphatidylcholine headgroups.
• The presence of negatively charged headgroups (phosphatidylglycerol, phosphatidylserine) was essential for quenching.
• Quenching extent showed a cooperative dependency on the number of negative headgroups and unbound 9-AA concentration, with quenched 9-AA proportional to 9-AA dimer-excimer complexes.

Conclusions:

• The fluorescence quenching of 9-AA is a sensitive indicator of transmembrane pH gradients in liposomes.
• Lipid headgroup charge is critical for 9-AA quenching, with negative charges facilitating the process.
• Membrane fluidity, influenced by lipid phase transitions, significantly affects the quenching efficiency.
• The observed cooperative dependency suggests a specific interaction mechanism between 9-AA and charged lipid headgroups within the membrane.