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Fluorescent Leakage Assay to Investigate Membrane Destabilization by Cell-Penetrating Peptide
Published on: December 19, 2020
Absorbance-based assay for membrane disruption by antimicrobial peptides and synthetic copolymers using
Laura B Zimmerman1, Brittany V Worley, Edmund F Palermo
1Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
Abstract:
A simple homogeneous assay for the detection of membrane permeabilization by antimicrobial peptides and synthetic copolymers is described. Liposomes encapsulating pyrroloquinoline quinone (PQQ), the prosthetic group of the apoenzyme glucose dehydrogenase (GDH), are used to detect membrane permeabilization by the antimicrobial peptides MSI-594 and MSI-78 as well as various synthetic antimicrobial copolymers in an optical microwell assay. PQQ-loaded liposomes and the peptide or copolymer are added to wells of a 96-well microtiter plate. If the integrity of the liposome is compromised, the PQQ encapsulated in the liposomes is released and available for activating the apoenzyme. The release of PQQ catalyzes a color change in the presence of apo-GDH, glucose, and the redox dye 1,6-dichlorophenol indophenol (DCPIP) that can be evaluated through a visual color change. For more quantitative measurements, the absorbance change over a 30min period was measured. The absorbance change is related to the activity and concentration for a given antimicrobial agent. Furthermore, by varying liposome compositions to include cholesterol, the potential toxicity of the peptide or polymer toward mammalian cells can be readily evaluated. The assay is simple and sensitive and will be useful for analyzing the membrane permeation/disruption properties of a host of antimicrobial peptides and synthetic polymers.
Insights
This study introduces a novel assay for detecting membrane permeabilization by antimicrobial peptides and synthetic polymers. The assay utilizes liposomes and a colorimetric reaction, offering a simple yet sensitive method for evaluating antimicrobial activity and potential toxicity.
Area of Science:
- Biochemistry
- Materials Science
- Pharmacology
Background:
- Membrane permeabilization is a key mechanism for antimicrobial agents.
- Assessing this property is crucial for developing new therapeutics.
- Existing methods can be complex or lack sensitivity.
Purpose of the Study:
- To develop a simple, homogeneous assay for detecting membrane permeabilization.
- To evaluate the efficacy of antimicrobial peptides and synthetic copolymers.
- To assess the potential toxicity of these agents towards mammalian cells.
Main Methods:
- Liposomes encapsulating pyrroloquinoline quinone (PQQ) were used.
- PQQ release upon liposome disruption indicates membrane permeabilization.
- A colorimetric reaction involving glucose dehydrogenase (GDH) and a redox dye (DCPIP) was employed.
- Absorbance measurements provided quantitative data.
Main Results:
- The assay successfully detected membrane permeabilization by known antimicrobial peptides (MSI-594, MSI-78) and synthetic copolymers.
- Quantitative measurements correlated with antimicrobial agent activity and concentration.
- Incorporating cholesterol into liposomes allowed for toxicity evaluation.
Conclusions:
- The developed assay is simple, sensitive, and effective for analyzing membrane permeation.
- It is suitable for a wide range of antimicrobial peptides and synthetic polymers.
- This assay can aid in the discovery and development of new antimicrobial agents.
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