Quantitative analysis of annexin V-membrane interaction by flow cytometry

Area of Science:

• Biochemistry
• Cell Biology
• Biophysics

Background:

• Phosphatidylserine (PS) exposure is a hallmark of apoptosis.
• Annexin V (anxV) is a protein that binds to PS.
• Quantifying anxV-membrane interactions is crucial for apoptosis detection.

Purpose of the Study:

• To construct and utilize a novel green fluorescent anxV-EGFP probe.
• To investigate the interaction between anxV and cell membranes under varying calcium and protein concentrations.
• To develop a mathematical model for anxV-membrane binding.

Main Methods:

• Construction of a fusion protein: enhanced green fluorescent protein (EGFP) fused to human annexin V (anxV).
• Flow cytometry (FCM) to analyze anxV-EGFP binding to membranes.
• Mathematical modeling to describe and quantify binding characteristics.

Main Results:

• The anxV-EGFP probe successfully allowed investigation of anxV-membrane interactions.
• A validated mathematical model quantifies the relationship between bound anxV, calcium concentration, and protein concentration.
• Binding analysis suggests a multi-step reaction mechanism for anxV-membrane interactions.

Conclusions:

• The developed anxV-EGFP probe serves as a valuable tool for studying membrane interactions.
• The mathematical model provides a quantitative framework for understanding anxV binding dynamics.
• This study offers insights for improving apoptosis detection methods using anxV.