Development and Optimization of a Bromothymol Blue-Based PLA2 Assay Involving POPC-Based Self-Assemblies

Area of Science:

• Biochemistry
• Enzymology
• Membrane Biophysics

Background:

• Phospholipase A2 (PLA2) enzymes are crucial for phospholipid hydrolysis at interfaces.
• Enzymatic activity is influenced by substrate assembly (micelles, liposomes) and composition.
• Direct quantification of enzyme-supramolecular assembly interactions is necessary.

Purpose of the Study:

• To develop a universal assay for quantifying PLA2 enzymatic activity on various phospholipid assemblies.
• To compare the kinetics of PLA2 isozymes IB and IIA on different supramolecular structures.
• To establish a method for screening PLA2 inhibitors.

Main Methods:

• Utilized the pH-sensitive dye bromothymol blue (BTB) for a novel assay.
• Employed 3-palmitoyl-2-oleoyl-sn-glycero-1-phosphocholine (POPC) self-assemblies (liposomes, mixed micelles with Triton X-100).
• Performed kinetic analysis and determined kinetic parameters using UV-VIS spectrophotometry.

Main Results:

• The BTB assay successfully quantified PLA2 activity on different POPC assemblies.
• Comparative kinetic analysis of PLA2 isozymes IB and IIA was performed.
• The assay demonstrated high accuracy for inhibitor screening.

Conclusions:

• A simple, universal, and accurate assay for PLA2 activity using BTB was developed.
• The assay is suitable for studying PLA2 kinetics on physiologically relevant supramolecular assemblies.
• This method facilitates screening of PLA2 enzymes, substrates, and inhibitors.