Aggregation studies on fluorescein-coupled cobra venom phospholipase A2

Area of Science:

• Biochemistry
• Enzymology
• Protein aggregation

Background:

• Phospholipase A2 (PLA2) from Indian cobra venom exhibits concentration-dependent aggregation.
• Previous studies suggested PLA2 might be active as a dimer or higher aggregate, but experimental challenges hindered determination of its aggregation state under assay conditions.

Purpose of the Study:

• To investigate the aggregation state of Indian cobra venom phospholipase A2 under actual assay conditions.
• To determine the influence of cofactors and substrates on PLA2 aggregation using fluorescence polarization.

Main Methods:

• Fluorescence polarization was employed using fluorescein isothiocyanate-labeled phospholipase A2 from Naja naja naja.
• Control experiments utilized labeled phospholipases A2 from rattlesnake and porcine pancreas with known aggregation states.
• The effect of divalent metal ions (Ca2+, Ba2+, Mn2+, Mg2+, Co2+) and lipid analogues (diheptanoylphosphatidylcholine, dodecylphosphocholine) on enzyme polarization was measured.

Main Results:

• Divalent metal ions Ca2+ and Ba2+ increased the polarization of the cobra venom enzyme, suggesting increased aggregation.
• The water-soluble substrate and lipid analogue, below their critical micelle concentrations, also increased enzyme polarization.
• Mn2+, Mg2+, and Co2+ ions did not significantly affect enzyme polarization.

Conclusions:

• Cofactors and substrates promote the aggregation of Indian cobra venom phospholipase A2 under assay conditions.
• The findings indicate that phospholipase A2 exists in an aggregated form during assays.
• Fluorescence polarization is a viable method for studying enzyme aggregation states.