Identification of Domain Formation During sPLA2 Activity by Biophotonics

The aim of this study was to identify the formation of membrane domains during the interfacial activation of pancreatic sPLA2 using fluorescence spectroscopy. The identification of phospholipase A2 (PLA2) activity by biphotonic methods is based on the behavior of fluorescent molecules present in the liposomal systems (enzyme-substrate). The liposomal system consists of a lipid membrane, which can be anything from a liposome to a cell membrane, and the PLA2 enzyme. Liposomes are biophysical models that can be used to study physicochemical processes, which boost the catalytic reaction at the heterogeneous interface between the aqueous phase (enzyme localization) and the lipid phase (substrate localization), for example, the study of membrane remodeling generated by the reaction products of PLA2. The PLA2 superfamily comprises a wide variety of enzymes that promote the hydrolysis of the sn-2 ester bond of phosphoglycerides. This enzyme is found in active form mainly in the pancreas but is distributed throughout the body in different organs and has been found to act during the effects of various chronic pathologies associated with some inflammatory responses. One of the fluorescent molecules used to identify the PLA2 catalysis reaction is C12-NBD-FA. The same molecule is also used as a monitor to detect the formation of domains during PLA2 activity, through the principle of the Forster resonance energy transfer (FRET) method, where a fluorescent acceptor molecule and a fluorescent donor molecule are chosen to measure the changes in fluorescence intensity in the system before and after the catalytic reaction. For this protocol, the fluorescent molecule octadecyl rhodamine chloride (C18-Rho) was chosen as the fluorescence acceptor and C12-NBD-FA as the donor to form the FRET pair. This protocol was performed on DPPC liposomes in gel or liquid crystalline state, and the changes in fluorescence were detected as a function of time at the donor emission length. The reversal in fluorescence that occurred while triggering the secretory PLA2 (sPLA2) activity was interpreted based on the change in transfer efficiency.