Abstract
The dimeric Crotalus atrox venom PLA2 is part of the secreted phospholipase A2 (PLA2) enzyme family that interacts at the lipid-solution interface to hydrolyze the sn-2 acyl ester bond of phospholipids. We have employed fluorescence correlation spectroscopy (FCS) to study the monomer-dimer equilibrium of the C. atrox venom PLA2 in solution, in the presence of urea, and in the presence of monomeric and micellar n-dodecylphosphocholine (C12-PN), a phosphatidylcholine analogue. Dilution experiments show that PLA2 is an extremely tight dimer, Kd < or = 0.01 nM, in solution. Urea was introduced to weaken the subunit's association, and an estimate for the PLA(2) dimer dissociation constant in buffer was obtained by linear extrapolation. The derived dissociation constant was at least several orders of magnitude greater than that suggested from the dilution experiments, indicating a complex interaction between urea and the PLA2 dimer. FCS data indicate that the PLA2 dimer begins to dissociate at 10 mM C12-PN in 10 mM Ca2+ and at 5 mM C12-PN in 1 mM EDTA. The PLA2 tryptophan fluorescence displayed spectral shifts and intensity changes upon interacting with C12-PN. On the basis of the FCS and tryptophan fluorescence results, we postulate an intermediate state where the two monomers are in loose interaction within a protein-lipid comicelle. As the concentration of C12-PN was increased, complete dissociation of the dimer was observed, inferred from the doubling of the particle number, and the average diffusion constant decreased to approximately 60 microm2/s, consistent with PLA2 associated with a C12-PN micelle. The presence of Ca2+ makes the comicelle intermediate more stable, retarding the separation of the monomers in the micellar suspension. Our data clearly indicate that PLA2, though a strong dimer in the absence of lipids, is dissociated by micellar C12-PN and supports the monomer hypothesis for PLA2 action.
Highlights
Studies on the C. atrox venom phospholipase A2 (PLA2) quickly identified this enzyme as a strong dimer, similar to another dimeric PLA2 isolated from Crotalus adamanteus venom [21,22,23]
To determine the solution dissociation constant for the C. atrox PLA2 dimer, we have measured the number of fluorescent particles in the excitation volume using the fluctuation amplitude in the fluorescence correlation spectroscopy (FCS) experiment as we dilute the enzyme solution
The primary focus of this work is on the dimer-monomer equilibrium of the strong dimer PLA2 from C. atrox venom
Summary
Studies on the C. atrox venom PLA2 quickly identified this enzyme as a strong dimer, similar to another dimeric PLA2 isolated from Crotalus adamanteus venom [21,22,23]. An alternative fluorescence technique that can provide information about monomer/dimer equilibrium and, at the same time, determine the mass of the protein/lipid aggregates is fluorescence correlation spectroscopy (FCS). This technique allows one to count the number of fluorophore-labeled proteins in a particle aggregate using the fluctuation of the fluorescence intensity. The comparison of dissociation curves of the number of particles and of tryptophan emission at different lipid concentrations in the presence and absence of Ca2+ allowed us to address the above questions and to propose a structural model for PLA2/lipid interactions. Our data indicate that the PLA2 dimer dissociates into monomers given sufficient micellar lipid and suggest that under assay conditions and in the presence of substrate the monomer form is the active enzyme form
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
