TIME-RESOLVED FLUORESCENCE ANISOTROPY OF FLUORESCENT-LABELED LYSOPHOSPHOLIPID AND TAURODEOXYCHOLATE AGGREGATES

Previous work from this laboratory demonstrated that the environment-s ensitive lysolipid N-(7-nitrobenz-2-oxa-1 diazol-4-yl)-monomyristoylph osphatidylethanolamine (N-NBD-MPE), at concentrations below its critic al micelle concentration (CMC(N-NBD-MPE) = 4 mu M), reached maximum fl uorescence yield upon the addition of taurodeoxycholate (TDC) at conce ntrations well below its CMC (CMC(TDC) = 2.5 mM). These data indicated the formation of micellar aggregates of the two amphiphiles at concen trations below both of their CMCs. In the present study, fluorescence lifetime and differential polarization measurements were made to deter mine the size of these aggregates. In the absence of TDC and at 0.5 mM TDC a single lifetime (tau) and rotational correlation time (phi) wer e measured for N-NBD-MPE at the submicellar concentration of 2 mu M, i ndicating a lack of interaction between the two molecules at this conc entration. Above 0.5 mM TDC, two discrete lifetimes were resolved. Bas ed on these lifetimes, two distinct rotational correlation times were established through polarization measurements. The shorter phi (0.19-0 .73 ns) was ascribed to local probe motions, whereas the longer phi wa s in a time range expected for global rotation of aggregates the size of simple bile salt micelles (3-6.5 ns). From the longer phi, molecula r volume and hydrodynamic radii were calculated, ranging from similar to 15 Angstrom at 1 mM to similar to 18 Angstrom at 5 mM TDC. These da ta support the conclusion that monomeric lysolipids in solution seed t he aggregation of numerous TDC molecules (aggregation number = 16 at 1 mM TDC) to form a TDC micelle with a lysolipid core at concentrations below which they both self-aggregate.