FLUORESCENCE STUDIES OF PHOSPHATIDYLCHOLINE MICELLE MIXING - RELEVANCE TO PHOSPHOLIPASE KINETICS

Two fluorescent micellar phospholipid probes 1-hexanoyl-2-(1-pyrenebut yroyl)phosphatidylcholine and -octanoyl-2-(1-pyrenebutyroyl)phosphatid ylcholine) have been synthesized, characterized, and used to monitor t he dynamics of lipid/amphiphile exchange in a variety of detergents an d phospholipid micelles using both steady-state and stopped-flow fluor escence techniques. The ratio of the pyrene monomer to excimer band is a good indicator of the extent of lipid mixing at equilibrium. Follow ing the time dependence of increase in the monomer band with stopped-f low methodology provides a rate constant for this exchange process (mo st systems were well fit with a single exponential). Short-chain pyren e-labeled phosphatidylcholine mixing with Triton X-100 micelles is ext remely fast and follows a concentration dependence indicative of the i mportance of micelle collisions for the exchange process. Submicellar amounts of Triton have no effect on the fluorescent dynamics of the pr obe molecule. Other detergents such as beta-octyl glucoside and deoxyc holate are also effective at higher concentrations, although significa nt differences exist in the extent of probe mixing. Short-chain diacyl phosphatidylcholine and lysophosphatidylcholine mixing rates are moder ately fast with mixing times that decrease as the hydrophobicity/chain length of the diluent matrix increases. The rate constants for lipid exchange can be compared to turnover rates of several phospholipases i n these assay systems. Anomalous mixing behavior of unusual micelle fo rming lipids [bolaforms and omega-carboxylate phosphatidylcholines [Le wis, K. A., Bian, J., Sweeny, A., and Roberts, M. F. (1994) Biochemist ry 29, 9962-9970] and polymerizable phosphatidylcholines [Soltys, C. E ., Bian, J., and Roberts, M. F. (1993) Biochemistry 32, 9545-9551] is particularly helpful in understanding kinetics of water-soluble phosph olipases on these systems.