Fluorescence studies of phosphatidylcholine micelle mixing: relevance to phospholipase kinetics

Two fluorescent micellar phospholipid probes (1-hexanoyl-2-(1-pyrenebutyroyl)phosphatidylcholine and 1-octanoyl-2-(1-pyrenebutyroyl)phosphatidylcholine) have been synthesized, characterized, and used to monitor the dynamics of lipid/amphiphile exchange in a variety of detergents and phospholipid micelles using both steady-state and stopped-flow fluorescence techniques. The ratio of the pyrene monomer to excimer band is a good indicator of the extent of lipid mixing at equilibrium. Following the time dependence of increase in the monomer band with stopped-flow methodology provides a rate constant for this exchange process (most systems were well fit with a single exponential). Short-chain pyrene-labeled phosphatidylcholine mixing with Triton X-100 micelles is extremely fast and follows a concentration dependence indicative of the importance of micelle collisions for the exchange process. Submicellar amounts of Triton have no effect on the fluorescent dynamics of the probe molecule. Other detergents such as beta-octyl glucoside and deoxycholate are also effective at higher concentrations, although significant differences exist in the extent of probe mixing. Short-chain diacylphosphatidylcholine and lysophosphatidylcholine mixing rates are moderately 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 in these assay systems. Anomalous mixing behavior of unusual micelle forming lipids [bolaforms and omega-carboxylate phosphatidylcholines [Lewis, K. A., Bian, J., Sweeny, A., & Roberts, M. F. (1994) Biochemistry 29, 9962-9970] and polymerizable phosphatidylcholines [Soltys, C. E., Bian, J., & Roberts, M. F. (1993) Biochemistry 32, 9545-9551] is particularly helpful in understanding kinetics of water-soluble phospholipases on these systems.