STUDY OF FAST REACTIONS USING COMPARTMENTALIZED PHOSPHOLIPID VESICLES

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The kinetics of some enzymatic and chemical reactions can be too fast to follow by conventional stopped-flow methods as the technique is often limited in time resolution by the mixing dead time of the instrument (about 1-2 milliseconds). We have been exploring the possibility of extending this mixing time constraint by use of loaded vesicle systems, which can be made to rupture by applying short (100-200 microseconds) and intense (up to 10 kV/cm) electric field pulses to initiate the reaction. To facilitate these studies, we have put together an electric-field jump apparatus (with rise and fall times of <1 microseconds) and constructed an optical cell that will allow signal detection of either fluorescence, optical density, or birefringence. Preliminary studies were done using vesicles prepared from the phospholipid surfactants, egg L-alpha-phosphatidylcholine (EPC) or Di-oleoylphosphatidylcholine (DOPC), sized to about 400 nanometer diameter by high pressure extrusion through polycarbonate filters. The model reaction systems studied were: (a) Ni(II) + Murexide (O.D. detection), (b) Ni(II) + Fluo-3:Ca(II) (fluorescence quenching), and (c) Fluo-3 + Ca(II) (fluorescence enhancement). The expected rate constants (1/sec) for these reactions are: 10e4 to 10e5 for reactions (a) and (b) and >10e8 for reaction (c). In all of these reactions, the fluorophores were encapsulated into the vesicles and the reacting ions kept in the external medium prior to the application of the external electric field. We have thus far obtained kinetic traces using the fluorescence detection system for reaction (b) and a step response for reaction (c). O.D. detection was not possible due to excessive light scattering. However, evaluation of the rate constants has been complicated due to the variability of the kinetic traces obtained. This is primarily caused by the heterogeneity in the size and lamellarity of the vesicle preparations. We are currently testing various other phospholipids and vesicles made from mixed phospholipids to solve this problem. The kinetic method developed here will ultimately be used to study biological reactions of relevance.