STUDY OF FAST REACTIONS USING COMPARTMENTALIZED PHOSPHOLIPID VESICLES

某些酶和化学反应的动力学可能太快 随后采用传统的停流方法，因为该技术通常 时间分辨率受限于仪器的混合死区时间 （大约1-2毫秒）。 我们一直在探索 通过使用加载的囊泡系统延长该混合时间限制， 可以通过施加短路（100-200微秒）使其破裂， 和强（高达10 kV/cm）电场脉冲，以启动 反应 为方便进行这些研究，我们已编制了一份 电场跳跃装置（上升和下降时间<1 微秒），并构建了一个光学细胞，将允许信号 荧光、光密度或双折射的检测。 使用由聚乙二醇制备的囊泡进行了初步研究。 磷脂表面活性剂，卵L-α-磷脂酰胆碱（EPC）或 二油酰磷脂酰胆碱（DOPC），尺寸约为400纳米 直径通过高压挤出通过聚碳酸酯过滤器。的 研究的模型反应体系为：（a）Ni（II）+紫脲酸铵（O.D. （B）Ni（II）+ Fluo-3：Ca（II）（荧光猝灭），和（c） Fluo-3 + Ca（II）（荧光增强）。 预期的速率常数 对于反应（a）和（B）为10 e4至10 e5 且对于反应（c）> 10 e8。 在所有这些反应中， 被封装到囊泡中，反应离子被保持在囊泡中。 在施加外部电场之前，外部介质。 迄今为止，我们已经获得了动力学痕迹使用荧光 反应（B）的检测系统和反应（c）的阶跃响应。 O.D.由于过度的光散射，检测是不可能的。 然而，速率常数的评估由于以下原因而变得复杂： 所获得的动力学轨迹的可变性。 这主要是 由囊泡大小和层状结构的不均匀性引起 准备工作 我们目前正在测试各种其他磷脂， 由混合磷脂制成的囊泡来解决这个问题。 的 动力学方法将最终用于研究 相关的生物反应

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.