Woolley P, Diebler H
Magdalene College, Cambridge, England.
Biophys Chem. 1979 Nov;10(3-4):305-18. doi: 10.1016/0301-4622(79)85018-8.
The kinetics of the binding of the fluorescence indicator N-phenyl naphthylamine to bilayer vesicles of C12-methyl-phosphatidic acid have been investigated by means of the temperature-jump relaxation technique utilizing fluorescence light detection. Single-exponential relaxation curves were observed, with time constants in the range 0.2-3 ms. The concentration dependence of the relaxation time yielded an apparent association rate constant (expressed in terms of monomeric phospholipid) of k(on) = 5 x 10(6) M(-1) s(-1) in aqueous solution at 25 degrees . The activation energy and viscosity dependence associated with the binding rate show that this process is actually diffusion-controlled. The theory of diffusion-controlled reactions then allows a determination of the average size of the bilayer vesicles and of the true rate constant for the association of the indicator molecules with the vesicles. Assuming spherical geometry for the vesicles, the values are: r(ves) = 190 A, which corresponds to 20000 lipid molecules per vesicle and k'(on) = 1 x 10(11) M(-1) s(-1) (25 degrees). The correctness of this size-determination was confirmed semi-quantitatively by electron microscopy. Since in fact a distribution of vesicle sizes must be present, a discussion is included of the relaxation function which the system is expected to take in the general case. Biological implications of diffusion control for the transport of non-polar substances and for lipid mixing are indicated.
利用荧光光检测的温度跃变弛豫技术，研究了荧光指示剂N-苯基萘胺与12-甲基磷脂酸双层囊泡结合的动力学。观察到单指数弛豫曲线，时间常数在0.2 - 3毫秒范围内。弛豫时间的浓度依赖性在25℃水溶液中得到了表观缔合速率常数（以单体磷脂表示）k(on) = 5×10⁶ M⁻¹ s⁻¹。与结合速率相关的活化能和粘度依赖性表明该过程实际上是扩散控制的。扩散控制反应理论进而允许确定双层囊泡的平均大小以及指示剂分子与囊泡缔合的真实速率常数。假设囊泡为球形几何形状，得到的值为：r(ves) = 190 Å，对应于每个囊泡20000个脂质分子，k'(on) = 1×10¹¹ M⁻¹ s⁻¹（25℃）。电子显微镜半定量地证实了这种大小测定的正确性。由于实际上必然存在囊泡大小的分布，因此还讨论了一般情况下系统预期呈现的弛豫函数。指出了扩散控制对非极性物质运输和脂质混合的生物学意义。
