Merkle H, Subczynski W K, Kusumi A
Biochim Biophys Acta. 1987 Feb 26;897(2):238-48. doi: 10.1016/0005-2736(87)90420-2.
Bimolecular collision rate of 8-anilinonaphthalene-1-sulfonic acid (ANS) and the nitroxide doxyl group attached to various carbons on stearic acid spin labels (n-SASL) in phosphatidylcholine-cholesterol membranes in the fluid phase was studied by observing dynamic quenching of ANS fluorescence by n-SASL's. The excited-state lifetime of ANS and its reduction by the n-SASL doxyl group were directly measured by the time-correlated single photon counting technique to observe only dynamic quenching separately from static quenching and were analyzed by using Stern-Volmer relations. The collision rate of ANS with the n-SASL doxyl group ranges between 1 X 10(7) and 6 X 10(7), and the extent of dynamic quenching by n-SASL is in the order of 5-much much greater than 6- greater than 7- less than 9- less than 10- less than 12- less than 16-SASL (less than 5-SASL) in dimyristoylphosphatidylcholine (DMPC) membranes. Collision rate of 16-SASL is only 10% less than that of 5-SASL. Since the naphthalene ring of ANS is located in the near-surface region of the membrane, these results indicate that the methyl terminal of SASL appears in the near surface area frequently, probably due to extensive gauche-trans isomerism of the methylene chain. The presence of 30 mol% cholesterol decreases the collision rate of ANS with 12- and 16-SASL doxyl groups but not with the 5-SASL doxyl group in DMPC membranes. On the other hand, in egg-yolk phosphatidylcholine membranes, inclusion of 30 mol% cholesterol does not affect the collision of ANS with either 5-SASL or 16-SASL doxyl groups, in agreement with our previous observation that alkyl chain unsaturation moderates cholesterol effects on lipid motion in the membrane (Kusumi et al., Biochim. Biophys. Acta 854, 307-317). It is suggested that dynamic quenching of ANS fluorescence by lipid-type spin labels is a useful new monitor of membrane fluidity that reports on various lipid mobilities in the membrane; a class of motion can be preferentially observed over others by selecting a proper spin label, i.e., rotational diffusion of lipid about its long axis and translational diffusion by using 5-SASL, wobbling motion of the lipid long axis by using 7-SASL or androstane spin label, and gauche-trans isomerism by using 16-SASL.
通过观察硬脂酸自旋标记物（n-SASL）对8-苯胺基萘-1-磺酸（ANS）荧光的动态猝灭，研究了在磷脂酰胆碱-胆固醇膜的流动相中，ANS与连接在硬脂酸自旋标记物不同碳原子上的氮氧自由基基团的双分子碰撞速率。通过时间相关单光子计数技术直接测量了ANS的激发态寿命及其被n-SASL氮氧自由基基团的还原，以仅观察与静态猝灭分开的动态猝灭，并使用Stern-Volmer关系进行分析。ANS与n-SASL氮氧自由基基团的碰撞速率在1×10⁷至6×10⁷之间，并且在二肉豆蔻酰磷脂酰胆碱（DMPC）膜中，n-SASL的动态猝灭程度顺序为5-远大于6-大于7-小于9-小于10-小于12-小于16-SASL（小于5-SASL）。16-SASL的碰撞速率仅比5-SASL低10%。由于ANS中的萘环位于膜的近表面区域，这些结果表明SASL的甲基末端频繁出现在近表面区域，这可能是由于亚甲基链广泛的gauche-trans异构化。在DMPC膜中，30 mol%胆固醇的存在降低了ANS与12-和16-SASL氮氧自由基基团的碰撞速率，但不影响与5-SASL氮氧自由基基团的碰撞速率。另一方面，在蛋黄磷脂酰胆碱膜中，包含30 mol%胆固醇不会影响ANS与5-SASL或16-SASL氮氧自由基基团的碰撞，这与我们之前的观察结果一致，即烷基链不饱和度会缓和胆固醇对膜中脂质运动的影响（Kusumi等人，生物化学与生物物理学报854，307-317）。有人提出，脂质型自旋标记物对ANS荧光的动态猝灭是一种有用的新的膜流动性监测方法，它可以报告膜中各种脂质的流动性；通过选择合适的自旋标记物，可以优先观察到一类运动，即使用5-SASL观察脂质绕其长轴的旋转扩散和平动扩散，使用7-SASL或雄甾烷自旋标记物观察脂质长轴的摆动运动，使用16-SASL观察gauche-trans异构化。
