PARALLAX METHOD FOR DIRECT MEASUREMENT OF MEMBRANE PENETRATION DEPTH UTILIZING FLUORESCENCE QUENCHING BY SPIN-LABELED PHOSPHOLIPIDS
PARALLAX METHOD FOR DIRECT MEASUREMENT OF MEMBRANE PENETRATION DEPTH UTILIZING FLUORESCENCE QUENCHING BY SPIN-LABELED PHOSPHOLIPIDS
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DOI：
10.1021/bi00375a006
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发表时间：
1987-01-13
期刊：
影响因子：
2.9
通讯作者：
LONDON, E
中科院分区：
文献类型：
--
作者：
CHATTOPADHYAY, A;LONDON, E
本报告描述了一种适合于确定嵌入膜中的各种荧光分子的深度的方法。该方法涉及确定的视差在荧光团的表观位置检测时，淬灭磷脂自旋标记在两个不同的深度进行比较。通过使用简单的代数表达式，该方法允许以埃为单位计算深度。此外，分析可以扩展到淬灭的能量转移受体或溴化探针在适当的条件下。应用该方法淬灭的7-硝基-2，1，3-苯并恶二唑-4-基（NBD）标记的脂质自旋标记的脂质位于三个不同的深度证明在模型膜。结果表明，NBD群的计算深度是自洽的程度上，他们是相同的，无论哪两个自旋标签已在一个特定的实验中使用。此外，计算的深度是独立的自旋标记浓度在膜内。1.ANG.，排除了由于自旋标记扰动而引起的主要影响。淬灭实验表明，头基标记的磷脂酰乙醇胺中的NBD基团的位置是在极性/烃界面和胆固醇的“尾巴”上的NBD标签是深埋。果然出乎意料的是，放置在磷脂酰胆碱的脂肪酰基链末端的NBD标记也靠近极性/烃界面。据推测，NBD基团的极性导致“环”回到连接到柔性酰基链的NBD基团的表面。
This report describes a method suitable for determining the depth of a wide variety of fluorescent molecules embedded in membranes. The method involves determination of the parallax in the apparent location of fluorophores detected when quenching by phospholipids spin-labeled at two different depths is compared. By use of straightforward algebraic expressions, the method allows calculation of depth in angstroms. Furthermore, the analysis can be extended to quenching by energy-transfer acceptors or brominated probes under appropriate conditions. Application of the method to quenching of 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD)-labeled lipids by spin-labeled lipids located at three different depths is demonstrated in model membranes. It is shown that the calculated depths of the NBD groups are self-consistent to the extent that they are the same no matter which two spin-labels have been used in a particular experiment. In addition, the calculated depth is independent of spin-label concentration in the membrane within .+-. 1 .ANG., ruling out major effects due to spin-label perturbation. The quenching experiments show that the location of the NBD group in head-group-labeled phosphatidylethanolamine is at the polar/hydrocarbon interface and that of an NBD label on the "tail" of cholesterol is deeply buried. as expected. Unexpectedly, NBD labels placed at the end of fatty acyl chains of phosphatidylcholines are also near the polar/hydrocarbon interface. Presumably, the polarity of the NBD group results in "looping" back to the surface of the NBD groups attached to flexible acyl chains.