Quenching of tryptophan phosphorescence in Escherichia coli alkaline phosphatase by long-range transfer mechanisms to external agents in the rapid-diffusion limit.
Quenching of tryptophan phosphorescence in Escherichia coli alkaline phosphatase by long-range transfer mechanisms to external agents in the rapid-diffusion limit.
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通过在快速扩散极限下向外部试剂的长程转移机制来猝灭大肠杆菌碱性磷酸酶中的色氨酸磷光。
DOI：
10.1021/bi00217a012
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发表时间：
1991
期刊：
影响因子：
2.9
通讯作者：
Gafni,A
中科院分区：
文献类型：
--
作者：
Mersol,JV;Steel,DG;Gafni,A
修订稿于 1990 年 10 月 2 日收到摘要：研究了几种自由扩散分子对大肠杆菌碱性磷酸酶的室温磷光的猝灭，每个分子的吸收光谱与该蛋白质的长寿命发射重叠，因此可以通过扩散增强的福斯特能量转移猝灭激发的三重态。由于淬灭速率对光谱重叠缺乏线性依赖性，还检测到了额外的非共振传递机制的存在。使用的猝灭剂是染料分子甲基红、甲基橙和2-[(4-羟基苯基)偶氮]苯甲酸，以及肌红蛋白、高铁肌红蛋白的嵌入血红素基团以及细胞色素c的还原和氧化形式。发现每个受体还原时猝灭大大减少，表明电子转移有效地发生从激发的色氨酸到受体的氧化形式。消除这种还原形式的电子转移提供了单独测量血红素蛋白的福斯特转移率的机会。当如此测量的肌红蛋白的转移速率常数应用于供体蛋白和受体蛋白均被视为球形且色氨酸和血红素基团均偏离中心的模型时（此处新提出的猝灭速率方程的模型），发现碱性磷酸酶表面下方的磷光色氨酸的深度为16μ。该值接近色氨酸 109（已知是碱性磷酸酶中的磷光残基）的深度，表明通过正确选择探针，该技术对于蛋白质结构研究中的距离测定确实很有价值。研究发现，根据细胞色素 c 数据计算出的距离在不同缓冲液中有所不同，并且还取决于缓冲液浓度，当 Tris-HCl 浓度从 50 mM 增加到 1 M 时，从 8 变为 12 Á。这反映了对细胞色素 c 模型的需要，该模型可以更好地代表其形状和静电特性。荧光猝灭是一种用于研究溶液中蛋白质结构的成熟且有用的光谱工具。然而，荧光的短衰减时间通常限制了这种方法对快速猝灭的适用性，因为后者过程必须与荧光竞争。最近，人们发现大量蛋白质中的色氨酸残基在分子氧被广泛去除的情况下会发出室温磷光（Saviotti & Galley，1974；Vanderkooi 等，1987；Papp & Vanderkooi，1989）。这种磷光的长衰减时间（毫秒到秒）允许通过对磷光衰减动力学的影响来研究在这种延长的时间尺度上发生的相互作用。特别令人感兴趣的是长程相互作用，因为它们允许研究埋藏在蛋白质内部的色氨酸残基，这些残基通常导致大多数蛋白质的磷光（Kai＆Imakubo，1979；Strambini＆Gonnelli，1985）。这些相互作用还使得人们能够使用溶液中游离且不与蛋白质结合的猝灭剂，从而
Revised Manuscript Received October 2, 1990 abstract: Quenching of the room-temperature phosphorescence of Escherichia coli alkaline phosphatase by several freely diffusing molecules was studied, each of whose absorption spectrum overlaps thelong-lived emission of this protein and which therefore can quench the excited triplet state by diffusion-enhanced Forster energy transfer. The presence of additional nonresonance transfer mechanisms was also detected, from a lack of linear dependence of quenching rate on spectral overlap. The quenching agentsused were the dye molecules methyl red, methyl orange, and2-[(4-hydroxyphenyl) azo] benzoic acid, as well as the embedded heme groups of myoglobin, metmyoglobin, and the reduced and oxidized forms of cytochrome c. Quenching was found to be greatly diminishedupon reduction of each acceptor, indicating that electron transfer occurs efficiently from theexcited tryptophan to the oxidized form of the acceptors. The elimination of this electron transfer in the reduced form affords the opportunity to separately measure the Forster transfer rates for the heme proteins. When the transfer rate constant thus measured for myoglobin is applied to a model where both donor and acceptor proteins are taken to be spherical with both tryptophan and the heme group placed off center (a model whose quenching rate equation is newly presented here), the depth of the phosphorescent tryptophan beneath the surface of alkaline phosphatase is found to be 16 Á. This value is close to the depth of tryptophan 109 (which is known to be the phosphorescent residue in alkaline phosphatase), showing that with properly chosen probesthis technique is indeed valuable for distance determinations in protein structure studies. The distance calculated from cytochrome c data was found to vary among different buffers and also to depend on buffer concentration, changing from 8 to 12 Á upon increase of Tris-HCl concentration from 50 mM to 1 M. This reflects the need for a model for cytochrome c which better represents its shape and electrostatic properties.Fluorescence quenching is an established and useful spec-troscopic tool for the study of protein structure in solution. However, the short decay time of fluorescence usually limits the applicability of this approach to rapid quenching since the latter process must compete with fluorescence. Recently, tryptophan residues in a large number of proteins have been found to emit room-temperature phosphorescence provided that molecular oxygen is extensively removed (Saviotti & Galley, 1974; Vanderkooi et al., 1987; Papp & Vanderkooi, 1989). The long decay time of this phosphorescence (milli-seconds to seconds) allows thestudy of interactions that occur on this extended time scale through their effects on the phosphorescence decay kinetics. Of particular interest are long-range interactions since these allow the study of trypto-phan residues which are buried inside the protein—residues which, as a rule, are responsible for most proteins’ phosphorescence (Kai & Imakubo, 1979; Strambini & Gonnelli, 1985). These interactions also enable one to use quenchers which are free in solution and not bound to the protein, thereby
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影响因子：
56.9
作者：
J. Vanderkooi;D. B. Calhoun;S. Englander
通讯作者：
J. Vanderkooi;D. B. Calhoun;S. Englander
DOI：
10.1016/1010-6030(89)87107-2
发表时间：
1989
期刊：
Bulletin of the Academy of Sciences of the USSR, Division of chemical science
影响因子：
--
作者：
Y. Malkin;T. Krasieva;V. A. Kuz’min
通讯作者：
V. A. Kuz’min
光响应 N 混合液晶，介电常数可实现 10^4 至 10^2 的剧烈变化
DOI：
--
发表时间：
2019
期刊：
影响因子：
--
作者：
森川響二朗;竹内智章;北森武彦;西川浩矢
通讯作者：
西川浩矢
吲哚核三重态寿命及其对溶剂微粘度的依赖性
影响因子：
2.8
作者：
G. Strambini;M. Gonnelli
通讯作者：
M. Gonnelli
大肠杆菌碱性磷酸酶的精制结构，分辨率为 2.8 A。
影响因子：
5.6
作者：
Sowadski,JM;Handschumacher,MD;Murthy,HM;Foster,BA;Wyckoff,HW
通讯作者：
Wyckoff,HW