Temperature dependence of spin-label intensity in solutions and its implication in spin-labeled erythrocyte membrane studies.
Temperature dependence of spin-label intensity in solutions and its implication in spin-labeled erythrocyte membrane studies.
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溶液中自旋标记强度的温度依赖性及其在自旋标记红细胞膜研究中的意义。
DOI：
10.1016/s0006-3495(83)84348-3
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发表时间：
1983
影响因子：
3.4
通讯作者：
Johnson,ME
中科院分区：
文献类型：
--
作者：
Fung,LW;Johnson,ME
亲爱的先生。用N-(L-氧基-2，2，6，6-四甲基-4-哌啶基)马来酰亚胺(MAL-6)标记的人红细胞膜蛋白具有多组分电子顺磁共振信号。这些薄膜样品的常规一次谐波(V_1)谱包含宽的和窄的线分量。广泛的成分是主要的信号，具有大的超精细分裂，来自被宿主蛋白强烈固定的标记；窄的成分，具有较小的超精细分裂，来自弱固定的标记。这两个信号的幅度，W和S，是方便的参数，W/S比已被许多工作人员用来分析电子顺磁共振谱(1-4)。这一比率在研究细胞内分子与细胞质一侧的膜表面结合方面非常有用，并已在本实验室用于研究在生理pH(5)下极低亲和力的血红蛋白膜结合。在定量上，我们用两态模型解释了从结合研究中得到的W/S值。然而，里夫金德和他的同事最近观察到，随着温度的升高，整合的EPR信号强度增加(6)，并提出存在一种状态，即由于偶极相互作用，在低温下EPR是静默的，但在较高温度时，EPR变得活跃。他们建议至少有三个州的模型更合适(6)。至少，对于自旋标记的红细胞研究，我们不同意这种解释。在这封信中，我们提供的数据表明，随着温度的增加，强度的增加主要与EPR腔灵敏度有关，而不是与EPR活性的束缚自旋标记浓度的增加有关。在EPR测量中，腔灵敏度取决于腔的Q、填充因子、样品的磁矩、影响噪声电平或信号电平的因素等。一般说来，腔体灵敏度是一个多功能量。绝对自旋浓度的测定是一项相当复杂的工作。例如，最近的工作表明，需要许多校正系数，包括石英杜瓦瓶插件的透镜效应和溶剂的透镜效应(8)，以比较有损溶液中的综合EPR信号强度。腔的Q还取决于腔内样品的介电常数。在这项研究中，我们保持了许多与腔体灵敏度有关的因素不变，只改变了温度和温度。
Dear Sir. Human erythrocyte membrane proteins alkylated with the nitroxide spin label, N-(l-oxyl-2, 2, 6, 6-tetramethyl 4-piperidinyl) maleimide (Mal-6), exhibit multicomponent electron paramagnetic resonance (EPR) signals. The conventional, first harmonic (V,) spectra of these membrane samples contain broad and narrow line components. The broad component, which is the major signal, and has a large hyperfine splitting, comes from labels that are strongly immobilized by the host proteins; the narrow component, with a smaller hyperfine splitting, comes from labels that are weakly immobilized. The amplitudes of these two signals, Wand S, are convenient parameters, and the W/S ratio has been used by many workers in the analysis of the EPR spectra (1-4). This ratio is very useful in studying intracellular molecules binding to membrane surfaces on the cytoplasmic side, and has been used in this laboratory to study a very low affinity hemoglobinmembrane association at physiological pH (5). Quantitatively, we have used a two-state model to interpret the W/S values obtained from the binding studies. However, Rifkind and co-workers have recently observed an increase in the integrated EPR signal intensity upon increasing temperature (6), and have suggested that there exists a state that is EPRsilent at low temperature due to dipolar interactions, but becomes EPR active at higher temperatures. They suggested that a model of at least three states is more appropriate (6). We disagree with this interpretation, at least, for spin-labeled erythrocyte studies. In this letter we present data showing that the increase in intensity upon increasing temperature is primarily related to EPR cavity sensitivity rather than to an increase in concentration of the bound spin labels that are EPR active.In an EPR measurement, the cavity sensitivity depends on the Q of the cavity, the filling factor, the magnetic moment of the sample, factors affecting either the noise level or the signal level, etc.(7). In general, the cavity sensitivity is a multifunctional quantity. The determination of absolute spin concentration is quite a complicated exercise. For example, recent work shows that many correction factors including the lens effect of a quartz Dewar flask insert and the lens effects of the solvent (8), are needed to compare integrated EPR signal intensities in lossy solutions. The Q of the cavity also depends on the dielectric constant of the sample inside the cavity. In this study, we have kept many factors related to cavity sensitivity constant, and have varied only the tem-