Fluorescence anisotropy measurements under oxygen quenching conditions as a method to quantify the depolarizing rotations of fluorophores. Application to diphenylhexatriene in isotropic solvents and in lipid bilayers.
Fluorescence anisotropy measurements under oxygen quenching conditions as a method to quantify the depolarizing rotations of fluorophores. Application to diphenylhexatriene in isotropic solvents and in lipid bilayers.
复制标题
氧猝灭条件下的荧光各向异性测量作为量化荧光团去偏振旋转的方法。
作者：
J. Lakowicz;F. Prendergast;D. Hogen
我们测量了 1,6-二苯基-1,3,5-己三烯 (DPH) 的荧光各向异性，因为其荧光寿命因氧猝灭而缩短。这些研究是对溶解在各向同性溶剂矿物油中的 DPH 以及嵌入二肉豆蔻酰-L-α-磷酸胆碱 (DMPC) 或二油酰-α-磷脂酰胆碱 (D) 磷脂囊泡中的 DPH 进行的。为了获得足够的氧气，必须使用。强度的显着变化是可逆的。为了控制所研究的系统，在这些条件下用氮气形成，arg最后-menti01 和各向异性用荧光团的氧 qut 观察到，其长度为 dl 弧度/秒，与 f 参数相比，提供了 m 荧光团环境的氧猝灭荧光寿命稳态荧光 z R 和 r ，对于 D P H 的温度，发现猝灭各向异性 me; > PC)，每个在几个温度下。淬火增加氧气淬火的压力导致 sig.y 和各向异性，并且这些影响 11 对于压力对等效实验的可能影响是 pern，或形成气相的氦气。 :d 条件下，与那些瘙痒相比，强度变化显着。消偏振旋转由其旋转速率（R）在各向异性中所描述，其时间为荧光寿命r。后者确保了 t 阻碍其旋转扩散的程度。荧光3提供了一种改变各向异性同时观察的方法，允许在两种不同的R值下对两种矿物油进行定量，从该溶液获得的R值与从稳态各向异性测量获得的值以及从差分偏振相位荧光测定获得的值完全一致（Lakowicz，J.R.等人（1979）Biochemisfry I8荧光各向异性测量已被可用于研究蛋白质-配体相互作用（Anderson & Weber，1965；Weber & Daniel，1966）、蛋白质-蛋白质相互作用（Levison 等人，1970；Dandliker & de Saussure，1970）和脂质双层的相变（Cogan 等人，1973；Shinitzky 等人，1971）。荧光发射速率和蛋白质旋转扩散速率或脂质双层中的荧光团旋转速率扩散速率通常对温度和溶剂粘度敏感，而荧光发射速率可能对这些因素不敏感：因此必须经常进行。
We have measured the fluorescence anisotropy of 1,6-diphenyl-l,3,5-hexatriene (DPH) as its fluorescence lifetime is decreased by oxygen quenching. Such studies were done on DPH dissolved in the isotropic solvent mineral oil and for DPH embedded in phospholipid vesicles of either dimyristoyl-L-a-phosphatic'vlcholine (DMPC) or dioleovl-ra-phosphatidylcholine (D . In order to obtain adeque oxygen had to be used. nificant changes in intens were reversible. To contr the systems under study formed with nitrogen, arg Under these last-menti01 and anisotropy were insig observed with oxygen qut of the fluorophore are dl radians/seconds and its li long compared with the f parameter provides a m fluorophore's environme Oxygen quenching of flu the fluorescence lifetimc steady-state fluorescence z R and r , . For D P H in peratures u e found that quenching-anisotropy me; >PC), each a t several temperatures. e quenching increased pressures of lxygen quenching resulted in sig.y and anisotropy, and these effects 11 for possible effects of pressure on equivalent experiments were pern, or helium forming the gas phase. :d conditions, changes in intensity iificant when compared with those iching. The depolarizing rotations scribed by its rotation rate ( R ) in iiting anisotropy at times which are Jorescence lifetime, r,. This latter asure of the degree to which the t hinders its rotational diffusion. rescence 3rovides a means to vary simulta :ous observation of the iisotropy allows quantitation of both mineral oil a t two different temhe values of R obtained from this urement agreed precisely with those ibtained from steady-state anisotropy measurements and with the values obtained from differential polarized phase fluorometry (Lakowicz, J. R., et al. (1979) Biochemisfry I8 Fluorescence anisotropy measurements have been useful in the investigation of protein--ligand interactions (Anderson & Weber, 1965; Weber & Daniel, 1966), protein-protein interactions (Levison et al.. 1970; Dandliker & de Saussure, 1970). and the phase transitions of lipid bilayers (Cogan et al.. 1973; Shinitzky et al.. 1971). This usefulness derives primarily from the similarity of the rates of fluorescence emission and the rates of rotational diffusion of proteins or the rates of fluorophore rotation in lipid bilayers. Diffusional rates are generally sensitive to temperature and solvent viscosity, whereas the rate of fluorescence emission can be insensitive to these factors: consequently one must frequently