Site-directed electrostatic measurements with a thiol-specific ph-sensitive nitroxide: differentiating local pK and polarity effects by high-field EPR.
Site-directed electrostatic measurements with a thiol-specific ph-sensitive nitroxide: differentiating local pK and polarity effects by high-field EPR.
复制标题
使用硫醇特异性 ph 敏感硝基氧进行定点静电测量：通过高场 EPR 区分局部 pK 和极性效应。
DOI：
--
复制
发表时间：
2004
影响因子：
15
通讯作者：
I. Grigor’ev
中科院分区：
文献类型：
--
作者：
A. Smirnov;A. Ruuge;V. Reznikov;M. Voinov;I. Grigor’ev
该通信描述了使用咪唑烷氮氧化物的甲硫基磺酸盐衍生物，甲硫基磺酸S-（1-氧基-2，2，3，5，5-五甲基-咪唑烷-4-基甲基）酯，IMTSL，通过电子顺磁共振进行肽的定点pKa测定。该自旋标记物共价连接到并入肽结构中的独特半胱氨酸的巯基。标记的叔胺氮N3容易参与质子交换反应，其通过氮氧部分的EPR谱的变化来监测。使用EPR在95 GHz（W-波段）的各向同性磁参数的氮氧化物，无论是AISO和GISO，在不同的极性和pH值的溶剂中进行校准。两个不同的线性相关性AISO和GISO之间的酸性和碱性形式的IMTSL观察，使之有可能区分从N3质子化氮氧化物EPR谱的局部极性的影响。层粘连蛋白B1链的合成P11肽片段的滴定说明了这种方法的实用性。
This communication describes the use of a methanethiosulfonate derivative of an imidazolidine nitroxide, methanethiosulfonic acid S-(1-oxyl-2,2,3,5,5-pentamethyl-imidazolidin-4-ylmethyl) ester, IMTSL, for site-directed pKa determination of peptides by electron paramagnetic resonance. This spin label is covalently attached to the thiol group of unique cysteines incorporated into peptide structures. The tertiary amine nitrogen N3 of the label readily participates in proton exchange reactions, which are monitored through changes in EPR spectra of nitroxide moiety. Using EPR at 95 GHz (W-band) isotropic magnetic parameters of this nitroxide, both Aiso and giso, were calibrated in solvents of different polarity and pH. Two different linear correlations between Aiso and giso for acidic and basic forms of IMTSL were observed, making it possible to differentiate effects of local polarity from N3 protonation on nitroxide EPR spectra. Titration of a synthetic P11 peptide fragment of the laminin B1 chain illustrates the utility of this method.