Practical conditions for photoaffinity labeling with 3-trifluoromethyl-3-phenyldiazirine photophore.
Practical conditions for photoaffinity labeling with 3-trifluoromethyl-3-phenyldiazirine photophore.
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DOI：
10.1016/j.ab.2005.10.015
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发表时间：
2006
影响因子：
2.9
通讯作者：
M. Hashimoto;Y. Hatanaka
中科院分区：
文献类型：
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作者：
M. Hashimoto;Y. Hatanaka
材料和方法TPD衍生物（1）和L-氨基酸氧化酶（5）和胰凝乳蛋白酶（6）的生物素化TPD配体先前已合成[8 - 10]。分别用UVP XX15（15W）和UVM-57（8W）灯在350和302 nm下进行照射。在JEOL JNM-ECA 500分光光度计上获得氧化氘中的19F-NMR，其中TFA作为内标（δ 0.00）。光标记蛋白质的化学发光检测和定量密度测定以与先前所述类似的方式进行[11]。结果和讨论TPD及其重氮异构体能够通过其特征UV光谱和19 F-NMR信号彼此区分[3 - 6]。光解过程总结见图1，A。重氮化合物的分光光度法检测在较低浓度（<1 mM）下是困难的，因为特征重氮吸收（400 nm）的摩尔消光系数比二氮丙啶吸收（350 nm）低10倍。此外，卡宾加合物通常没有特征吸收。与此相反，由TPD的光解产生的所有化合物都具有三氟甲基基团，其适合于估计每种化合物的浓度。
Materials and methods The TPD derivative (1) and biotinylated TPD ligands for L-amino acid oxidase (5) and chymotrypsin (6) were synthesized previously [8-10]. Irradiation was performed at 350 and 302 nm with a UVP XX15 (15W) and UVM-57 (8W) lamp, respectively. 19 F-NMR in deuterium oxide was obtained on a JEOL JNM-ECA500 spectrophotometer with TFA as the internal standard (δ 0.00). Chemiluminescent detection and quantitative densitometry of photolabeled proteins was performed in a similar manner as described previously [11]. Results and discussionsTPD and its diazo isomer are able to be differentiated from each other by their characteristic UV spectra and 19 F-NMR signals [3-6]. The photolytic process is summarized in Fig 1, A. The spectrophotometric detection of diazo compounds is difficult at lower concentrations (< 1 mM), because the molar extinction coefficient of characteristic diazo absorption (400 nm) is 10 times lower than that of diazirine absorption (350 nm). Furthermore, carbene adducts usually have no characteristic absorption. In contrast to this, all compounds generated from the photolysis of TPD have a trifluoromethyl group that is suitable for the estimation of each