Tryptophan fluorescence of terminal deoxynucleotidyl transferase: effects of quenchers on time-resolved emission spectra.
Tryptophan fluorescence of terminal deoxynucleotidyl transferase: effects of quenchers on time-resolved emission spectra.
复制标题
末端脱氧核苷酸转移酶的色氨酸荧光：猝灭剂对时间分辨发射光谱的影响。
DOI：
10.1021/bi00346a034
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发表时间：
1985
期刊：
影响因子：
2.9
通讯作者：
Barkley,MD
中科院分区：
文献类型：
--
作者：
Robbins,DJ;DeibelJr,MR;Barkley,MD
肯塔基大学医学中心生物化学系，列克星敦，肯塔基州 40536-0084 收到 1984 年 12 月 27 日 摘要：末端脱氧核苷酸转移酶 (EC 2.7. 7.31) 是一种不需要模板的真核 DNA 聚合酶。通过荧光研究小牛胸腺末端转移酶中的色氨酸环境。通过时间分辨发射光谱分离这种多色氨酸酶的异质发射。纳秒荧光在 296 nm 激发下衰减，并且通过全局分析对各种发射波长进行去卷积，假设寿命而不是相对权重因子与发射波长无关。数据符合三个寿命指数 r¡= 1.4 ns、2= 4.5 ns 和 3= 7.7 ns。三种成分相应的衰变相关发射光谱在约 328、335 和 345 nm 处具有最大值。在稳态和时间分辨实验中检查了单个色氨酸环境对极性和非极性荧光猝灭剂的可及性。在存在碘化物和丙烯酰胺的情况下，稳态发射光谱移至蓝色。然而，在低猝灭剂浓度下，7.7 ns 组件（最大 345 nm）的发射几乎不受影响，表明这种亲水性色氨酸环境被隐藏在蛋白质内。另一方面，三氯乙醇存在下稳态发射光谱的红移表明 1.4-ns 成分（最大 328 nm）是暴露的疏水性色氨酸环境。结果与末端转移酶蛋白的由内而外模型一致，其中疏水性较强的色氨酸靠近表面，而亲水性最强的色氨酸位于核心。
Department of Biochemistry, University of Kentucky Medical Center, Lexington, Kentucky 40536-0084 Received December 27, 1984 abstract: Terminal deoxynucleotidyltransferase (EC 2.7. 7.31) is a eucaryotic DNA polymerase that does not require a template. The tryptophan environments in calf thymus terminal transferase were investigated by fluorescence. The heterogeneous emission from this multitryptophan enzyme was separated by time-resolved emission spectroscopy. Nanosecond fluorescence decays at 296-nmexcitation and various emission wavelengths were deconvolved by global analysis, assuming that the lifetimes but not the relative weighting factors were independent of emission wavelength. The data were fit to three exponentials of lifetimes r¡= 1.4 ns, 2= 4.5 ns, and 3= 7.7 ns. The corresponding decay-associated emission spectra of the three components had maxima at about 328, 335, and 345 nm. The accessibility of individual tryptophan environments to polar and nonpolar fluorescence quenchers was examined in steady-state and time-resolved experiments. In the presence of iodide and acrylamide, the steady-state emission spectra shift to the blue. However, at low quencher concentrations, the emission from the 7.7-ns component (maximum 345 nm) is hardly affected, suggesting that this hydrophilic tryptophan environment is buried within the protein. On the other hand, the red shift in the steady-state emission spectrum in the presence of trichloroethanol indicates that the 1.4-ns component (maximum 328 nm) is an exposed hydrophobic tryptophan environment. The results are consistent with an inside-out model for terminal transferase protein, with the more hydrophobic tryptophan (s) near the surface and the most hydrophilic tryptophan (s) in the core.