Ratiometric analysis of fluorescence lifetime for probing binding sites in albumin with near-infrared fluorescent molecular probes
Ratiometric analysis of fluorescence lifetime for probing binding sites in albumin with near-infrared fluorescent molecular probes
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DOI：
10.1111/j.1751-1097.2007.00173.x
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发表时间：
2007-11-01
影响因子：
3.3
通讯作者：
Achilefu, Samuel
中科院分区：
文献类型：
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作者：
Berezin, Mikhail Y.;Lee, Hyeran;Achilefu, Samuel
许多疾病已与白蛋白的异常构象有关，白蛋白是血液中主要的细胞外蛋白。当前的蛋白质结构分析需要纯孤立的样品，从而限制了它们用于血液中的白蛋白分析。在这项研究中，我们报告了一种通过使用近红外（NIR）多胸蛋白染料的荧光寿命特性的高通量结构相关分析的新方法。基于分子建模，多胸染料与白蛋白上极性不同的两个结合位点结合。结果，NIR分子探针表现出两个不同的寿命，具有两个相应的荧光分数贡献。分数贡献以及单个荧光寿命的分布代表了通过比率分析表征白蛋白结构的独特参数。在筛选一个小型NIR多叶氨酸染料的小库后，我们鉴定并使用了具有最佳荧光寿命特性的多胸染料来评估商业上牛血清白蛋白中与结构相关的差异作为模型系统。结果表明，NIR染料的寿命变化反映了白蛋白第三纪结构的扰动，而通过不同方法制备的白蛋白的第三级结构略有变化。由于NIR地区的血液吸收光的吸收降低，因此开发的方法可用于确定全血白蛋白的结构变化，而无需事先分离纯蛋白。
A number of diseases have been linked to abnormal conformation of albumin, a major extracellular protein in blood. Current protein structural analysis requires pure isolated samples, thereby limiting their use for albumin analysis in blood. In this study, we report a new approach for high-throughput structure-related analysis of albumin by using the fluorescence lifetime properties of near-infrared (NIR) polymethine dyes. Based on molecular modeling, polymethine dyes are bound to two binding sites with different polarities on albumin. As a result, an NIR molecular probe exhibits two distinct lifetimes with two corresponding fluorescent fractional contributions. The distribution of fractional contributions along with individual fluorescence lifetimes represents unique parameters for characterizing albumin architecture by ratiometric analysis. After screening a small library of NIR polymethine dyes, we identified and used a polymethine dye with optimal fluorescence lifetime properties to assess structure-related differences in commercially available bovine serum albumin as model systems. The results show that changes in the lifetime of NIR dyes reflect the perturbation of the tertiary structures of albumin and that albumin prepared by different methods has slightly altered tertiary structures. Because of the reduced absorption of light by blood in the NIR region, the method developed can be used to determine structural changes in albumin in whole blood without prior isolation of the pure protein.