Berezin Mikhail Y, Lee Hyeran, Akers Walter, Nikiforovich Gregory, Achilefu Samuel
Department of Radiology, Washington University School of Medicine, St. Louis, MO, USA.
Photochem Photobiol. 2007 Nov-Dec;83(6):1371-8. doi: 10.1111/j.1751-1097.2007.00173.x.
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.
许多疾病都与血液中主要的细胞外蛋白白蛋白的异常构象有关。目前的蛋白质结构分析需要纯的分离样本，因此限制了其在血液中白蛋白分析的应用。在本研究中，我们报告了一种利用近红外（NIR）聚甲炔染料的荧光寿命特性对白蛋白进行高通量结构相关分析的新方法。基于分子建模，聚甲炔染料与白蛋白上两个不同极性的结合位点结合。结果，一个近红外分子探针呈现出两个不同的寿命以及两个相应的荧光分数贡献。分数贡献的分布以及各个荧光寿命代表了通过比率分析表征白蛋白结构的独特参数。在筛选了一个近红外聚甲炔染料的小型文库后，我们鉴定并使用了一种具有最佳荧光寿命特性的聚甲炔染料，以评估市售牛血清白蛋白作为模型系统的结构相关差异。结果表明，近红外染料寿命的变化反映了白蛋白三级结构的扰动，并且不同方法制备的白蛋白三级结构略有改变。由于血液在近红外区域对光的吸收减少，所开发的方法可用于在不预先分离纯蛋白的情况下测定全血中白蛋白的结构变化。
