The Biophysical Interdisciplinary Schottenstein Center for the Research and Technology of the Cellome, Physics Department, Bar Ilan University, Ramat-Gan, 5290002 Israel.
Photochem Photobiol Sci. 2018 Oct 10;17(10):1417-1428. doi: 10.1039/c7pp00455a.
The present study investigates the fluorescence properties of BO21 and their dependence on various intracellular conditions. The results obtained with cell-free solutions indicate that the influences of pH and temperature on the fluorescence spectra are negligible, while viscosity, various proteins and heparin have significant influence. In the presence of heparin, a red shift of the emission spectrum (from 515 to 550 nm) is observed, suggesting that this shift cannot simply be attributed to electrostatic interaction between BO21 and the polyanionic heparin, but rather to aggregation of BO21 on the polyanion. In water, the quantum yield of BO21 was found to be 1000 times lower than that of fluorescein, yet surprisingly its fluorescence polarization (FP) was found to be about 40 times higher (FP = 0.470), even though both have similar structures and molecular weights. A thorough analytical and experimental investigation of these phenomena indicates that the very high FP of BO21 in water is a consequence of its very short lifetime. However, upon the addition of heparin to aqueous BO21, the fluorescence lifetime (FLT) of BO21 increases from τ = 10.35 to 56.5 ps, with a consequent dramatic drop in its fluorescence polarization from 0.470 to 0.230. From its behavior in aqueous glycerol solution, it is hypothesized, with support from theoretical calculations, that BO21 is a molecular rotor. Using these properties, BO21 may be a good candidate as a sensor, for example, of heparin levels in blood or of intracellular viscosity.
本研究考察了 BO21 的荧光性质及其对各种细胞内条件的依赖性。无细胞溶液的结果表明，pH 值和温度对荧光光谱的影响可以忽略不计，而粘度、各种蛋白质和肝素则有显著影响。在肝素存在的情况下，发射光谱发生红移（从 515nm 移至 550nm），这表明这种位移不能简单地归因于 BO21 与带负电荷的肝素之间的静电相互作用，而是 BO21 在带负电荷的肝素上的聚集。在水中，BO21 的量子产率发现比荧光素低 1000 倍，但令人惊讶的是，它的荧光偏振（FP）却高约 40 倍（FP = 0.470），尽管两者具有相似的结构和分子量。对这些现象的深入分析和实验研究表明，BO21 在水中非常高的 FP 是其非常短的寿命的结果。然而，当向水性 BO21 中加入肝素时，BO21 的荧光寿命（FLT）从 τ = 10.35 增加到 56.5 ps，其荧光偏振度从 0.470 急剧下降至 0.230。从它在水性甘油溶液中的行为，可以假设，在理论计算的支持下，BO21 是一种分子转子。利用这些性质，BO21 可能是一种很好的传感器候选物，例如，用于检测血液中的肝素水平或细胞内粘度。
