Oral Radiation Oncology, Department of Oral Restitution, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan.
Biochem Biophys Res Commun. 2012 May 18;421(4):855-9. doi: 10.1016/j.bbrc.2012.04.102. Epub 2012 Apr 25.
Although oxygen is required for functional chromophore formation during the maturation process of fluorescent proteins, the effects of hypoxia on their fluorescence have rarely been studied in mammalian cells. We recently reported that severe hypoxia (pO(2)<0.1%) abrogates fluorescence from the fluorescent ubiquitination-based cell cycle indicator (Fucci) expressed in HeLa cells. Fucci is a system for visualizing cell cycle progression in live cells using red (monomeric Kusabira Orange 2, mKO(2)) and green (monomeric Azami Green, mAG) fluorescent proteins. In this study, taking advantage of the system, we attempted to determine the dependence on oxygen tension (pO(2)) of these two fluorescent proteins during the maturation process. The oxygen tension at which the number of fluorescence-positive cells was reduced by 50% (pO(2)·50) was 0.9% and 0.3% for mKO2 and mAG, respectively. Furthermore, we measured fluorescence recovery kinetics after reoxygenation in cells treated at two different pO(2) levels, and observed that mKO2 exhibits slower kinetics of oxidation than mAG. Thus, we demonstrate that mKO2 exhibits a stronger dependence on oxygen tension than mAG, as well as the usefulness of this novel method to produce varying levels of hypoxic conditions.
尽管氧气是荧光蛋白成熟过程中功能性发色团形成所必需的，但在哺乳动物细胞中，缺氧对其荧光的影响很少被研究。我们最近报道，严重缺氧（pO2<0.1%）会消除 HeLa 细胞中表达的荧光素酶基细胞周期指示剂（Fucci）的荧光。Fucci 是一种使用红色（单体 Kusabira Orange 2，mKO2）和绿色（单体 Azami Green，mAG）荧光蛋白在活细胞中可视化细胞周期进程的系统。在这项研究中，我们利用该系统试图确定这两种荧光蛋白在成熟过程中对氧张力（pO2）的依赖性。使荧光阳性细胞数量减少 50%的氧张力（pO2·50）分别为 0.9%和 0.3%，适用于 mKO2和 mAG。此外，我们测量了在两个不同 pO2水平下处理的细胞再氧合后的荧光恢复动力学，并观察到 mKO2的氧化动力学比 mAG 慢。因此，我们证明 mKO2比 mAG 对氧张力的依赖性更强，并且这种新方法对于产生不同程度的缺氧条件非常有用。
