Belousov Vsevolod V, Fradkov Arkady F, Lukyanov Konstantin A, Staroverov Dmitry B, Shakhbazov Konstantin S, Terskikh Alexey V, Lukyanov Sergey
Laboratory of Genes for Regeneration, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya Street 16/10, Moscow 117997, Russia.
Nat Methods. 2006 Apr;3(4):281-6. doi: 10.1038/nmeth866.
We developed a genetically encoded, highly specific fluorescent probe for detecting hydrogen peroxide (H(2)O(2)) inside living cells. This probe, named HyPer, consists of circularly permuted yellow fluorescent protein (cpYFP) inserted into the regulatory domain of the prokaryotic H(2)O(2)-sensing protein, OxyR. Using HyPer we monitored H(2)O(2) production at the single-cell level in the cytoplasm and mitochondria of HeLa cells treated with Apo2L/TRAIL. We found that an increase in H(2)O(2) occurs in the cytoplasm in parallel with a drop in the mitochondrial transmembrane potential (DeltaPsi) and a change in cell shape. We also observed local bursts in mitochondrial H(2)O(2) production during DeltaPsi oscillations in apoptotic HeLa cells. Moreover, sensitivity of the probe was sufficient to observe H(2)O(2) increase upon physiological stimulation. Using HyPer we detected temporal increase in H(2)O(2) in the cytoplasm of PC-12 cells stimulated with nerve growth factor.
我们开发了一种基因编码的、高度特异性的荧光探针，用于检测活细胞内的过氧化氢（H₂O₂）。这种名为HyPer的探针由插入原核H₂O₂传感蛋白OxyR调节结构域的环状排列黄色荧光蛋白（cpYFP）组成。利用HyPer，我们在单细胞水平上监测了用Apo2L/TRAIL处理的HeLa细胞的细胞质和线粒体中H₂O₂的产生情况。我们发现，细胞质中H₂O₂的增加与线粒体跨膜电位（ΔΨ）的下降以及细胞形态的变化同时发生。我们还观察到凋亡HeLa细胞在ΔΨ振荡期间线粒体H₂O₂产生的局部爆发。此外，该探针的灵敏度足以观察到生理刺激后H₂O₂的增加。利用HyPer，我们检测到神经生长因子刺激的PC-12细胞细胞质中H₂O₂的瞬时增加。
