DEVD-NucView488: a novel class of enzyme substrates for real-time detection of caspase-3 activity in live cells
DEVD-NucView488: a novel class of enzyme substrates for real-time detection of caspase-3 activity in live cells
复制标题
DOI：
10.1096/fj.07-099234
复制
发表时间：
2008-07-01
期刊：
影响因子：
4.8
通讯作者：
Firestone, Gary L.
中科院分区：
文献类型：
--
作者：
Cen, Hui;Mao, Fei;Firestone, Gary L.
细胞内酶活性的活细胞检测要求底物是细胞可渗透的，并且所产生的产物易于检测并保留在细胞中。我们的目标是创造一种新的荧光底物，可用于实时检测活细胞中的凋亡。我们已经合成了一种高度细胞渗透性的caspase-3底物，DEVD NucView 488，通过连接荧光DNA结合染料的caspase-3识别序列，使染料无功能。在底物裂解时，染料被释放并在与DNA结合时变得高度荧光。DEVD-NucView 488在活细胞群中检测到caspase-3激活要早得多，并且与目前可用的其他凋亡试剂相比具有更高的灵敏度。此外，用DEVD-NucView 488孵育的细胞没有表现出毒性和正常的凋亡进展。DEVD-NucView 488是caspase-3激活动力学研究的理想底物，因为它实时检测caspase-3活性，还有效地标记caspase-3激活细胞核中的DNA，用于凋亡形态的实时荧光可视化。该荧光底物的设计中所采用的策略可以应用于未来的努力，以开发用于检测实时细胞内酶活性的底物。
Live-cell detection of intracellular enzyme activity requires that substrates are cell-permeable and that the generated products are easily detected and retained in cells. Our objective was to create a novel fluorogenic substrate that could be used for real-time detection of apoptosis in living cells. We have synthesized a highly cell-permeable caspase-3 substrate, DEVD-NucView488, by linking a fluorogenic DNA-binding dye to the caspase-3 recognition sequence that renders the dye nonfunctional. On substrate cleavage, the dye is released and becomes highly fluorescent on binding to DNA. DEVD-NucView488 detected caspase-3 activation within a live-cell population much earlier and with higher sensitivity compared with other apoptosis reagents that are currently available. Furthermore, cells incubated with DEVD-NucView488 exhibited no toxicity and normal apoptotic progression. DEVD-NucView488 is an ideal substrate for kinetic studies of caspase-3 activation because it detects caspase-3 activity in real-time and also efficiently labels DNA in nuclei of caspase-3-activated cells for real-time fluorescent visualization of apoptotic morphology. The strategy utilized in the design of this fluorogenic substrate can be applied in future endeavors to develop substrates for detecting real-time intracellular enzyme activity.