School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai 201203, China.
Analyst. 2012 Nov 7;137(21):5097-104. doi: 10.1039/c2an35822k. Epub 2012 Sep 12.
A novel fluorescence derivatization method combined with HPLC was developed to detect the activity of caspase-3 and -8 in two cell lines (Hela cells and A549 cells) which were activated by low temperature-assisted ultraviolet irradiation (LT-UV), mitomycin C (MMC) and camptothecin during the apoptosis, respectively. Two peptide substrates for either caspase-3 or -8 were designed, of which peptide fragments were obtained by enzymatic modification, followed by fluorescence derivatization. A single fluorescent product was formed when a peptide was heated at 120 °C for 10 min in a neutral aqueous medium (pH 7.0) containing catechol, sodium periodate and sodium borate. Commercial kits for detecting the activity of caspase-3 and -8 were used as a control. The relative activity of the caspases detected by fluorescence derivatization was similar to that obtained by commercial kits, which indicated that the novel method is reliable. The activity assays of recombinant human caspases showed that the novel method provided higher selectivity than that of commercial kits, which proved it to be more accurate for determining the activity of caspases in apoptosis.
建立了一种新的荧光衍生化方法，结合 HPLC 检测两种细胞系（Hela 细胞和 A549 细胞）中 caspase-3 和 -8 的活性，这两种细胞系分别通过低温辅助紫外线照射（LT-UV）、丝裂霉素 C（MMC）和喜树碱在凋亡过程中被激活。设计了两种针对 caspase-3 或 -8 的肽底物，其中肽片段通过酶修饰获得，然后进行荧光衍生化。当在中性水性介质（pH 7.0）中加热含有儿茶酚、高碘酸钠和硼酸钠的肽 120°C 10 分钟时，会形成单一的荧光产物。商业试剂盒用于检测 caspase-3 和 -8 的活性作为对照。通过荧光衍生化检测到的 caspase 的相对活性与商业试剂盒获得的活性相似，这表明该新方法是可靠的。重组人 caspase 的活性测定表明，与商业试剂盒相比，该新方法提供了更高的选择性，这证明它在确定凋亡中 caspase 的活性方面更准确。
