Faculty of Pharmaceutical Sciences, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8521, Japan.
Anal Biochem. 2013 Feb 15;433(2):79-85. doi: 10.1016/j.ab.2012.10.018. Epub 2012 Oct 22.
Caspases are the key regulatory factors of apoptosis and are also found to be involved in inflammatory cytokinesis. Sensitive and selective determination of caspases has significant importance in evaluation of apoptosis, disease diagnosis, and drug development. Here, we developed an assay method for the determination of caspase activity. This method is based on a novel fluorescence (FL) reaction selective for N-terminal Ser-containing peptides. FL derivatization of peptides requires heating in the presence of catechol, HEPES buffer (pH 7.5), and sodium periodate. Under optimized conditions, the reaction showed a unique sequence preference for N-terminal Ser-containing peptides, and a lower detection limit (signal/noise [S/N] = 3) of approximately 0.1 μM was obtained for SKTS and SSNSF. Acetylated substrates were enzymatically cleaved to produce N-terminal Ser-containing peptides, which were selectively converted to FL compounds. The enzyme activities were simultaneously determined as low as 2 U (4.3 nM) caspase-3 and 2.5 U (3.3 nM) caspase-8 by high-performance liquid chromatography (HPLC) with FL detection. The proposed assay method does not require any labeled substrates and can be applied to evaluate cell-based apoptosis and also to study apoptosis inhibitors or inducers.
半胱天冬酶是细胞凋亡的关键调节因子，也被发现参与炎症细胞分裂。在评估细胞凋亡、疾病诊断和药物开发方面，对细胞凋亡的灵敏和选择性检测具有重要意义。在此，我们开发了一种用于检测半胱天冬酶活性的方法。该方法基于对 N 端含有丝氨酸的肽段具有选择性的新型荧光（FL）反应。FL 衍生化肽段需要在儿茶酚、HEPES 缓冲液（pH7.5）和高碘酸钠存在下加热。在优化条件下，该反应对 N 端含有丝氨酸的肽段具有独特的序列偏好性，对于 SKTS 和 SSNSF，检测限（信号/噪声[S/N] = 3）约为 0.1 μM。乙酰化的底物被酶切产生 N 端含有丝氨酸的肽段，这些肽段被选择性地转化为 FL 化合物。通过高效液相色谱（HPLC）与 FL 检测，该方法可以同时测定低至 2U（4.3nM）的 caspase-3 和 2.5U（3.3nM）的 caspase-8 的酶活性。该方法不需要任何标记的底物，可用于评估基于细胞的细胞凋亡，也可用于研究凋亡抑制剂或诱导剂。
