A novel fluorescence reaction for N-terminal Ser-containing peptides and its application to assay caspase activity

• Published in: Analytical biochemistry Vol. 433; no. 2; pp. 79 - 85
• Main Authors: Rahman, Mohammed Shafikur, Kabashima, Tsutomu, Yasmin, Hasina, Shibata, Takayuki, Kai, Masaaki
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.02.2013
• Subjects: apoptosis; Caspase; Caspase 3 - chemistry; Caspase 8 - chemistry; caspase-3; caspase-8; catechol; cytokinesis; derivatization; detection limit; disease diagnosis; drugs; enzyme activity; Fluorescence; Fluorometric assay; heat; HEPES; high performance liquid chromatography; Humans; N-terminal Ser-containing peptides; peptides; Peptides - chemical synthesis; Peptides - chemistry; Sensitivity and Specificity; Serine - chemistry; sodium; Caspase; HEPES; N-terminal Ser-containing peptides; Fluorometric assay
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2012.10.018

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 0.1μM 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 2U (4.3nM) caspase-3 and 2.5U (3.3nM) 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.