A novel fluorescence derivatization method combined with HPLC for determining the activities of endogenous caspase

Abstract

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.