A SENSITIVE, CONTINUOUSLY RECORDING FLUOROGENIC ASSAY FOR CALPAIN

We have developed a sensitive and continuously recording fluorogenic a ssay for the thiol protease calpain. This assay uses the dipeptide sub strate Suc-Leu-Tyr-4-Methoxy-2-Naphthylamine (Suc-LY-MNA) in Tris buff er (pH 7.5) in the presence of 0.1% CHAPS. The assay is linear over a wide range of enzyme concentration and is capable of detecting 10 pico molar calpain making it more sensitive than any previously published m ethod. Moreover, this assay gives a rate that is linear for over ten m inutes making it useful for mechanistic studies of inhibitors. This as say can be easily adapted to a 96-well plate format facilitating the l arge scale screening of inhibitors. (C) 1998 Academic Press.