AN ULTRASENSITIVE, CONTINUOUS FLUOROMETRIC ASSAY FOR CALPAIN ACTIVITY

A rapid, continuous assay for calcium-activated neutral protease activ ity is described. This assay is based on monitoring the elevation in f luorescence intensity that occurs upon calpainolytic digestion of dich lorotriazinylamino-fluorescein-labeled microtubule-associated protein 2. Tedious separation of peptide products from the protein substrate i n this rapid assay is unnecessary, which thus offers two remarkable ad vantages over conventional caseinolytic assay procedures: (i) it raise s sensitivity of detection by about three orders of magnitude, allowin g the quantitative determination of calpain in the high picogram range in 10 min; and (ii) it permits a continuous detection of activity, wh ich may prove invaluable in enzyme-mechanism studies that require pre- steady-state measurements. Other features and advantages of the assay, along with its limitations, are discussed in detail. (C) 1995 Academi c Press, Inc.