An ultrasensitive, continuous fluorometric assay for calpain activity.

TitleAn ultrasensitive, continuous fluorometric assay for calpain activity.
Publication TypeJournal Article
Year of Publication1995
AuthorsTompa, P., E. Schád, A. Baki, A. Alexa, J. Batke, and P. Friedrich
JournalAnal Biochem
Date Published1995 Jul 1
KeywordsAnimals, Calpain, Factor Xa, Fluoresceins, Fluorometry, Hydrolysis, Microtubule-Associated Proteins, Prothrombin, Rats, Rats, Wistar, Sensitivity and Specificity

A rapid, continuous assay for calcium-activated neutral protease activity is described. This assay is based on monitoring the elevation in fluorescence intensity that occurs upon calpainolytic digestion of dichlorotriazinylamino-fluorescein-labeled microtubule-associated protein 2. Tedious separation of peptide products from the protein substrate in this rapid assay is unnecessary, which thus offers two remarkable advantages over conventional caseinolytic assay procedures: (i) it raises sensitivity of detection by about three orders of magnitude, allowing the quantitative determination of calpain in the high picogram range in 10 min; and (ii) it permits a continuous detection of activity, which 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.

Alternate JournalAnal. Biochem.
PubMed ID8572308