|Title||An ultrasensitive, continuous fluorometric assay for calpain activity.|
|Publication Type||Journal Article|
|Year of Publication||1995|
|Authors||Tompa, P., E. Schád, A. Baki, A. Alexa, J. Batke, and P. Friedrich|
|Date Published||1995 Jul 1|
|Keywords||Animals, 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 Journal||Anal. Biochem.|