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 |
Journal | Anal Biochem |
Volume | 228 |
Issue | 2 |
Pagination | 287-93 |
Date Published | 1995 Jul 1 |
ISSN | 0003-2697 |
Keywords | Animals, Calpain, Factor Xa, Fluoresceins, Fluorometry, Hydrolysis, Microtubule-Associated Proteins, Prothrombin, Rats, Rats, Wistar, Sensitivity and Specificity |
Abstract | 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. |
DOI | 10.1006/abio.1995.1352 |
Alternate Journal | Anal. Biochem. |
PubMed ID | 8572308 |
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