calpain and 5-((4-6-dichloro-1-3-5-triazin-2-yl)amino)fluorescein

Topics: Animals; Calpain; Fluoresceins; Fluorescent Dyes; In Vitro Techniques; Microtubule-Associated Proteins; Sensitivity and Specificity; Spectrometry, Fluorescence; Substrate Specificity

Topics: Autolysis; Calpain; Enzyme Activation; Erythrocytes; Fluoresceins; Fluorescent Dyes; Humans; In Vitro Techniques; Isoenzymes; Kinetics; Microtubule-Associated Proteins; Protein Structure, Quaternary; Substrate 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.

Topics: Animals; Calpain; Factor Xa; Fluoresceins; Fluorometry; Hydrolysis; Microtubule-Associated Proteins; Prothrombin; Rats; Rats, Wistar; Sensitivity and Specificity