An ultrasensitive, continuous fluorometric assay for calpain activity.
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.
Tompa
P
P
Institute of Enzymology, Hungarian Academy of Sciences, Budapest, Hungary.
Schád
E
E
Baki
A
A
Alexa
A
A
Batke
J
J
Friedrich
P
P
D000818:Animals; D002154:Calpain / Q000032:analysis*; D015951:Factor Xa / Q000737:chemistry; D005452:Fluoresceins; D005470:Fluorometry; D006868:Hydrolysis; D008869:Microtubule-Associated Proteins / Q000737:chemistry*; D011516:Prothrombin / Q000737:chemistry; D051381:Rats; D017208:Rats, Wistar; D012680:Sensitivity and Specificity
D005452:Fluoresceins; D008869:Microtubule-Associated Proteins; C039083:5-((4,6-dichloro-1,3,5-triazin-2-yl)amino)fluorescein; D011516:Prothrombin; D015951:Factor Xa; D002154:Calpain