A continuous method for measuring calpain activity.
A simplified methodology for assaying the caseinolysis by calpains was developed. This methodology, including the incubation of calpain with casein and direct measurement of the absorbance at 500 nm, is based on the turbidity of the reaction mixture caused by the aggregation of hydrolysates during the reaction. Unlike the typical caseinolysis assay, this novel assay does not need to separate the substrate from hydrolysates and can be continuously monitored in visible wavelength range. The activity of calpain is expressed by the maximum reaction velocity (delta A500/min) at 25 degrees C).
Jiang
S T
ST
Department of Marine Food Science, National Taiwan Ocean University, Keelung, Taiwan, Republic of China. sjiang@ind.ntou.edu.tw
Wang
J H
JH
Chang
T
T
Chen
C S
CS
D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002154:Calpain / Q000378:metabolism*; D002364:Caseins / Q000378:metabolism; D002417:Cattle; D004789:Enzyme Activation; D005450:Fluorescamine / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D012394:Rosaniline Dyes; D012996:Solutions; D013054:Spectrophotometry, Atomic; D014238:Trichloroacetic Acid
D002135:Calcium-Binding Proteins; D002364:Caseins; D007527:Isoenzymes; D012394:Rosaniline Dyes; D012996:Solutions; D005450:Fluorescamine; D014238:Trichloroacetic Acid; C048139:Coomassie blue; C033668:calpastatin; D002154:Calpain