A sensitive, continuously recording fluorogenic assay for calpain.
We have developed a sensitive and continuously recording fluorogenic assay for the thiol protease calpain. This assay uses the dipeptide substrate Suc-Leu-Tyr-4-Methoxy-2-Naphthylamine (Suc-LY-MNA) in Tris buffer (pH 7.5) in the presence of 0.1% CHAPS. The assay is linear over a wide range of enzyme concentration and is capable of detecting 10 picomolar calpain making it more sensitive than any previously published method. Moreover, this assay gives a rate that is linear for over ten minutes making it useful for mechanistic studies of inhibitors. This assay can be easily adapted to a 96-well plate format facilitating the large scale screening of inhibitors.
Mallya
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Cephalon Inc., 145 Brandywine Parkway, West Chester, Pennsylvania, 19380, USA.
Meyer
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Bozyczko-Coyne
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Siman
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Ator
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D002154:Calpain / Q000032:analysis*; D002793:Cholic Acids / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D004151:Dipeptides / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000032:analysis; D004912:Erythrocytes / Q000201:enzymology*; D005470:Fluorometry; D006801:Humans; D007700:Kinetics; D009281:Naphthalenes / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012680:Sensitivity and Specificity; D013379:Substrate Specificity
D002793:Cholic Acids; D003902:Detergents; D004151:Dipeptides; D004791:Enzyme Inhibitors; D009281:Naphthalenes; D011994:Recombinant Proteins; D002154:Calpain; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate