Journal
ANALYTICAL BIOCHEMISTRY
Volume 394, Issue 1, Pages 68-74Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2009.07.012
Keywords
Competitive substrates; Fluorescent substrates; Caspase specificity; 2-Aminoacridone; HPLC
Funding
- Counsel of Medical Science at Medical University Sofia
- National Science Fund of Bulgaria [BYX-10/2005]
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The specificity of 10 recombinant caspases was investigated using a set of competitive substrates. The caspase activity was determined by high-performance liquid chromatography using highly fluorescent peptides containing 2-aminoacridone (AMAC) as reporting group. The sequences of the used substrates were designed according to literature data for being specific for 10 of the caspases. The described approach allows the concentration changes of several substrates to be monitored simultaneously in a single sample. Because the substrates are in competitive conditions, the preferences of particular caspases to given peptide sequences are most clearly demonstrated. In the studied competitive assay conditions, all tested caspases except caspase 2 exhibit activity toward more than one substrate. None of the used peptide sequences was found to be highly specific for a defined caspase. The results obtained indicate that there is well-expressed group specificity among the caspases. (C) 2009 Elsevier Inc. All rights reserved.
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