A protease assay using time-resolved lanthanide luminescence from an engineered calcium binding protein substrate
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I D Clark1, J P MacManus, A G Szabo
1Institute for Biological Sciences, National Research Council, Ottawa, Ontario, Canada.
Objectives:
The objective of this work was to demonstrate the utility of luminescence from lanthanides bound to a mutant of the Ca2+ binding protein, oncomodulin, to monitor protease activity.
Design And Methods:
A mutant of oncomodulin with a cysteine residue at position 57 located in the CD binding loop was conjugated to a salicylic acid group. The luminescence of Tb3+ resulting from electronic energy transfer from the salicylic acid group was monitored using time resolved lanthanide luminescence in the presence of proteolytic enzymes.
Results:
Low detection limits for subtilisin (150 pg), chymotrypsin (2.5 ng), cathepsin B (3.5 ng), and HIV-1 protease (25 ng) were found.
Conclusion:
The simplicity of the assay coupled with its high level of sensitivity make it useful for the detection of protease at very low concentrations.
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