A protease assay using time-resolved lanthanide luminescence from an engineered calcium binding protein substrate.
OBJECTIVE
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.


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.


CONCLUSIONS
The simplicity of the assay coupled with its high level of sensitivity make it useful for the detection of protease at very low concentrations.
Clark
I D
ID
Institute for Biological Sciences, National Research Council, Ottawa, Ontario, Canada.
MacManus
J P
JP
Szabo
A G
AG
D000818:Animals; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D002401:Cathepsin B / Q000032:analysis; D002918:Chymotrypsin / Q000032:analysis; D010450:Endopeptidases / Q000032:analysis*; D016333:HIV Protease / Q000032:analysis; D008163:Luminescent Measurements; D008674:Metals, Rare Earth / Q000737:chemistry*; D015202:Protein Engineering; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D013381:Subtilisins / Q000032:analysis; D013997:Time Factors
D002135:Calcium-Binding Proteins; D008674:Metals, Rare Earth; C034733:oncomodulin; D010450:Endopeptidases; D013381:Subtilisins; D002918:Chymotrypsin; D002401:Cathepsin B; D016333:HIV Protease