Measurement of specific protease activity utilizing fluorescence polarization.
A fluorescence polarization assay was designed to measure proteolytic cleavage of a specific peptide substrate for human cytomegalovirus protease. The peptide substrate was derivatized by biotinylation of a gamma-aminobutyric acid-modified amino-terminus and labeled with 5-(4,6-dichlorotriazinyl)aminofluorescein at the carboxy-terminus. Incubation of this substrate with recombinant human cytomegalovirus protease and subsequent addition of egg white avidin produced a polarization signal that was proportional to the relative amounts of cleaved and uncleaved substrate. The uncleaved substrate produced a high polarization value upon binding to avidin, whereas the cleaved, low-molecular-weight fluorescently tagged peptide that cannot bind to avidin produced a low polarization value. The inhibitory activity of a 3,4-dichloroisocoumarin against the protease was evaluated by comparing the change in polarization with a noninhibited control. The fluorescence polarization protease assay does not suffer from interference due to the presence of absorptive interferants making this a convenient, homogenous assay for high throughput screening.
Levine
L M
LM
Monsanto Corporate Research, St. Louis, Missouri, USA.
Michener
M L
ML
Toth
M V
MV
Holwerda
B C
BC
D000595:Amino Acid Sequence; D001360:Avidin; D001710:Biotin; D003587:Cytomegalovirus / Q000201:enzymology; D010450:Endopeptidases / Q000032:analysis* / Q000378:metabolism; D005069:Evaluation Studies as Topic; D019793:Fluorescein; D005452:Fluoresceins; D005454:Fluorescence Polarization / Q000379:methods*; D006801:Humans; D007700:Kinetics; D015394:Molecular Structure; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D005452:Fluoresceins; D009842:Oligopeptides; D001360:Avidin; D001710:Biotin; D010450:Endopeptidases; D019793:Fluorescein