A quantitative continuous enzyme assay of intramolecularly quenched fluorogenic phospholipase substrates for molecular imaging.
There has been recent growth in the development of activatable near-infrared (NIR) fluorescent probes for molecular imaging, generally designed by placing fluorochromes on a cleavable substrate in close proximity to one another, such that they self-quench, but fluoresce on separation via enzymatic cleavage of the substrate. Although these probes offer excellent contrast, the detection of enzyme activity has largely only been described qualitatively. In order to assess the effectiveness of a probe, it is useful to have a quantitative measure, such as the enzyme-substrate kinetic parameters. We have developed an assay to determine kinetic parameters and applied it to an intramolecularly quenched molecule, Pyro-PtdEtn-BHQ, a NIR fluorescent probe specific to phosphatidylcholine-specific phospholipase C. The development of this assay includes corrections for intermolecular quenching, calibration, optimization of reaction mixtures, and determination of kinetic and inhibition parameters. This assay can easily be extended to analyze and compare the efficiency of other fluorescent activatable phospholipase probes as suitable molecular imaging agents.
Department of Radiology, University of Pennsylvania, Philadelphia, PA 19104, USA.
Theresa M
TM
Mawn
Anatoliy V
AV
Popov
E James
EJ
Delikatny
D000818:Animals; D001409:Bacillus cereus; D002645:Chickens; D004531:Eggs; D057075:Enzyme Assays / Q000379:methods*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D057054:Molecular Imaging / Q000379:methods; D010713:Phosphatidylcholines / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D010738:Type C Phospholipases / Q000737:chemistry* / Q000378:metabolism
D005456:Fluorescent Dyes; D010713:Phosphatidylcholines; D010738:Type C Phospholipases; C105598:phosphatidylcholine-specific phospholipase C