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US 6,632,630 B2Granted
Monooxygenase assays
AssigneeAclara Biosciences, Inc.
InventorsSharat Singh, Ahmed Chenna, Hossein Salimi-Moosari, Ian Gibbons
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Abstract
Cytochrome P-450 assay methods and kits for the methods are provided employing a cytochrome P-450 enzyme, substrates characterized by having an oxidizable methylene group oxidized to an aldehyde and a fluorescent hydrazine. A fluorescent hydrazine is added to the reaction mixture and the resulting hydrazone analyzed by capillary electrophoresis. The method finds use in evaluating compounds for enzyme modulating activity.
Claims13
An assay for determining cytochrome P-450 enzyme activity comprising:
- combining under conditions for P-450 enzyme activity, a P-450 enzyme, a hydrogen donor cofactor, and at least one substrate characterized by having an ether group bonded to a methylene group, whereby said methylene group is enzymatically oxidized to an aldehyde group;
- adding a fluorescent hydrazine to react with said aldehyde group of each of said at least one substrate to form a hydrazone;
- quenching enzyme activity with a quencher;
- and
- assaying for said hydrazone using capillary electrophoresis, whereby separate peaks are obtained for each at least one substrate.
2 more elements
An assay for determining cytochrome P-450 enzyme activity comprising:
- combining under conditions for P-450 enzyme activity comprising a pH in the range of about 6-8 and a temperature in the range of about 20 to 50° C., a P-450 enzyme, NADPH, and at least one substrate characterized by having an ether group bonded to a methylene group, whereby said methylene group is enzymatically oxidized to an aldehyde group;
- adding a fluorescent hydrazine to react with said aldehyde group of each of said at least one substrates to form a hydrazone;
- quenching enzyme activity with a quencher;
- and
- assaying for said hydrazone using capillary electrophoresis, whereby separate peaks are obtained for each substrate.
2 more elements
Dependencies are inferred from the claim text.