Assays for detecting modified compounds
Provided are methods and compositions which are useful for separating, isolating, detecting, and quantifying compounds of interest which have been modified chemically, enzymatically or catalytically from other compounds which have not been so modified. The modifications may take the form of functional groups which are gained, lost or retained by the compounds of interest.
1. A reagent for determining the presence of an enzyme that adds or removes a charged functional group to an uncharged moiety on a substrate or quantifying the activity of said enzyme in a sample, the reagent comprising:
the substrate to which a first member and a second member of an energy transfer pair are covalently bound at a distance such that a FRET interaction does not occur unless the charged functional group is present,
wherein the second member of the energy transfer pair has a charge opposite to the charge of the functional group, and
wherein the presence of the charged functional group on the substrate causes noncovalent binding of the second member of the energy transfer pair to the charged functional group, bringing the first member and the second member of the energy transfer pair close enough so that a FRET interaction occurs.
2. The reagent of claim 1 , wherein the charged functional group is negatively charged.
3. The reagent of claim 2 , wherein the negatively charged functional group is a phosphate, carboxyl, sulfate, nitro, or acyl group.
4. The reagent of claim 1 , wherein the second member of the energy transfer pair further comprises a chelator that binds a metal ion that binds to the functional group.
5. The reagent of claim 4 , wherein the functional group is a phosphate group.
6. A reagent for determining the presence of an enzyme that adds or removes an uncharged functional group to a charged moiety on a substrate or quantifying the activity of said enzyme in a sample, the reagent comprising:
the substrate to which a first member and a second member of an energy transfer pair are covalently bound at a distance such that a FRET interaction does not occur unless the uncharged functional group is absent,
wherein the second member of the energy transfer pair has a charge opposite to the charge of the moiety, and
wherein the absence of the uncharged functional group from the substrate causes noncovalent binding of the second member of the energy transfer pair to the charged moiety, bringing the first member and the second member of the energy transfer pair close enough so that a FRET interaction occurs.
7. The reagent of claim 6 , wherein the uncharged functional group is a methyl or an acetyl group.
8. The reagent of claim 6 , wherein the charged moiety is positively charged.
9. The reagent of claim 8 , wherein the positively charged moiety is an amino group.
10. The reagent of claim 1 , wherein the substrate is a synthetic compound.
11. The reagent of claim 1 , wherein the substrate comprises the charged functional group added to the uncharged moiety.
12. The reagent of claim 11 , wherein the substrate is a synthetic compound.
13. The reagent of claim 11 , wherein the functional group is negatively charged.
14. The reagent of claim 13 , wherein the negatively charged functional group is a phosphate, carboxyl, sulfate, nitro, or acyl group.
15. The reagent of claim 11 , wherein the second member of the energy transfer pair further comprises a chelator that binds a metal ion that binds to the functional group.
16. The reagent of claim 15 , wherein the functional group is a phosphate group.
17. The reagent of claim 6 , wherein the substrate is a synthetic compound.
18. The reagent of claim 6 , wherein the substrate comprises the uncharged functional group added to the uncharged moiety.
19. The reagent of claim 18 , wherein the substrate is a synthetic compound.
20. The reagent of claim 18 , wherein the uncharged functional group is a methyl or an acetyl group.
21. The reagent of claim 18 , wherein the charged moiety is positively charged.
22. The reagent of claim 21 , wherein the positively charged moiety is an amino group.
23. A test composition comprising:
the reagent of claim 1 ; and
a test sample,
wherein the reagent is in contact with the test sample.
24. A test composition comprising:
the reagent of claim 6 ; and
a test sample,
wherein the reagent is in contact with the test sample.