Methods for measuring ADP
This invention relates to assays for detecting and measuring ADP. In particular, this invention provides homogeneous luminescent assays that detect ADP generation and measures ADP accumulation based on enzymatic coupling reactions. The assays of the present invention can be applied to all types of kinases and other ADP-generating enzymes, are antibody free, beads free, radioisotope free, and compatible with commonly used kinase buffers.
1. A method of determining ADP, comprising:
a) to a sample suspected of comprising ADP, adding a first reagent comprising components of an enzyme coupling scheme for producing NADH or NADPH, to produce an assay mixture;
b) incubating said assay mixture for sufficient time for the reactions of said enzyme coupling scheme to occur to produce said NADH or NADPH;
c) adding to said assay mixture a second reagent to drive the generation of light emission, said second reagent comprising:
(i) FMN:NADH oxidoreductase, FMN, luciferase and RCHO or
(ii) diaphorase and Resazurin;
d) determining light emission generated from step c) as a measure of the amount of ADP in said sample.
2. The method according to claim 1 , wherein said first reagent comprises components selected from the group consisting of:
(1) pyruvate kinase, pyruvate dehydrogenase, PEP and NAD;
(2) Acetyl-CoA Synthetase, pyruvate dehydrogenase or ketoglutarate dehydrogenase, phosphate, acetyl-CoA, pyruvate or a-ketoglutarate, and NAD;
(3) Succinyl-CoA Synthetase, Ketoglutarate dehydrogenase or pyruvate dehydrogenase, phosphate, succinyl-CoA, a-ketoglutarate or pyruvate, and NAD;
(4) Acetyl-CoA Synthetase, CoA-glutathionine reductase, phosphate, acetyl-CoA, glutathione and NADP; and
(5) Succinyl-CoA Synthetase, CoA-glutathionine reductase, phosphate, succinyl-CoA, glutathione and NADP.