Bioluminescent succinate detection assay

A method for detecting or determining the presence or amount of succinate in a sample, the method comprising:

• (a) contacting the sample with a first detection reagent to form a first reaction mixture;
• (b) contacting the first reaction mixture with a second detection reagent to form a second reaction mixture;
• and
• (c) detecting luminescence in the second reaction mixture, thereby detecting or determining the presence or amount of succinate in the sample;
• wherein if succinate is present in the sample, the first detection reagent and the second detection reagent converts the succinate to ATP.

A method for detecting or determining the presence or amount of a succinate forming enzyme in a sample or the activity of a succinate forming enzyme, the method comprising:

• (a) contacting the sample with a peptide, protein, or non-protein substrate, 2-oxoglutarate, Fe(II), and ascorbate to form a succinate reaction mixture, wherein succinate is formed if the sample comprises a succinate forming enzyme;
• (b) contacting the succinate reaction mixture with a first detection reagent to form a first reaction mixture, wherein the succinate formed in step (a) is converted to succinyl-CoA;
• (c) contacting the first reaction mixture with a second detection reagent to form a second reaction mixture;
• and
• (b) detecting luminescence in the second reaction mixture, thereby detecting or determining the presence or amount of succinate forming enzyme or succinate forming enzyme activity in the sample,
• wherein:
• (i) the first detection reagent comprises 3-oxoacid CoA-transferase (SCOT), inorganic phosphate, and acetoacetyl-CoA and the second detection reagent comprises succinyl-CoA ligase (SCS), ADP, a bioluminescent enzyme, and a luciferin substrate;
• (ii) the first detection reagent comprises 3-oxoacid CoA-transferase (SCOT), inorganic phosphate, acetoacetyl-CoA, succinyl-CoA ligase (SCS), ADP, magnesium chloride, and the second detection reagent comprises a bioluminescent enzyme, and a luciferin substrate;
• (iii) the first detection reagent comprises GDP forming succinyl-CoA ligase (SCS-GDP), GTP, and coenzyme A and the second detection reagent comprises guanylate kinase (GMPK), ADP, a bioluminescent enzyme, and a luciferin substrate;
• (iv) the first detection reagent comprises ADP forming succinyl-CoA ligase (SCS-ADP), ATP, and coenzyme A and the second detection reagent comprises an ATP depletion reagent and an ADP-to-ATP conversion/detection reagent, wherein the first reaction mixture is contacted sequentially with the ATP depletion reagent and the ADP-to-ATP conversion/detection reagent, wherein the ATP depletion reagent comprises adenylate cyclase and pyrophosphatase, and wherein the ADP-to-ATP conversion/detection reagent comprises pyruvate kinase, phosphoenolpyruvate, a bioluminescent enzyme, and a luciferin substrate;
• or
• (v) the first detection reagent comprises ADP forming succinyl-CoA ligase (SCS-ADP), ATP, and coenzyme A, and the second detection reagent comprises an ATP depletion reagent and subsequently an ADP-to-ATP conversion/detection reagent, wherein the first reaction mixture is contacted sequentially with the ATP depletion reagent and the ADP-to-ATP conversion/detection reagent;
• and a bioluminescent enzyme, and a luciferin substrate.

A method for determining whether a compound modulates 2-oxoglutarate oxygenase activity in a sample, the method comprising:

• (a) contacting the sample with the compound to form a test sample, wherein the sample comprises a 2-oxoglutarate oxygenase;
• (b) contacting the test sample with a peptide, protein, or non-protein substrate, 2-oxoglutarate, Fe(II), and ascorbate to form a succinate reaction mixture;
• (c) contacting the succinate reaction mixture with:
• a first detection reagent to form a first reaction mixture;
• (d) contacting the first reaction mixture with a ATP detection reagent or a second detection reagent to form a second reaction mixture;
• (e) contacting the second reaction mixture with a ADP-to-ATP conversion/detection reagent to form a third reaction mixture if a second detection reagent is used in step (d);
• (f) detecting luminescence in the second reaction mixture of step (d) or the third reaction mixture of step (e);
• and
• (g) comparing the luminescence in the second reaction mixture of step (d) or the third reaction mixture of step (e) to luminescence in a control sample, wherein if the luminescence in the second reaction mixture of step (d) or the third reaction mixture of step (e) is different from the luminescence in a control sample, the compound is identified as being a modulator of 2-oxoglutarate oxygenase activity.