Assay Method Information

Assay Name:  ICL Enzyme Assay
Description:  Isocitrate lyase activity was determined at 37°C by measuringthe formation of glyoxylate phenylhydrazone at 324 nm [Bai et al., Drug Dev. Res., 67:818-23]. The reaction mixture contained 100 μL of 0.5 mM potassium phosphate buffer, 1.2 μL of 1 mM magnesium chloride, 24 μL of 100 mM 2-mercaptoethanol, 7 μL of 4 mM phenylhydrazine hydrochloride, 6 μL of 50 mM trisodium isocitric acid, and ICL enzyme (usually 3-6 μL). This mixture was made up to 200 μL with MilliQ water (water that has been purified and deionized to a high degree by a water purification system manufactured by Millipore Corporation). At the end of the 10th minute this reaction mixture was made up to 1 mL and ultraviolet (UV) absorbance was measured at 324 nM, which served as a control. For the test compounds, 3 μL of 100 mM 3-nitropropionic acid (3-NPA) was used, and in the case of the candidate molecules, 10 μL of 10 mM concentration was added to the abovementioned reaction mixture. At the end of the 10th minute this reaction mixture was made up to 1 mL and UV absorbance was measured at 324 nM, which served as the test.
Affinity data for this assay
 

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