Citrate synthase activity

Citrate synthase (CS) activity was determined using a colorimetric plate-based assay in which CoA-SH, a byproduct formed by the CS-mediated reaction of oxaloacetate and acetyl-CoA, interacts with 5′, 5′-Dithiobis 2-nitrobenzoic acid (DTNB) to form TNB (OD: 412 nm). Assay buffer consisted of Buffer C (105 mM potassium-MES, 30 mM KCl, 10 mM KH₂PO₄, 5 mM MgCl₂, and 1 mM EGTA; pH = 7.2) supplemented with DTNB (0.2 mM) and acetyl-CoA (0.5 mM). A 96-well round bottom plate was loaded with assay buffer (200 µL/well), the permeabilizing agent alamethicin (0.03 mg/mL), and isolated mitochondria (10 µg/well) and then incubated at 37 °C for 5 min to deplete endogenous substrates. The assay was initiated by the addition of oxaloacetate (1 mM) to sample wells, with absorbance at 412 nm recorded every 30 s for 20 min. The mitochondrial suspension was also added to one control well per sample to account for nonspecific activity, which was later subtracted from the sample rate. CS activity was determined using the Beer-Lambert Law and the molar absorption coefficient of TNB (13.6 mM/cm).