Citrate synthase activity assay kit

Citrate synthase (CS) activity was determined using a citrate synthase activity assay kit (Solarbio, China). According to the instruction of the kit, the protein concentration was determined after cell lysis. Then, 7 μL protein was mixed with other reagents in the kit and put into a slit quartz cuvette. The initial absorbance A1 was recorded at 412 nm for 10 seconds. The whole quartz cuvette was then put into a 37 °C water bath. After 2 minutes of reaction, it was taken out, and the absorbance A2 was recorded at 412 nm, so the OD value was calculated Δ A=A2-A1. CS enzyme activity was calculated according to the formula provided with the assay kit.

To measure the activity of CS activity, the heart tissue of mice was weighed and placed in an ice-cold 20 mmol/L Tris hydrochloride buffer (pH 7.8) containing 0.2% Triton X-100 and protease inhibitor cocktail. The tissue-to-buffer ratio was 1:20 (w:v). The heart was finely cut with scissors and then homogenized. The obtained homogenate was centrifuged at 13,000× g for 10 min at 4°C. The supernatant was collected and determined by measuring coenzyme A formation at 412 nm with Citrate Synthase Activity Assay Kit (Solarbio BC 1060, China) according to the manufacturer's instruction.