Citrate Synthase Activity Assay: A Colorimetric Assay to Measure Mitochondrial Citrate Synthase Activity in Drosophila Tissue Homogenate

Within the mitochondria, citrate synthase, localized in the matrix of the inner mitochondrial membrane, is the rate-limiting enzyme that catalyzes the reaction between acetyl coenzyme A and oxaloacetic acid to form citrate, the first step of the citric acid cycle. 

To analyze citrate synthase activity, transfer adult Drosophila fly thoraxes abundant in mitochondria into a tube filled with a chilled extraction buffer containing ethylenediaminetetraacetic acid, EDTA, and a non-ionic detergent.

Homogenize the thorax tissues on ice to prevent heat-mediated protein denaturation. The applied shear force, in conjunction with the detergents, lyses the tissue and releases intracellular components, including mitochondria.

Further, the rupture of the inner mitochondrial membrane leads to the release of mitochondrial matrix proteins, including citrate synthase. EDTA binds to divalent cations and inhibits protease activity, thereby preventing protein degradation.

Dilute the homogenate using extraction buffer and add an appropriate volume into the reaction mixture containing wells of the multi-well plate. The reaction mixture comprises acetyl coenzyme A, oxaloacetic acid, and dithionitrobenzoic acid.

Citrate synthase in the homogenate catalyzes the reaction between oxaloacetic acid and acetyl coenzyme A, forming citrate and coenzyme A with a thiol group. The thiol group reacts with the colorless dithionitrobenzoic acid, forming a yellow product, thionitrobenzoic acid.

Using a microplate reader, measure the absorbance of the yellow-colored solution. The absorbance could be related to citrate synthase activity in the tissue homogenate.