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Citrate Synthase Assay

Citrate Synthase Assay
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Cat. #: CB007

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Citrate Synthase Assay Kit

Cat. #: CB007

(250 tests in 96-well plate)

Description

Citrate synthase (E.c. 4.1.3.7) is a pace-maker enzyme in the Krebs cycle (citric acid cycle or tricarboxylic acid cycle, TCA). Citrate synthase, CS, has a molecular weight of 51,709 Da, with gene map locus 12q13.2-q13.3. CS is localized in the mitochondrial matrix, but is nuclear encoded, synthesized on cytoplasmic ribosomes and transported into the mitochondrial matrix. CS, therefore, is commonly used as a quantitative marker enzyme for the content of intact mitochondria. CS catalyzes the reaction of two-carbon acetyl CoA with four-carbon oxaloacetate to form six-carbon citrate, thus regenerating coenzyme A.

Acetyl-CoA + oxalacetate + H2O → citrate + CoA-SH

This colorimetric assay is based on the reaction between 5’, 5’-Dithiobis 2-nitrobenzoic acid (DTNB)

and CoA-SH to form TNB, which exhibits maximum absorbance at 412 nm:

CoA-SH + DTNB → TNB + CoA-S-S-TNB

The intensity of the absorbance is proportional to the citrate synthase activity. This enzyme is an exclusive marker of the mitochondrial matrix1.

Reagents and buffers

Tris-HCl buffer (40 mM, pH 8.1): Assay buffer.

Tris–HCl buffer (0.1 M, pH 7.0): For Citrate Synthase standard preparation.

Triethanolamine-HCl buffer (0.5 M, pH 8.0) + EDTA (5 mM): 8.06 g triethanolamine/100 ml a.d., adjust pH with 37% HCl, add 186.1 mg EDTA. pH does not change after addition of EDTA.

Triton X-100 (10% solution): Reagent solution is 100%, add 90 ml a.d. to 10 g (ca. 10 ml) Triton X-100.

Triton X-100 is viscous and sticky. Weigh on balance in a beaker and dissolve by stirring.

Prepare 12.2 mM acetyl-CoA, store at -20 °C

25 mg acetyl CoA + 2.5 ml a.d., make aliquots of 250 µl and store at -20 °C. Store on ice during

measurement, freeze it again after the experiment.

Prepare fresh every day

Triethanolamine-HCl-buffer (0.1 M, pH 8.0): 1 ml of 0.5 M triethanolamine-HCl-buffer of pH 8.0 + 4 ml

Oxalacetate (10 mM, pH 8.0): 6.6 mg oxalacetate + 5 ml of 0.1 M triethanolamine-HCl-buffer of pH 8.0.

DTNB (1.01 mM, pH 8.1): 2 mg DTNB + 5 ml of 1 M Tris-HCl-buffer of pH 8.1.

Sample Preparation

1. Isolated mitochondria

10-25 µl mitochondrial suspension (5 mg/ml) is used for each spectrophotometric measurement.

2. Suspended cells

For typical cells (HUVEC, lymphocytes) at 1-2.106 cells/ml, take replicates of 110 µl samples into Eppendorf tubes, freeze in liquid nitrogen, and store until measurement.

3. As a standard, citrate synthase is (8.6 mg prot./ml) diluted 1:500 in 0.1 M Tris-HCl buffer, pH 7.0 (RT),  this yields a final protein concentration of 0.0172 

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