I would like to extract and quantify acyl-CoA from E. coli. But it's difficult to extract because acyl-CoA are easily degraded by endogenous acyl-CoA hydrolase.
Perhaps you could use an inhibitor of acyl-CoA hydrolase during extraction. According to the BRENDA database (http://www.brenda-enzymes.org/enzyme.php?ecno=3.1.2.20#INHIBITORS), some inhibitors are diethyldicarbonate, CoA, and diisopropylfluorophosphate.
Hi Zhou Wei,
What do you exactly mean by "acyl coA" is it acetyl-coenzyme A ? or is it fatty acyl coenzyme A ??
Acetyl coenzyme A can be monitored directly with a SIgma assay kit whose reference is MAK039. For 100 assays, it costs 350 euros.
For fatty acyl coenzyme A, a global method for the determination of tissue level of all the acyl-CoA as well as the free CoA has been described (See the joined paper). The principle of that determination is the cleavage of the thio-ester bond of acyl-CoA after incubation with sodium tetrahydroborate (NaBH4) and derivatization of the free sulphydryl groups with afluorescent agent. The separation of the fluorescent derived coenzyme A adducts is achieved on a reversed-phase HPLC column. The detection limit is said to be lower that 3 pmol. A precise determination of the acyl-CoA pool is thus possible with only 50 mg of fresh tissue. The acid-soluble CoASH is also measured on an aliquot of the aqueous acid extract prior any cleavage treatment.
Best regards,
Pr Philippe Urban
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