Add time:08/25/2019         Source:sciencedirect.com

An assay for 3-oxoacyl-coenzyme A (3-oxoacyl-CoA) thiolases is described. The reaction utilizes acetyldithio-CoA as the nucleophile and variable chain length saturated acyl-CoA's as the electrophiles. The properties of the 3-oxoacyl-CoA dithioester product, notably a pKa of 6.6 ± 0.1 and an extinction coefficient of 21,600 cm−1m−1 for the enethiolate at 357 nm, make it possible to spectrophotometrically follow the reaction in the thermodynamically unfavorable carbon-carbon bondforming direction. These properties eliminate both the background decomposition and the dependence on Mg2+, chain length, and pH that complicate assays with 3-oxoacyl-CoA substrates. Purified thiolase I from pig liver was 140-fold more active with butyryl-CoA as the electrophile than with acetyl-CoA and 38-fold more reactive with hexanoyl-CoA than with myristoyl-CoA. Beef liver homogenate showed a much greater relative activity with myristoyl-CoA as the electrophile than either purified pig heart thiolase I or pig heart homogenate. The analysis of the separation of thiolases by anion-exchange chromatography is simplified and further suggests the existence of isozymes with varying chain length specifities.

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