A SIMPLE ENZYMATIC METHOD FOR THE PREPARATION OF RADIOLABELED ERUCOYL-COA AND OTHER LONG-CHAIN FATTY ACYL-COAS AND THEIR CHARACTERIZATION BY MASS-SPECTROMETRY

A simple two-step method for the biosynthesis of radiolabeled erucoyl-coenzyme A of high specific activity and other long-chain fatty acyl-coenzyme A (acyl-CoA) thioesters is reported. 1-14C-labeled erucic and oleic acids, as well as unlabeled ricinoleic and nervonic acids, were incubated at 35°C with coenzyme A in the presence of ATP, MgCl2, and acyl-CoA synthetase (EC 6.2.1.3) from Pseudomonas spp. to yield the corresponding CoA thioesters. Following incubation, each thioester was purified by rapid passage through a disposable reverse-phase C18 extraction column. The overall yields were >90% and the purities >95%, based on the distribution of radioactivity, and chromatographic and spectral properties. Fast ion bombardment-mass spectrometry was employed to confirm the structures of the various acyl-CoAs. © 1990.