A sensitive and robust method for quantification of intracellular short-chain coenzyme A esters.
A procedure for the analysis of short-chain intracellular coenzyme A (CoA) esters and adenine nucleotide pools in microbial cells is described. The simultaneous isolation of bacterial cells from media, quenching of their metabolism, and extraction of metabolites was accomplished by centrifugation of cells through a layer of silicone oil into a denser solution of trichloroacetic acid. The acid was neutralized by extraction into Freon containing tri-n-octylamine to provide a salt-free solution of cell metabolites. After high-performance liquid chromatography separation, CoA, CoA esters, and adenine-containing nucleotides were derivatized by postcolumn reaction with bromoacetaldehyde to form the fluorescent 1,N6-ethenoadenine adducts which were analyzed by a fluorescence detector at picomolar levels.
Shimazu
Mark
M
Kosan Biosciences, Inc., 3832 Bay Center Place, Hayward, CA 94545, USA.
Vetcher
Leandro
L
Galazzo
Jorge L
JL
Licari
Peter
P
Santi
Daniel V
DV
D002851:Chromatography, High Pressure Liquid; D003065:Coenzyme A / Q000032:analysis* / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D004952:Esters; D013997:Time Factors
D004952:Esters; D003065:Coenzyme A