Simultaneous quantification of coenzyme A and its salvage pathway intermediates in in vitro and whole cell-sourced samples

Abstract

We report a method for the simultaneous quantification of the essential metabolic cofactor coenzyme A (CoA) and its thiol-bearing precursors-including 4 0 -phosphopantetheine, which was recently shown to play a potentially important role as nexus metabolite in CoA biology-with pmol sensitivity. This sensitivity is gained by making use of an established thiol-derivatisation agent that produces a fluorophore upon labeling, which is subsequently separated by HPLC and quantified by fluorescence detection. While previous reports have made use of a similar strategy to quantify CoA, very few have extended the method to the CoA biosynthetic intermediates (some of which occur at levels much lower than CoA) or have critically evaluated its analytical performance. In this study we addressed these shortcomings, and also overcame the difficulty associated with the independent confirmation of the concentrations of the analytical standards used for quantification. The method's utility is showcased through time-course analyses of in vitro reconstituted enzyme reactions and by analysis of extracts from Escherichia coli and Staphylococcus aureus, demonstrating its potential in advancing studies of CoA biosynthesis and CoA-dependent biology in a wide range of systems.

Vnn1 pantetheinase limits the Warburg effect and sarcoma growth by rescuing mitochondrial activity

“…Quantification of CoA was performed as described ( Goosen & Strauss, 2017 ). Tissue extracts were prepared following a modification of a protocol described ( Siudeja et al, 2011 ).…”

Thiophosphate Analogs of Coenzyme A and Its Precursors—Synthesis, Stability, and Biomimetic Potential

“…Since C5a, C6a, and C6b were prepared in insufficient yield, their precursor molecules on the dpCoA level were used instead in these experiments. Potential substrates were incubated with fetal bovine serum or human Nudt7, and the conversion was analyzed after conjugation to a fluorescent dye followed by RP-HPLC separation ( Figure S2 ) [ 33 ].…”

Harnessing the Vnn1 pantetheinase pathway boosts short chain fatty acids production and mucosal protection in colitis

“…Finally, 3.15 µL of 10 mM N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide (in 100% acetonitrile) was added to bring the total volume up to 70 µL and the total acetonitrile concentration to 30%. CoA level was determined by high-performance liquid chromatography and quantified as described 30…”