High performance liquid chromatography stability study of malonyl‐coenzyme A, using statistical experimental designs

Abstract

Malonyl-CoA is a biochemically important compound, formed by an acetyl-coenzyme A carboxylase catalysed reaction. The stability of this short-chain coenzyme A derivative under various experimental conditions is discussed in this article. High-performance liquid chromatography was used for the analysis of the reaction mixture because of its excellent selectivity and sufficient sensitivity. Several variables were investigated as possible stability-influencing factors: pH, magnesium and buffer concentration, reaction temperature and time. The Plackett-Burman screening design was first used for selecting the most important variables, with which a central composite design was constructed. In this way, a response surface was obtained with the percentage remaining malonyl-CoA as a function of magnesium concentration, reaction temperature and time. The usefulness of this approach is demonstrated by obtaining kinetic data from the mathematical function and by the evaluation of the stopping of reaction procedure in the activity assay of acetyl-coenzyme A carboxylase.

Time‐resolved multiparameter analytics on a cell‐free production platform for acyl‐CoA precursors

“…The analysis of the “pure” purchased malonyl‐CoA reveals about 4% of free CoA and 3% acetyl‐CoA as impurities. Furthermore, malonyl‐CoA is known to undergo hydrolytic decomposition, leading to the spontaneous formation of CoA 26 (Figure S3B ). Taking hydrolysis measurements as a reference, we found that ∼ 6% of malonyl‐CoA degrades during the reaction time releasing CoA, which becomes available for MatB.…”

Dephospho-CoA kinase provides a rapid and sensitive radiochemical assay for coenzyme A and its thioesters

“…Although we originally used the Arctic shrimp enzyme because it was readily inactivated by heating, this property was not of advantage because even the mild heat treatment needed to inactivate the enzyme resulted in some loss of malonyl-CoA, the most labile of the thioesters [7;13]. Although the stability of CoA thioesters to alkaline conditions is markedly decreased in the presence of magnesium ion [24], a component of the Arctic shrimp phosphatase buffer, an enzyme having a lower pH optimum seemed a better choice. After we had established our assay a new phosphatase from Antarctic shrimp became commercially available.…”