Inhibition of enzyme activity

Inhibition of malate dehydrogenase activity was determined using a commercial kit (Sigma-Aldrich Chemie GmbH). All reagents (Malate dehydrogenase, Oxaloacetic acid, β-Nicotinamic adenine dinucleotide (reduced form)) were from Sigma-Aldrich Chemie GmbH and the enzymatic assay carried out essentially according to Sigma-Aldrich (http://www.sigmaaldrich.com/technical-documents/protocols/biology/_enzymatic-assay-of-malic-dehydrogenase.html). In short, the substrates oxaloacetic acid and nicotinamide adenine dinucleotide (NADH, reduced form) were mixed in phosphate buffer at room temperature. Different concentrations of preservatives were thoroughly mixed with the reagents. The reaction was started by addition of enzyme and the reduction in absorbance at 340 nm was followed for up to 10 min as NADH was oxidised to NAD⁺. The reaction speed was calculated from slope of the absorbance curves. The different concentrations were tested using three parallels for each biological replicate. Similar results were obtained on two different days.