Carbonic anhydrase II enzyme assay

Esterase activity of carbonic anhydrase II was measured by monitoring the release of 4-nitrophenol at A348 nm in a Thermo Scientific Helios Epsilon spectrophotometer using standard methods [46]. Cuvettes contained 900 μl of 15 mM Tris Sulphate Buffer, pH 7.6 at 0°C (Sigma), 500 μl of 3 mM 4-nitrophenyl-acetate (Sigma) and 30 μg/μl of mitochondrial sample (3 mitochondrial preparations and 3 replicates for each condition, 4-11 weeks and 78 weeks). The rate of change in absorbance of the assay was plotted. Unpaired t-tests (GraphPad Prism) were carried out at each time point.