2.8. Carbonic anhydrase assay

CA activity assay was a modification of the procedure described by Capasso et al. ³³ . Briefly, the hydratase assay was performed at 0 °C using CO₂ as substrate following the pH variation due to the catalyzed conversion of CO₂ to bicarbonate. Bromothymol blue was used as pH indicator. The production of hydrogen ions during the CO₂ hydration reaction lowers the pH of the solution leading to a colour transition of the dye. The time required for the colour change is inversely proportional to the amount of CA present in the sample. The Wilbur–Anderson units (WAU) were calculated according to the following definition: one WAU of CA activity is defined as the ratio (T ₀ − T)/T, where T ₀ (the time needed for the pH indicator color change for the uncatalyzed reaction) and T (the time needed for the pH indicator color change for the catalyzed reaction) are recorded as the time (in seconds) required for the pH to drop from 8.3 to the transition point of the dye (pH 6.8) in a control buffer and the presence of enzyme, respectively.