3.3. Esterase Activity Assay

For measuring the esterase activity in cell lysates, pellets overexpressing hCAs were lysed by protein extraction reagent (BugBuster^®, Millipore, Burlington, MA, USA). After clarification by high-speed centrifugation, the soluble fraction was diluted ten times in esterase assay buffer (25 mM Tris pH 8.0, 75 mM NaCl, 0.02 mM ZnSO₄) and left at room temperature for one hour. The hCA concentrations in cell lysates were measured by a comparison to a standard protein (31 kDa, 0.5 µg) on SDS-PAGE. The lysates were then further diluted to 0.2 µM hCAII or 2.0 µM hCAI. For the measurement of the esterase activity of purified proteins, tag-free hCAs were dialyzed into the esterase assay buffer and diluted to 0.3 µM.

The substrate p-nitrophenyl acetate (pNPA) was diluted from stock in ethanol to 0.48 mM or 0.6 mM by esterase assay buffer immediately before the assay. Fifty µL of cell lysates or purified proteins were added into a well (96-well plate, SARSTEDT, Sarstedt, Germany) containing 50 µL of diluted substrate with or without acetazolamide. Triplicate measurements were recorded for each condition. Ten-times-diluted protein extraction reagent was used as a blank at the same reaction time as the hCAII reaction mixture to correct for the delay in measurements. The absorbance at 405 nm was recorded using a plate reader (SPECTROstar Nano, BMG LABTECH, Mornington, VIC, Australia). The absorbance was converted into product concentration by a linear calibration curve generated from 0 to 0.2 mM pNP in the same esterase assay buffer at pH 8.0 and 25 °C (Figure S9). The absorbance measurements were transposed and corrected in GraphPad Prism 9.3.1.