A new method for estimating external carbonic anhydrase activity in macroalgae

The effects of external carbonic anhydrase (CA, E.C 4.2.1.1) on HCO3- and CO2 use under disequilibrium conditions were examined in 14 species of macroalgae. CO2 was added to the algae in synthetic seawater free from inorganic carbon and buffered to pH 8.7. This resulted in a transient O-2 evolution which was similar for most of the species tested: an initial rapid increase in the rate was followed by a gradual decrease, approaching a steady state within 10 min. The initial high rate of O-2 evolution was attributed to diffusive entry of CO2 into the cells and the steady state to use of HCO3-. An enhancement of CO2 diffusive entry was obtained with acetazolamide, an inhibitor of external CA activity. Using this enhancement, a variable was developed to quantify the degree to which CO2 entry into the cell was prevented by the external CA. This variable, CA (%), was used as a measure of the external CA activity of the alga. Comparative measurements of the external CA activity using a potentiometric method revealed that the new method was able to detect low levels of external CA activity, where the potentiometric method failed. These two different methods could be used together to increase the reliability of the measurements. The new method was useful for red and brown macroalgae and for those green macroalgae that lacked direct HCO3- uptake. Thus pi-eminent external CA activity was found for Valonia utricularis (green), Halopteris scoparia (brown), and Osmunda pinnatifida (red), where the potentiometric method failed, or nearly failed, to indicate any external CA activity. Direct uptake of HCO3- interfered with the new method and the degree of this interference was dependent on the magnitude of the uptake.