A NEW METHOD FOR ESTIMATING EXTERNAL,CARBONIC ANHYDRASE ACTMTY IN MACROALGAE1,2

The effects of external carbonic anhydrase (CA, E.C. 4.2.1.1) on HCO^?₃and CO₂use under disequilibnum 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 O₂ 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 O₂evolution was attributed to diffusive entry of CO₂into the cells and the steady state to use of HCO^?₃. An enhancement of CO₂diffusive entry was obtained with acetazolamide, an inhibitor of external CA activity. Using this enhancement, a variable was developed to quantify the degree to which CO₂entry 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 potentiomtric 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 HCO₃uptake. Thus prominent 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 HCO^?₃interfered with the new method and the degree of this interference was dependent on the magnitude of the uptake.