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1. Details are given of an electrometric method for measuring the activity of isoenzymes of carbonic anhydrase (EC 4.2.1.1) in catalysing the hydration of carbon dioxide under different conditions at 0° C. In the method, a measured volume of water saturated with carbon dioxide at a known partial pressure and appropriate temperature is introduced into a buffered solution. Using a sensitive electrometer and recording instrument, the subsequent change in hydrogen ion concentration is recorded as a function of time. Under the conditions of assay, the pH change induced in the presence of substrate is very small (pH < 0·05 units) and the period of observation need not exceed 10 sec.

2. For enzymes isolated from guinea-pig tissues, it is found that the specific activity of the `high activity' isoenzyme (carbonic anhydrase C, carbonic anhydrase II, HACA) is about eighteen times that of the `low activity' counterpart (carbonic anhydrase B, carbonic anhydrase I, LACA) when measured at 0° C, pH 7·2, and ionic strength 0·19. Under the same conditions, the K_m was found to be 10 mM for the `high activity' isoenzyme and 23 mM for the `low activity' isoenzyme. No differences were found between the equivalent kinetic parameters of the corresponding isoenzymes isolated from different tissues.

3. The isoenzymes isolated from guinea-pig tissues are found to be inhibited by acetazolamide in a non-competitive manner. It is also found that the `high activity' isoenzyme is many times more sensitive to this inhibitor than is the `low activity' isoenzyme. Evidence is presented which indicates that one acetazolamide binding site is present on each molecule of either isoenzyme.

4. While chloride ions specifically inhibit the `low activity' component of guinea-pig carbonic anhydrase (I<sub>0·5</sub> = 40 mM), acetate, butyrate and pyruvate inhibit both isoenzymes. Under the conditions employed, acetate and pyruvate are more strongly inhibitory to the `low activity' isoenzyme than to the `high activity' isoenzyme, while butyrate is more strongly inhibitory to the `high activity' isoenzyme.

5. The findings are discussed with particular reference to the physiological significance of the presence of the isoenzymes in the gastro-intestinal tract. Also considered are possible relationships between the distribution of the `low activity' isoenzyme in these tissues and the transport and metabolism of products of fermentation occurring in the intestinal lumen.