Electrometric and colorimetric determination of carbonic anhydrase.

The activity of carbonic anhydrase has been estimated by three procedures: manometric (l-3), calorimetric (2, 4-8), and electrometric (9). For investigations of the enzyme concentration of tissues the calorimetric method has often been used in preference to the manometric technique because of its relative simplicity. A disadvantage of the calorimetric method has been the inhibitory effects of the indicator (10) and of the carbonate-bicarbonate buffer, an effect also true of phosphate buffer in the manometric method, though to a lesser degree (8). Roughton and Booth (8) have found that Verona1 buffer is without noticeable effect on carbonic anhydrase and have suggested a calorimetric method which employs this buffer with brom-thymol blue. This method permits the measurement of enzyme activity in terms of moles of CO2 hydrated, allowance being made for inhibition by the indicator. However, the influence of the indicator on the enzyme activity in this system has not been studied. This paper describes a new electrometric method which is here employed for evaluation of the inhibitory effect of indicators on carbonic anhydrase preparations from various sources. A modification of the veronal calorimetric method suitable for routine determinations of carbonic anhydrase in tissues is also presented together with data on its reliability.