Determination of Carbonic Anhydrase Activity

Abstract

IN a recent paper, Wilbur and Anderson¹ have compared the electrometric and colorimetric methods of determining carbonic anhydrase activity. They have confirmed an observation first made by Kiese and Hastings² that indicators can inhibit the enzyme and introduce a source of error into the Philpot and Brinkman techniques. They maintain, therefore, the superiority of the electrometric method. However, it must be pointed out that any procedure which is based on measurements of the time taken to bring about a given change in pH, in the presence and absence of the enzyme, is much less reliable than the original manometric method of Meldrum and Roughton³. Experiments carried out in this laboratory have indicated that the electrometric method of following the pH changes is unsatisfactory when different substances are being tested for activating effects. Some of the errors are almost certainly due to interference with the rapid establishment of equilibrium between the glass electrode and the medium. Besides, there is always the possibility that the effects of activators and inhibitors depend on the pH at any given instant. For example, in a 4-ml. solution containing 4·5 units of carbonic anhydrase (activity = 160 units per mgm.), bromothymol blue at a concentration of 2·5 mgm. per cent inhibits by more than 45 per cent at pH 6·8 but only by 25 per cent at pH 7·5. It is suggested, therefore, that the 'boat' method must remain the standard procedure, particularly in work dealing with activators and inhibitors.