Measurement of carbonic anhydrase activity of blood at body temperature.

Most measurements of blood carbonic anhydrase activity recorded in the literature have been made by the method of Meldrum and Roughton (1) or some modification of it. These procedures are carried out in a cold water bath at 10” or below. This temperature is disadvantageous technically, and, moreover, affords no direct information as to the activity of the enzyme at mammalian body temperature; it is well known that low temperatures exaggerate the apparent activity of the enzyme markedly (l-4). In addition, the enzyme, under some circumstances, may be partially inactivated at low temperature (2,5,6). Methods based on that of Meldrum and Roughton (1) are unsatisfactory, also, in that violent shaking is required to counteract the retarding effect of cold on the diffusion of the evolved gas; variations in the rate and angle of rotation of the shaker used give rise to apparent variations in enzyme activity. Differences in size and shape of the vessels used also cause large differences in the values obtained (7). Mitchell, Pozzani, and Fessenden (8) have shown also that calculation of enzyme activity by means of the formula of Meldrum and Roughton (1) is not valid mathematically and yields misleadingly high values of enzyme activity. Mitchell et al. found that the catalyzed and the uncatalyzed reactions used in the estimation of carbonic anhydrase activity were unimolecular in character; accordingly they introduced the utilization of the principle of Guggenheim (9), based on velocity constants, in the measurement of activity of the enzyme. The present method is a modification of the procedure of Mitchell et al. (8), designed to estimate carbonic anhydrase activity in blood at 37”. It is to be noted that the velocity constant of the reaction is only one of several variables in the method; so that the values obtained are related to, but not the same as, the velocity constant.