The recent review on this subject by Davis (117) makes it possible to reduce this section to a brief critical outline of the types of methods used for determination of carbonic anhydrase and its inhibitors. A few papers published since Davis' review are mentioned individually. All methods depend on either the evolution or absorption of CO2 or on a pH change. It is obvious that a wide variety of physical techniques and chemical principles is applicable. A. Manometric methods These were developed by Meldrum and Roughton (350) at the time they discovered the enzyme and were refined in later years by Roughton and colleagues. The basic reaction is evolution of CO2 from NaHCOs-buffer mixtures or uptake of CO2 by buffer solutions. A bibliography and description are given in reference 117. These methods have yielded a great deal of useful information. In recent years, however, their use has lessened, for at least three reasons: rapid and vigorous shaking has frequently damaged the enzyme; the methods are somewhat cumbersome; and they are not suitable for rapid flow measurements. An inherent advantage of the manometric principle, however, is its potential for extreme sensitivity. Using the Cartesian diver, it has been possible to detect carbonic anhydrase in single red cells, glia, and epithelia of choroid plexus (17 1). B. pH methods T. H. MAREN Vohme 47 activity, it must be shown that the procedure is run more rapidly than the uncatalyzed rate. For the dehydration reaction, the pH is critical in calculating this rate. The implication in Hausler's (203) and Hansson's (198) methods is that at the surface and at the critical areas of the section, evolution of CO2 markedly elevates the pH, slowing the uncatalyzed rate. .This pH is not known, but if it were 7.4, the half-time for dehydration is 3 min; if pH were 8.4, it is 30 min. In the Hansson method (198) sections float for only 2-10 min, so that it is reasonable to suppose that enzyme activity is involved. Hansson (198) was also the first to show inhibition of the reaction at low (0.3 PM) concentrations of acetazolamide, ruling out complexation of cobalt present (1.3 mu). The drug at 20 mg/kg given 30 min before autopsy also inhibited staining. It is anticipated that the application of this new method will permit recognition of carbonic anhydrase in specific secretory sites, as well as in places (i.e. skin) where in the chemical method the mass of tissue may obscure enzyme in a relatively few critical cells. The nature of the cobalt deposit in the incubated tissue sections is at present unknown. Hansson (198), using a phosphate-bicarbonate medium quite different from that of Hausler, studied the uptake of 6oCo and 32P into sections during incubation and found the tissue content of both isotopes increased. From this he concluded that the deposit was a basic cobalt phosphate salt. By analogy, the deposit in Hausler's method may be a cobalt bicarbonate complex. Hansson (197) has demonstrated the enzyme in red cells by fluorescent antibodies; it would be interesting if this ...
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