COMPARISON OF O-18 EXCHANGE AND PH STOP-FLOW ASSAYS FOR CARBONIC-ANHYDRASE

The hydration velocity of CO2 (0.002 M) catalyzed by bovine carbonic anhydrase (BCA) was measured at 25°C and pH 7.4 by three different techniques: two initial-rate (steady-state) stop-flow methods, one using a glass pH electrode (in Hannover, method 1) and one using spectrophotometric measurements of a pH indicator (in Philadelphia, method 2), and an exchange method in which the disappearance of C18O16O from a bicarbonate solution was determined at equilibrium (in Philadelphia, method 3). The Michaelis-Menten constant (K(m)) and the inhibition constants for chloride (K(i,Cl)) and ethoxzolamide (K(i,ez)) were the same for methods 1, 2, and 3. The turnover numbers were 270,000, 400,000, and 555,000 s-1 by methods 1, 2, and 3, respectively. Values for CO2 hydration velocity measured by methods 2 and 3 on the same solution of BCA at the same time were the same K(m), maximal reaction velocity (V(max)), K(i,ez), and K(i,Cl) obtained from normal human hemolysate at 37°C and pH 7.2 by methods 2 and 3 were the same. K(m) and V(max) of the carbonic anhydrase isozyme CA III of homogenate from rabbit soleus were also identical by methods 1 and 3. According to Michaelis-Menten theory, the values of K(m) and V(max) obtained by method 3 should have been significantly smaller than those obtained by methods 1 and 2. We conclude that the catalytic step itself is apparently not rate limiting under physiological conditions and that method 3 can be used to obtain Michaelis-Menten characteristics of carbonic anhydrase.