The importance of carbonic anhydrase B and C for the unloading of CO2 by the human erythrocyte.
Carbonic anhydrase (carbonate dehydratase, EC 4.2.1.1) isoenzymes HCA B and HCA C from human erythrocytes were purified by affinity chromatography and characterized kinetically at 37 degrees C in 25 mM N-methylimidazole buffer, I = 0.15, pH 7.1, using a pH-indicator stopped-flow technique. The rate constants for the uncatalyzed hydration of CO2 and dehydration of H2CO3 were 0.12 . s-1 and 58 . s-1, respectively, Km and turnover numbers in the hydration reaction were 14.0 mM and 19.1 . 10(5) . s-1 for HCAC and 3.3 mM and 0.56 . 10(5) . s-1 for HCA B. Km and turnover numbers in the dehydration reaction were 70 mM and 5 . 10(5) . s-1 for HCA C and 16.8 mM and 0.27 . 10(5) . s-1 for HCA B. Ki for chloride was 14 mM for HCA B and greater than 200 mM for HCA C. Ki for acetazolamide was 0.9 microM against HCA B and 16 nM against HCA C. The rates of hydration of CO2 in hemolysates with known concentrations of HCA B and HCA C, and in mixtures of purified HCA B and HCA C with concentrations near those in the erythrocyte, were similar to the rates calculated from the kinetic parameters of each isoenzyme. HCA B is 86% inhibited by chloride in vivo. This strong inhibition, together with the low specific activity, explains why HCA B only accounts for 10% of the total carbonic anhydrase activity of the erythrocyte, en spite the cellular concentration HCA B is 8 times higher than that of HCA C. HCA B and HCA C can together accelerate the intracellular CO2-reaction 23 000-fold, which gives a margin of 25-fold over physiological needs in hard work and 50-fold at rest. Perceptible physiological effects on respiration should therefore be seen when total carbonic anhydrase activity of erythrocytes is 96 to 98% inhibited. These degrees of inhibition are achieved when plasma concentrations of acetazolamide reach 2 and 5 microM, respectively.
D001639:Bicarbonates / Q000097:blood; D002245:Carbon Dioxide / Q000097:blood*; D002257:Carbonic Anhydrase Inhibitors; D002256:Carbonic Anhydrases / Q000097:blood*; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D013997:Time Factors
D001639:Bicarbonates; D002257:Carbonic Anhydrase Inhibitors; D002245:Carbon Dioxide; D002256:Carbonic Anhydrases