Influence of oxygenation, pH, and 2, 3-diphosphyglycerate on carbon dioxide exchange.

he majority of carbon dioxide in blood is trans-Tported in the form of bicarbonate, and only relatively small amounts exist as dissolved Con or carbamate. The latter compound is formed by the combination of free carbon dioxide with uncharged amino groups of hemoglobin. Hb=NH,+=Hb-NH2 + H + The role of carbamate in carbon dioxide exchange is more important than would be expected from its small concentration because it is oxylabile, ie, significantly greater amounts of carbamate have been demonstrated in reduced hemoglobin solutions than in oxygenated solutions.' This has led Roughton2 to postulate that changes in carbamate concentration account for 70 percent of the Haldane effect, the difference in carbon dioxide content between oxy-generated and reduced blood at the same PCOZ.~ Recently, Bauer has reported that 2,3-diphospho-glycerate (DPG), which is normally present in high concentration inside the human erythrocyte, markedly diminishes the oxylabile transport of carbon dioxidee3 Kilmartin and Rossi-Bernardi4 have clearly demonstrated that carbamate formation occurs at the N-terminal amino groups of both the a and P chains of hemoglobin. Current evidence suggests that DPG binds to the hemoglobin molecule near the central cavity and involves, among several sites, the N-terminal amino groups of both P chainsU5 Presumably the decrease in oxylabile COz seen in the presence of DPG is a result of competition between DPG and COz for these sites. Unfortunately, the direct chemical measurement of COz bound as carbamate is quite d a c u l t and relatively inaccurate.' We have approached the problem by studying the Haldane reaction with the continuous flow reaction techniquee6 The time course of changes in PCO~ and Po2 during the oxygenation of the reduced erythrocytes at 37OC was monitored following mixing of a suspension of reduced red cells with an oxygenated buffer having the same pH and Pcoz. Since total carbon dioxide content in the mixture was held constant, any increase in measured Pcoz following oxygenation reflected a similar decrease in the sum of bicar-bonate and carbamate. In our apparatus the reaction could be monitored either in the presence or absence of acetazolamide*" during the same experiment. When this reaction occurs in the presence of acetazolamide, the catalysis of bicarbonate to free CO2 by carbonic anhydrase is inhibited so that during the first few seconds following oxygenation, only changes in carbamate concentration are seen. This technique permits separation of the relative contributions of bicarbonate and carbamate to the carbon dioxide …