Identification of the high and low affinity CO₂-binding sites of human haemoglobin

Abstract

THE unloading of oxygen in the tissues is facilitated by lowering the oxygen affinity of haemoglobin (Hb) by both CO₂ and diphosphoglycerate (DPG)¹. This occurs because CO₂ and DPG are oxygen-linked, that is, they are bound more firmly to deoxyhaemoglobin than to oxyhaemoglobin^2,3. The binding of CO₂ to Hb takes place by a reaction of CO₂ with the α-amino groups to form carbamino compounds^4,5, and binding curves can be measured by rather laborious methods^4,6. Perrella et al.⁷ have modified these methods so that much smaller amounts of Hb, such as the specifically carbamylated Hbs⁸, can be used. In this method, Hb equilibrated with CO₂ is rapidly taken to pH 11 to stabilise the carbamino CO₂ and BioRad AG 1×8 resin added to remove carbonate and bicarbonate ions. The carbamino CO₂ is displaced from the Hb by acidification and measured in a Van Slyke apparatus. DPG-free human deoxyhaemoglobin gave diphasic CO₂ binding curves⁷ showing that the affinities of the α and β chain α-amino groups for CO₂ are different, but it was impossible to establish which group had the higher affinity.