The measurement of the intrinsic alkaline Bohr effect of various human haemoglobins by isoelectric focusing.

We have used isoelectric focusing to measure the differences between the pI values of various normal and mutant human haemoglobins when completely deoxygenated and when fully liganded with CO. It was assumed that the DeltapI(deox.-ox.) values might correspond quantitatively to the intrinsic alkaline Bohr effect, as most of the anionic cofactors of the haemoglobin molecule are ;stripped' off during the electrophoretic process. In haemoglobins known to exhibit a normal Bohr coefficient (DeltalogP(50)/DeltapH) in solutions, the DeltapI(deox.-ox.) values are lower the higher their respective pI(ox.) values. This indicates that for any particular haemoglobin the DeltapI(deox.-ox.) value accounts for the difference in surface charges at the pH of its pI value. This was confirmed by measuring, by the direct-titration technique, the difference in pH of deoxy and fully liganded haemoglobin A(0) (alpha(2)beta(2)) solutions in conditions approximating those of the isoelectric focusing, i.e. at 5 degrees C and very low concentration of KCl. The variation of the DeltapH(deox.-ox.) curve as a function of pH (ox.) was similar to the isoelectric-focusing curve relating the variation of DeltapI(deox.-ox.) versus pI(ox.) in various haemoglobins with Bohr factor identical with that of haemoglobin A(0). In haemoglobin A(0) the DeltapI(deox.-ox.) value is 0.17 pH unit, which corresponds to a difference of 1.20 positive charges between the oxy and deoxy states of the tetrameric haemoglobin. This value compares favourably with the values of the intrinsic Bohr effect estimated in back-titration experiments. The DeltapI(deox.-ox.) values of mutant or chemically modified haemoglobins carrying an abnormality at the N- or C-terminus of the alpha-chains are decreased by 30% compared with the DeltapI value measured in haemoglobin A(0). When the C-terminus of the beta-chains is altered, as in Hb Nancy (alpha(2)beta(Tyr-145-->Asp) (2)), we observed a 70% decrease in the DeltapI value compared with that measured in haemoglobin A(0). These values are in close agreement with the estimated respective roles of the two major Bohr groups, Val-1alpha and His-146beta, at the origin of the intrinsic alkaline Bohr effect [Kilmartin, Fogg, Luzzana & Rossi-Bernardi (1973) J. Biol. Chem.248, 7039-7043; Perutz, Kilmartin, Nishikura, Fogg, Butler & Rollema (1980) J. Mol. Biol.138, 649-670]. In other mutant haemoglobins it is demonstrated also that the DeltapI(deox.-ox.) value may be decreased or even suppressed when the substitution affects residues involved in the stability of the tetramer. These results support the interpretation proposed by Perutz, Kilmartin, Nishikura, Fogg, Butler & Rollema [(1980), J. Mol. Biol.138, 649-670] for the mechanism of the alkaline Bohr effect, and also indicate that the transition between the two quaternary configurations is a prerequisite for the full expression of the alkaline Bohr effect.

The exchange of bone CO2 in vivo.

Bone 14CO2 specific activities have been measured in 10 male rats perfused for 30, 60 or 120 minutes with [14C]bicarbonate. A steady blood 14CO2 specific activity is observed from the 30th minute, whereas bone 14CO2 specific activity increased linearly with time. About 7-10% of the total activity infused may be recovered in the skeleton at any given time. Bone samples heated to constant weight lost 15% of their total CO2 content and more than 50% of the 14CO2 activity. This indicates that 14CO2 present in bone is almost exclusively located in a bicarbonate pool which may be considered as the rapidly exchangeable bone CO2 fraction. The rate of exchange between bone and blood CO2 is estimated at a maximum of 15% of the total CO2 production. It is postulated that blood flow to the skeleton is the limiting factor for bone CO2 exchange. These results lead to the conclusion that the large bone CO2 store cannot play a significant role in the buffering of extracellular fluids in acute acid-base abnormalities.

The bone CO2 compartment: evidence for a bicarbonate pool.

CO2 was measured in rat cortical bone samples with an accurate manometric technique. Bone CO2 content increased with the age of the animals. When heated to constant weight, bone samples lost 15.5% of their initial fresh CO2 content, indicating that CO2 exists in several chemical forms in the bone CO2 pool. Bone samples were exposed in vitro to atmospheres of increasing PCO2. Bone CO2 increased by 15% of its original content when exposed for 60 min. to pure CO2. CO2 uptake by bone in vitro was shown to be time- and PCO2-dependent, evoking a saturating mechanism and requiring the presence of water in bone. These findings lead to the conclusion that the bone CO2 compartment consists of 30% bicarbonate and 70% carbonate. The exact location of the bicarbonate pool and its availability as a buffer store for the whole organism are discussed.

[Current conception of the Bohr effect]. / Conception actuelle de l'effet Bohr

The molecular mechanism of the Bohr effect is explained according to the molecular model proposed by Perutz et al. The Bohr effect is due to changes in the pK of specific carboxyl and amino groups of the four globin chains following the transition between the deoxy and oxy conformations of the molecule. Carbon dioxide binds to the N terminal valine of the 4 monomers to form carbamino compounds. This carbaminoformation depends upon pH, PCO2 and predominates on deoxygenated haemoglobin. It is lowered when O2 binds to the heme groups (O2 linked carbamino compounds). Through the carbamino compounds Carbon dioxide lowers both the affinity of haemoglobin for O2 and the Bohr effect. Diphosphoglycerate also binds to the haemoglogin molecule. This organophosphate lowers the affinity for O2 but increases the Bohr effect. In whole blood, the Bohr effect is therefore dependent upon pH, O2 saturation, PCO2 and DPG concentration into the red blood cells.