L.H.M. Janssen

The interaction of chloride ions with human hemoglobin

Abstract Studying the effect of KCl on the Bohr effect of human hemoglobin, it appeared that at low Cl− concentration the alkaline Bohr effect is considerably smaller than it is at a Cl− ion concentration near 0.1 M. The data show that at least part of the Bohr effect, that thus far could not be attributed to a particular residue in hemoglobin, is due to interaction of hemoglobin with anions. The effect of KCl on the Bohr effect shows a striking similarity with the effect of 2,3-diphosphoglycerate (DPG) on the Bohr effect. Based on this a mechanism is proposed which satisfactorily explains the observed salt effect.

H+ titration studies of human hemoglobin

Abstract The normal and differential titration curves of human Hb and HbO 2 , of the isolated α- and β-chains, of apohemoglobin and of HbO 2 with the SH groups blocked by iodoacetamide, were determined and analysed. 1. 1. From the differential titration curves of the α-chains and of Hb and HbO 2 it can be concluded that all carboxyl groups and all Lys and Arg residues are titratable in these molecules. 2. 2. Seven His residues are found to be titratable in the α- and 6 in the β-chains. The titration curve of the α-chains could be calculated with only one p K value for the His residues, whereas p K values varying from 5.6 to 8 were necessary to obtain a good fit for the β-chains. 3. 3. Assuming that the acid Bohr groups are carboxyl groups 20 His residues appeared to be titratable in Hb and HbO 2 , which means that probably 6 His residues become titratable upon dissociation of hemoglobin into its subunits. 4. 4. In HbO 2 the p K of the β 93 SH groups was found to be 9.9. 5. 5. By blocking the reactive SH groups with iodoacetamide, 2 His residues become titratable which are masked in HbO 2 . 6. 6. On removing the heme from hemoglobin probably 8 His residues become unmasked. Assuming that in globin all 6 SH groups are titratable, their p K is found near 9.

Effect of 2,3-diphosphoglycerate on the Bohr effect of human adult hemoglobin

Abstract The effect of 2,3-diphosphoglycerate (DPG) on the Bohr effect of human hemoglobin has been studied by means of hydrogen ion titration techniques. The results indicate a) that both the acid and the alkaline Bohr effect are equally affected, b) that the DPG binding to deoxyhemoglobin (Hb) is much stronger than to carboxyhemoglobin (HbCO) and c) that Hb binds effectively one DPG molecule. The effect on the Bohr effect can roughly be described by assuming that upon binding two groups per tetramer change their pK from 6.8 to 7.8 and two others from 6.8 to 5.8. These groups very probably are the imidazole groups of the two histidines H21 (143)β and the two phosphate groups of DPG (second dissociation). From the experiments a value for the dissociation constant K of the Hb-DPG complex of about 10 −5 M −1 could be estimated at pH 6.2 and pH 7.5.

Study of the Bohr groups of bovine hemoglobin

Abstract The Bohr effect of bovine hemoglobin and of bovine hemoglobin in which protonic groups were substituted by reaction with FDNB was studied by means of normal, differential and difference titration curves. A number of results were obtained by comparison of calculated curves with the experimental curves. This study revealed the following facts. 1. 1. The presence of the normal and acid Bohr groups is easily seen from the differential titration curves of Hb and HbO2 respectively. 2. 2. An analysis of these curves shows that only 18 of the 32 histidine residues are titratable and that 4 of these 18 residues have an abnormally high p K of about 8 in Hb as well as in HbO2. 3. 3. From a comparison of the differential and difference titration curves of substituted hemoglobin with those of unsubstituted hemoglobin, it is concluded that 2 of the 4 α-amino groups are alkaline Bohr groups.

Comparison of the oxygen and proton binding behavior of human hemoglobin A and A2

Abstract The proton and oxygen affinity was studied of both human hemoglobin A and A2 over the pH region 5.5–9.0. This study revealed the following: 1. 1. The Bohr effect of hemoglobin A is identical to that of hemoglobin A2. 2. 2. There is no difference in the oxygen affinity between the two hemoglobins. 3. 3. The oxygen binding curves of hemoglobin A and A2 are identically affected by 2,3-diphosphoglycerate. 4. 4. His G19(117)β in hemoglobin A has an abnormally high p K value of about 7.8 in both oxy- and deoxyhemoglobin.

Some aspects of cooperativity in human hemoglobin

Abstract The cooperativity in hemoglobin can be described by the Hill parameter n , the free energy of interaction Δ F 1 and the allosteric free energy Δ F A . By this latter is meant here the free energy change associated with the transition from the deoxy to the oxy conformation in hemoglobin. In this paper some general relations between n , Δ F 1 and Δ F A are given. A method is presented by which Δ F A can be calculated from oxygenation data.

Influence of chemical modifications of the reactive SH groups on the proton binding behaviour of human and horse hemoglobin

Abstract From a proton-binding study of human HbCO with the reactive β93 SH groups substituted by iodoacetamide, we found that in this derivative two imidazole groups more appear to be titratable than in HbCO. In order to investigate the specificity of iodoacetamide in this respect, we have studied the influence of two other commonly used SH reagents, viz. N -ethylmaleimide and p -mercuribenzoate. 1. 1. Reaction of N -ethylmaleimide and p -mercuribenzoate with human HbCO does not lead to the unmasking of two imidazole groups. 2. 2. Upon blocking the SH groups with p -mercuribenzoate, two groups from the neutral region change their p K from 7 to 8. 3. 3. Horse HbCO behaves analogously to human HbCO. The p K SH however seems to be higher than in human HbCO.