G.A.J. Van Os

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.

The dissociation and association behaviour of yeast ribosomes

Abstract 1. 1. The dissociation of yeast ribosomes has been studied quantitatively as a function of bound Mg 2+ and as a function of bound H + . Our results strongly suggested that the degree of dissociation was a function of the total electrical charge only. It seemed irrelevant whether this charge was located on the RNA chains or on the proteins. 2. 2. Association could occur when a sufficient fraction of the charge had been neutralised, either by increasing the Mg 2+ concentration or by decreasing the pH. As a consequence there seems no reason to believe that Mg 2+ has a direct binding function in holding the ribosomal subunits together. 3. 3. Since association took place at a much higher Mg 2+ concentration than the dissociation does, the dissociation-association reaction is not a real equilibrium reaction. Because no alteration in the ribosomal structure, besides the dissociation into subunits could be detected, the difference in the dissociation and the association behaviour is probably not caused by changes of the ribosomal structure. It is shown that a combination of long-range repulsion forces and short-range attraction forces can explain this hysteresis. 4. 4. Evidence is presented that there is some kind of heterogeneity in the ribosome population.

Relationship between the conformation of bovine serum albumin and its digestion by pepsin

Abstract 1. 1.The digestion of bovine serum albumin by pepsin in the pH region of the Normal-Fast transition was studied by means of electrophoresis on polyacrylamide gels. The results suggest a close relationship between the conformation of the albumin molecule and its digestion by pepsin. 2. 2.At pH values just above 3.6 ten well-defined fragments were found. These features might support a model of albumin in which there are only a few, possibly three sites, available for hydrolysis by pepsin. 3. 3.At pH values below 3.6 the more complex electrophoretic patterns suggest the existence of more sites, probably as a consequence of unfolding of the albumin molecule.

The Bohr effect of the isolated α and ß chains of human hemoglobin

Although the Bohr effect (i.e. the linkage between oxygen and proton binding sites) has been discovered already in 1904 [l] , still the molecular mechanism of this effect is not completely understood. A number of groups, responsible for a part of the Bohr effect, has been identified [2]. Recently we have shown that part of the Bohr effect is not an intrinsic property of the hemoglobin tetramer, but due to a difference in interaction of chloride ions with oxy and deoxyhemoglobin [ 31. A question which is still unanswered is whether the Bohr effect is related to the change in quaternary structure only or also to the change in tertiary structure of the subunits. Experiments carried out in our laboratory on the Bohr effect of valency hybrids (submitted for publication) suggest that the Bohr effect is related to the state of ligation rather than to the change in quaternary structure. This observation is in accordance with the results of kinetic studies on the rate of proton release upon ligand binding [4-61 which suggest a relation between the Bohr effect and changes in tertiary structure of the subunits. If so, then it becomes difficult to understand why the isolated (II and fl.chains do not show any Bohr effect at all as has been suggested in oxygenation studies [7,8]. We present therefore a study of the Bohr effect of the (Y and /_I chains of hemoglobin using the very sensitive pHstat equipment we constructed [9]. This method is more suited to observe small effects than measuring oxygen binding curves. Horse heart myoglobin was studies for comparison.

The viscosity change during the polymerization of nucleoside diphosphates.

During the polymerization of nucleoside diphosphates to polynucleotides catalysed by polynucleotide phosphorylase the viscosity of the reaction mixture increases, reaches a maximum and then decreases. Although this effect has already been observed by Beers in 1957 [l] , it is still not clearly understood. Recently Harvey et al. [2] suggested that the maximum in the viscosity could be explained by the existence of a slowly dissociating complex between polynucleotide phosphorylase and growing polynucleotide chains. In this paper it will be shown that the decrease in viscosity can also be interpreted as a thermal degradation of the newly synthesized polynucleotides.

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.