S.H. de Bruin

Structure, kinetics and thermodynamics of DNA hairpin fragments in solution.

The hairpin-to-coil equilibrium of the hexadecadeoxynucleotide d(ATCCTATTTTTAGGAT) was extensively studied by means of NMR, T-jump and UV. The thermodynamic and kinetic parameters for this equilibrium were determined, yielding a consistent picture of the dynamical behavior of this hairpin structure, which is shown to be a clear example of a situation in which the linebroadening of the imino proton resonances is not determined by the lifetime of the double helix. A comparative study of the homologous hairpins in which the size of the loop was elongated from 4 to 7 thymidine residues shows a monotonous decrease in Tm for the hairpin-to-coil transitions. This finding is in contrast with the view that the stability of hairpins reaches a maximum with a loop size of 6-7 residues. The NMR results indicate that the accessibility of the thymine bases in the loop towards solvent molecules or complementary nucleotides greatly depends on the size of the loop.

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.

Two‐dimensional Fourier transform 1H NMR studies of ribosomal protein E‐L30

Two‐dimensional Fourier transform 1H NMR spectra of ribosomal protein L30 of Escherichia coli MRE 600 were recorded at 500 MHz both in H2O and in 2H2O. From the available data we infer (i) the existence of one or more hydrophobic domains in the molecule, (ii) at least two helical regions and (iii) the presence of an antiparallel β‐sheet involving two threonines.

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.

Loopstructures in synthetic oligonucleotides. Hairpin stability and structure studied as a function of loop elongation

The formation of hairpin structures in the homologous, (partly) self-complementary DNA fragments d(ATCCTATnTAGGAT), n = 0–7, was studied by means of nuclear magnetic resonance, T-jump and ultra-violet techniques. It is shown that all compounds in the series may adopt hairpin-like conformations, albeit for n < 3 this only occurs to a significant amount at relatively low concentrations (∼ 10μM). For the present series of oligonucleotides, hairpin formation is accompanied by an apparent loop enthalpy significantly different from zero. The stability of the DNA hairpins turns out to be at its maximum for loop lengths of four or five residues, whereas earlier experiments (Tinocoet al., 1973) indicated that loop lengths of six to seven residues are most favourable for RNA hairpins. This is explained by considering the difference in geometry of A-RNA and B-DNA helices.

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.

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.