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S H de Bruin

Publications and source records attributed to S H de Bruin.

At least 19 recordsLinked to original sources
PubMed1983

High-resolution proton magnetic resonance studies of the 3'-terminal colicin fragment of 16 S ribosomal RNA from Escherichia coli. Assignment of iminoproton resonances by nuclear Overhauser effect experiments and the influence of adenine dimethylation on the hairpin conformation.

The "colicin" fragments comprising the 49 3'-terminal nucleotides of 16 S ribosomal RNA have been isolated from wild-type Escherichia coli and from a kasugamycin-resistant mutant that lacks methylation of two geminal adenine residues. Proton nuclear magnetic resonance (n.m.r.) spectra (500 MHz) were recorded at various temperatures. The low-field resonances arising from the hydrogen-bonded iminoprotons of paired bases were assigned using the nuclear Overhauser effect (n.o.e.). Crucial to the interpretation of the spectra are the resonances that originate from the two hydrogen-bonded iminoprotons of a U X G basepair. Combined with temperature-jump relaxation kinetics experiments the n.o.e.s lead to the conclusion that a conserved A X U/U X G junction in the hairpin is a thermolabile dislocation in the helix. The n.m.r. spectra of the wild-type and mutant fragment are only different with respect to the iminoproton resonances of the two base-pairs adjoining the hairpin loop. The spectra recorded at various temperatures tend to indicate that dimethylation of the adenosines labilizes these base-pairs, but no definitive conclusions are drawn. The results confirm our previous views that dimethylation of the adenosine residues affects the conformation of the hairpin loop.

Adenine↗
PubMed1983

500-MHz 1H-NMR studies of ribosomal proteins isolated from 70-S ribosomes of Escherichia coli.

A method for the large-scale isolation of ribosomal proteins is described avoiding pre-separation of 30-S and 50-S subunits. Five proteins isolated in this way were studied with high-resolution 1H NMR at 500 MHz. These are S21, L18, L25, L30 and L33. The results show that L18, L25 and L30 exhibit tertiary structure in solution and indications for secondary structure in S21 are found. Protein L33 appears to be a random coil. Several resonances in the 1H NMR spectra are assigned to particular protons of amino acid residues, e.g. the aromatic ring protons of tyrosines and histidines, and epsilon-protons of lysines.

Aniline Compounds↗
PubMed1983

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.

Base Sequence↗
PubMed1981

Equilibrium aspects of the binding of myo-inositol hexakisphosphate to human hemoglobin as studied by 31P NMR and pH-stat techniques.

The interaction of myo-inositol hexakisphosphate (P6-inositol) with human hemoglobin has been studied as a function of pH using pH-stat techniques and 31P NMR. With the pH-stat method the following data were obtained: the association constants for the P6-inositol/deoxyhemoglobin and P6-inositol/carboxyhemoglobin complexes at alkaline and acid pH respectively and the proton absorption curves associated with the protein/phosphate interaction for both complexes from pH 5.5 to pH 9. From these data affinities of P6-inositol towards deoxyhemoglobin (Hb) and carboxyhemoglobin (HbCO) have been calculated as a function of pH. The shape of the proton absorption curves was found to be strongly dependent on the ligation state of the hemoglobin molecule. The pH dependence of the 31P NMR spectra of P6-inositol bound to Hb or HbCO provides a monitor for the proton-binding behaviour of the phosphate groups of P6-inositol when present in the central cavity of the protein. It appears that this behaviour is only slightly dependent on the ligation state of the hemoglobin molecule. The NMR spectral data were interpreted in terms of a model which takes into account the electrostatic interaction between the phosphate groups within the P6-inositol molecule as well as the electrostatic interaction between the phosphate groups and positively charged groups on the protein. To account for the discrepancy between the pH-stat and 31P NMR results, i.e. a strong dependence of the proton-absorption curves and a weak dependence of the proton-binding behaviour of P6-inositol on the ligation state of the protein respectively, it is proposed that a conformational change takes place in HbCO upon P6-inositol binding.

Carboxyhemoglobin↗
PubMed1981

31P NMR study of the kinetics of binding of myo-inositol hexakisphosphate to human hemoglobin. Observation of fast-exchange kinetics in high-affinity systems.

The association and dissociation kinetics of the complexes of myo-inositol hexakisphosphate (P6-inositol) with deoxyhemoglobin (Hb) and carboxyhemoglobin (HbCO) have been investigated by 31P NMR between pH 6.8 and pH 5.5. These complexes represent high-affinity systems with binding constants varying between 10(5) M-1 and 2 X 10(9) M-1. 31P NMR spectra of P6-inositol were recorded in the presence of hemoglobin as a function of the P6-inositol/hemoglobin molar ratio. It appeared that the exchange of the polyphosphate molecule between the solution and the central cavity binding site is fast on the NMR time scale. This observation cannot be reconciled with a single-step binding mechanism of P6-inositol to hemoglobin. Analysis of the spectra revealed the occurrence of additional binding of P6-inositol to both Hb and HbCO. This binding was also observed in pH-state experiments performed at low ionic strength. 31P NMR experiments carried out with hemoglobin of which the alpha-chain N termini were carbamylated, strongly suggest that these termini constitute the additional binding site for P6-inositol. A model is proposed which accounts for the enhancement of exchange kinetics in these high-affinity systems. In this model a rapid migration is assumed for P6-inositol between the central cavity binding site and an entry/leaving site on the hemoglobin molecule. Based on this model 31P NMR linewidths and chemical shift patterns for this three-site exchange problem were calculated.

Carboxyhemoglobin↗
PubMed1979

The binding of chloride ions to ligated and unligated human hemoglobin and its influence on the Bohr effect.

The contribution of the interaction of chloride ions with deoxy and oxyhemoglobin to the Bohr effect can be described by a simple binding model. Applying this model to experiment data reveals that at physiological pH and ionic strength about half of the release of Bohr protons is due to a difference in chloride ion binding to deoxy- and oxyhemoglobin. The chloride-independent part of the Bohr effect corresponds with the shift in pK which His-146 beta shows upon oxygenation. The proton absorptioon by hemoglobin observed upon oxygenation below pH 6 is apparently due to a chloride-ion-induced proton uptake, which is larger for oxyhemoglobin than for deoxyhemoglobin. The analysis of the experimental data indicates the existence of only two oxygen-linked chloride ion binding sites in both deoxy and oxyhemoglobin. In deoxyhemoglobin the binding sites most likely consist of Val-1 alpha of one chain and Arg-141 alpha of the partner chain. The sites in oxyhemoglobin consist of groups with a pK value in the neutral pH range; they do not contain lysyl or arginyl residues.

Binding Sites↗
PubMed1978

The spin-state transition of the hemochrome non-equilibrium conformation in partially reduced human methemoglobin. A pulse-radiolysis study of aqueous-methanol solutions of methemoglobin.

The effect of external parameters on the relaxation process of the hemochrome-type non-equilibrium conformation in partially reduced methemoglobin has been investigated. The relaxation of the intermediate ferrous low-spin state to the high-spin equilibrium conformation of hemoglobin appears to be facilitated particularly by protons and phosphate ions. In addition to studying the spin-state transition in aquomethemoglobin we have also studied it in complexes of the heme group in methemoglobin with fluoride, azide and cyanide anions.

Computers↗
PubMed1978

Kinetics of carbon monoxide binding to fully and partially reduced human hemoglobin valency hybrids.

The kinetics of carbon monoxide binding following fast reduction of the valency hybrids alpha2+betaCO2 and alphaCO2beta+2 by hydrated electrons have been studied at different degrees of reduction. The results show that at pH 6.0 and 7.0 reduction of one heme group yields a species which reacts fast with carbon monoxide (rate constant of the order of 10(6) M-1S-1). At pH 6.0 the intermediates alphaCO2beta2 and alpha2betaCO2 bind carbon monoxide with a rate characteristic of the T state. At pH 7.0 alphaCO2beta2 is for the greater part in the T state, while in the case of alpha2betaCO2 the R and the T state are about equally populated.

Carbon Monoxide↗
PubMed1977

An investigation of heavy meromyosin-ADP binding equilibria by proton release measurements.

The interaction of magnesium-ADP with skeletal muscle heavy meromyosin has been studied by measuring the accompanying release of protons. Total pH changes of the order of 0.03 were involved, and measurements were performed with a discrimination of some ten-thousandths of a pH unit. At pH 8.0 and 25 degrees C about 0.5 mol of protons per mol of heavy meromyosin is released at saturation. A stoichiometry of binding close to 2 mol of ADP per mol of protein was found, with a binding constant, obtained from the proton release titration curve (pH 8.0, 25 degrees C), of 2 X 10(5) M-1. At 5 degrees C the release of protons per mole is slightly greater, and the binding constant is somewhat increased, reflecting a negative enthalpy of binding. Similar proton release behavior is observed in the presence of manganous ions in place of magnesium. The liberation of protons is thus unrelated to the temperature-dependent isomerization of myosin in the presence of substrate. Alkylation of a reactive thiol group (SH1) does not change the proton liberation at pH 8.0. From the pH dependence of proton release, the association constant of heavy meromyosin with magnesium-ADP at other pH values can be inferred and shows an appreciable rise as the pH increases. The pH-proton release profile also allows the pK of the ionizing groups perturbed by the ligand to be deduced. At least two groups ionizing above pH 7 and one below are involved. Their pK's in the unperturbed state are assigned as 8.5, 9.3, and about 6.6, respectively; they are displaced in the complex to about 8.0, 9.1, and 6.3. A relation to the pH-activity profile of myosin ATPase is indicated. The pH-proton release profile is somewhat changed when the SH1 group is alkylated. Measurements with potassium-ADP, in the absence of magnesium, show that at pH 8.0 there is no proton release but rather a sizeable proton absorption (about 0.5 mol of protons per mol of heavy meromyosin). The association constant derived from the titration curves (pH 8.0, 25 degrees C) is 3 X 10(4) M-1.

Adenosine Diphosphate↗
PubMed1977

The CO and NO Bohr effect of human hemoglobin with and without inositolhexaphosphate.

Using NO and CO as ligands the Bohr effect of human hemoglobin has been measured with and without inositolhexophosphate. It appears that in the absence and presence of inositolhexaphosphate hemoglobin shows a distinct ligand specificity with respect to the Bohr effect. Ligation with NO is accompanied by release of a larger number of Bohr effect. It is shown that this latter result is due to the fact that the number of protons taken up upon binding of inositolhexaphosphate to ligated hemoglobin is larger for HbNO than for HbCO. It is suggested that this additional proton uptake is partially due to a restoration of the saltbridge between His 146beta and Asp 94beta upon addition of IHP.

Carboxyhemoglobin↗
PubMed1977

Heterogeneity in the kinetics of oxygen binding to partially reduced human methemoglobin. A pulse-radiolysis study of oxygenated solutions of methemoglobin.

The pulse-radiolysis technique has been introduced because it permits a rapid reduction (in a few microseconds) of one heme group of the methemoglobin tetramer by hydrated electrons. The kinetics of the binding of oxygen to this particular valence intermediate (Hb3+) with one reduced alpha or beta subunit has been studied. It appears that the hydrated electrons preferentially reduce one type of subunit of methemoglobin at acid and neutral pH-values as is shown by the biphasic behaviour of Hb3+ on oxygenation. The second-order on-rate constants measured for the binding of oxygen to Hb3+ are 14 +/- 3 mM-1 ms-1 and 56 +/- 9 mM-1 ms-1, respectively. The relative contribution of the faster fraction is about 0.63 +/- 0.08 of the total oxygenation process. A comparison of the kinetic absorbance difference spectrum for the reduction of methemoglobin with the static difference spectrum of deoxyhemoglobin and methemoglobin in the Soret-region revealed a decreased absorbance of the unliganded subunit of Hb3+ at 430 nm. This fact suggests that Hb3+ is in the relaxed quaternary conformation, which is in agreement with the observed on-rate constants.

Humans↗
PubMed1975

The effect of potassium chloride on the Bohr effect of human hemoglobin.

The normal and differential titration curves of liganded and unliganded hemoglobin were measured at various KCl concentrations (0.1 to 2.0 M). In this range of KCl concentrations, the curves for deoxyhemoglobin showed no salt-induced pK changes of titratable groups. In the same salt concentration range oxyhemoglobin showed a marked change in titration behavior which could only be accounted for by a salt-induced increase in pK of some titratable groups. These results show that the suppression of the alkaline Bohr effect by high concentrations of neutral univalent salt is not caused by a weakening of the salt bridges in deoxyhemoglobin but is due to an interaction of chloride ions with oxyhemoglobin. Measurements of the Bohr effect at various KCl concentrations showed that at low chloride ion concentration (5 times 10-3 M) the alkaline Bohr effect is smaller than at a concentration of 0.1 M. This observation indicates that at a chloride ion concentration of 0.1 M, part of the alkaline Bohr effect is due to an interaction of chloride ions with hemoglobin. Furthermore, at low concentrations of chloride ions the acid Bohr effect has almost vanished. This result suggests that part of the acid Bohr effect arises from an interaction of chloride ions with oxyhemoglobin. The dependence of the Bohr effect upon the chloride ion concentration can be explained by assuming specific binding of chloride ions to both oxy- and deoxyhemoglobin, with deoxyhemoglobin having the highest affinity.

Binding Sites↗