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1.
Enzymatic reduction of spin-labeled ferrihemoglobin   总被引:1,自引:0,他引:1  
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2.
Measurement of the dissociation constants of ferrihemoglobin by light scattering indicates that the quaternary structure is altered by the type of heme ligand. Fluoromethemoglobin and aquomethemoglobin, high spin derivatives with weak ligands, have tetramer-dimer dissociation constants of 80 and 50 muM, respectively. For low spin cyanmethemoglobin the dissociation constants were 1 muM (pH 6.0) and 3 muM (pH 9.0) under the general conditions of 0.1 ionic strength and 25 degrees. Of the ferrihemoglobins studied, alkaline methemoglobin (pH 9.0) has the lowest dissociation constant (0.2 muM). Dissociation constants of mixtures of alkaline and fluoromethemoglobin were significantly higher than that of the alkaline form alone. At pH 9.0 the 55 and 78% fluoride-bound derivatives had tetramer-dimer dissociation constants of 0.7 and 2 muM, respectively. The cyanmethemoglobin quaternary conformation was found to be less affected by pH than the fluoromethemoglobin and aquomethemoglobin conformations. Measurement of the dissociation constant (0.2 muM) for aquomethemoglobin-inositol hexaphosphate indicates stabilization of the tetramer by this organic phosphate. The extent of stabilization by inositol hexaphosphate does not appear to be that found for deoxyhemoglobin as suggested by Perutz (Perutz, M. F. (1972) Nature 237, 495-499) even though inducement of higher spin and iron-heme plane displacement may occur.  相似文献   

3.
A C Anusiem 《Biopolymers》1975,14(6):1305-1307
Measurements of the magnetic susceptibility of guinea pig, human A, and pigeon ferrihemoglobins as a function of pH at 30°C demonstrate maxima very near the extrema found for the standard enthalpies and standard entropies of ligand binding, i.e., at the so-called “characteristic pH” values, which are very different for these three species. Ligand binding is dependent on spin state, at least through the characteristic pH behavior, which is not yet understood.  相似文献   

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X-ray diffraction studies have shown that hemoglobin has two predominant interfaces in the tetramer at which dissociation to dimers could occur. These interfaces have been designed as alpha1-beta1 and alpha1-beta2. There are 2 tyrosyl residues and 1 tryptophanyl residue in the alpha1-beta2- interface but only 1 tyrosyl residue in the alpha1-beta1 interface exposed to the solvent are perturbed. The ultraviolet difference spectrum between ferrihemoglobin dissociated in 1 M NaClO4 and undissociated hemoglobin revealed two negative peaks, one at 292.5 nm and another at 285 nm. This difference spectrum is due to tyrosyl and tryptophanyl residues which reside on the plane of cleavage and were exposed to 1 M NaClO4 upon dissociation. Hence, dissociation must have occurred along the alpha1-beta2 interface to yield alpha1 beta1 dimers. The deltaF degrees value extrapolated to zero salt concentration calculated on the basis of difference spectroscopy and sedimentation velocity experiments is 8.6 plus or minus 0.7 kcal per mol at pH 7.1 (K equals 4.5 times 10-7.  相似文献   

6.
Reactivity of crystalline ferrihemoglobin towards azide   总被引:1,自引:0,他引:1  
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7.
A C Anusiem 《Biopolymers》1975,14(6):1293-1304
The molar magnetic susceptibility of ferrihemoglobin solutions, as a function of pH and temperature using the nmr technique of Evans, in water and 5% t-butanol has been determined. Results suggest that within a small pH region, the conventional analysis of magnetic susceptibility in terms of high-spin and low-spin contributions breaks down. The implication of this is discussed in terms of possible conformational change involving two subspecies. The effect of t-butanol is also discussed in terms of the t-butanol effect on water.  相似文献   

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There is evidence that suggests that two forms of ferrihemoglobin may exist under various experimental conditions. We have studied the spectral changes brought about by the use of glycerol-water mixtures as a solvent for ferrihemoglobin. Our results indicate that in the presence of substantial amounts of glycerol a form of acid ferrihemoglobin spectrally resembling that of the imidazole complex of ferrihemoglobin is stabilized. A quantitative analysis of the spectral changes suggests that the effect of glycerol on ferrihemoglobin can in large part be ascribed to its effect on the activity of the solvent water.  相似文献   

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The final step in the erythrocyte methemoglobin reduction pathway, the transfer of an electron from cytochrome b5, to methemoglobin, has been studied using magnetic circular dichroism spectroscopy. Spectral analysis allowed us to determine accurately the concentration of each redox species in mixtures of the two heme-proteins and to follow simultaneously the kinetics of the appearance or disappearance of each of these species during reduction reactions. Our analysis detected a substantial increase in the high-spin hydroxymethemoglobin species in the partially reduced bovine hemoglobin tetramer. This species was sensitive to the degree of reduction and pH, and was spectrally similar to fluoride methemoglobin. At pH 7.8. 100% of the hydroxide component of methemoglobin was in the high-spin form when two or more subunits were in the ferrous form. Kinetic analysis of bovine methemoglobin reduction yielded values for the apparent first-order rates for the tetrameric species possessing four, three, two, and one ferric subunit. Further analysis showed that the reduction kinetics can also be described by an equilibrium state, pure competitive inhibition model for enzyme catalysis in which ferrous and ferric subunits of hemoglobin compete for cytochrome b5 This analysis generated a KD that depends on ionic strength and hemoglobin tetramer conformation, a Vmax that was independent of these factors, and an inhibition constant that was equal to Kd. This model is consistent with the hypothesis that the reduction of methemoglobin can be separated into two steps, the ionic interaction between cytochrome b5 and hemoglobin and the electron transfer.  相似文献   

13.
The acid-alkaline pH-jump in suspension of crystalline sheep hemoglobin has been studied in the range of 5.95 to 8.94. Crystals suspended in 3.8 M Cs2SO4 show a rapid optical transition of half-time equal to or less than 2 ms. As the ammonia concentration is increased in the Cs2SO4-suspended crystals, a second optical transition is observed as a pseudo-first-order reaction, with a rate constant of between 10 and 15 s-1. The alkaline-acid pH-jump proceeds through a very rapid shift of the alkaline-acid equilibrium and is followed by a first-order dissociation constant between 9 and 12 s-1. The dissociation of the ammonia is biphasic, and the ratio between the fast and slow phases is 9.  相似文献   

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At pH 6.5 in a 0.05 M bis-Tris-0.1 M Cl? buffer, tetra aquo ferrihemoglobin A (HbA+) binds CN? with a Hill coefficient of n = 1.4. The Hill coefficient increases slightly and the average CN? affinity decreases in the presence of excess spin labeled triphosphate (SLTP). This is probably the result of the finding that the SLTP exhibits a twofold higher affinity for HbA+ than for tetra cyano HbA+. Over the course of heme saturation with CN?, a certain fraction of the SLTP is specifically released. This shows linkage between organic phosphate binding and heme ligation. These findings bear a marked resemblance to the ligand binding phenomena in hemoglobin A (HbA) and provide good evidence that under these experimental conditions, HbA+ is undergoing a quaternary conformation change as the hemes become saturated.  相似文献   

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We have carried out a detailed study of ligand binding of ferrihemoglobins under various conditions. Our results show that n varies with time and that this variation is paralleled by changes in the spectrum of methemoglobin. This suggests some form of structural perturbation. The time-dependent value of n is discussed in terms of the observed spectral changes accompanying prolonged equilibration.  相似文献   

20.
A method is described for the electrochemical reduction of pteroylpentaglutamate which results in high yields of the fully reduced tetrahydro derivative.  相似文献   

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