Magnetic and Spectral Properties of Carp Carbonmonoxyhemoglobin. Competitive Effects of Chloride Ions and Inositol Hexakisphosphate

Cerdonio, M.; Morante, Silvia; Vitale, S.; Dalvit, Claudio; Russu, Irina M.; Ho, Chien; Young, Alice De; Noble, Robert W.

doi:10.1111/j.1432-1033.1983.tb07384.x

Cited by 14 publications

(19 citation statements)

References 23 publications

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“…Taken all together the present data do not allow any low-lying triplet state with an energy less than 1100 cm-' above the singlet ground state. In our more recent publication concerning carp HbCO we reported changes in NMR and Soret spectra that seemed to parallel our susceptibility results (5). Such spectral changes do occur, but their apparent relationship to our susceptibility data was an unfortunate result of pure chance.…”

supporting

confidence: 84%

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Reexamination of the evidence for paramagnetism in oxy- and carbonmonoxyhemoglobins.

Cerdonio¹,

Morante²,

Torresani³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

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Experiments have been carried out in an attempt to confirm previous reports of paramagnetism in the oxy-and carbonmonoxy derivatives of human and carp hemoglobin. When care is taken to ensure complete saturation of the hemoglobins with ligand and the diamagnetic contributions of all of the buffers are carefully evaluated, these hemoglobin derivatives are found to have the same gram susceptibilities as exhibited by a set of metal free proteins, suggesting that they are fully diamagnetic.In previous articles (1-5), we reported evidence of the existence of a paramagnetic contribution to the magnetic susceptibilities of the carbonmonoxy and oxy derivatives of human and carp hemoglobins. In response to the controversy surrounding our observations and the findings of others (6, 7), we have repeated the experiments with additional controls. We wish to report that there is no longer any reason to suspect that these derivatives are paramagnetic at room temperature. Using a few metal-free proteins we have been able to establish a diamagnetic reference for protein solutions. We find that the magnetic properties of oxy-and carbonmonoxyhemoglobins correspond to such a diamagnetic reference. This brings our observations into agreement with those of refs. 6 and 7.The methods of sample preparation, the instrumentation, the overall precision, and the data reduction were the same as in our most recent papers (see ref. 5). However, we took greater care to be certain that the hemoglobins were fully saturated with the ligand in the highly concentrated solutions used in those experiments. We did this after some observations of our own and discussions with Joe Savicki and George Lang aroused the suspicion that partial deoxygenation could have been the source of our previous findings. For every protein sample the diamagnetic contribution of the buffer was determined independently. These determinations led us to revise the values of the buffer corrections at high salt concentrations. Our results are shown in Fig. 1 Fig. 1. We obtain for the common Xg the value -(0.587 + 0.005) x 10-6 ml/g. We have also measured the susceptibilities of solutions of human HbO2 and HbCO and carp HbCO at high salt concentrations (-350 mM NaCl). The Xm. values for all of these derivatives fall, within error limits, on the same straight line when plotted as a function of molar heme concentration with the intercept at zero concentration equal to the solvent Xm., Using the same value for the partial volume of Hb as that used for the low-ionic- strength results, we calculate XHb to be -(0.593 + 0.006) x 10-6 ml/g. It is evident that no significant difference exists between XHb at low and high salt concentrations. This value is consistent with our earlier results obtained with human HbCO at low salt concentrations but not with the results we reported for this derivative at high ionic strength in ref. 3. Finally, we have also redone our studies in the frozen state. Fully liganded human HbCO and HbO2 samples were frozen, by immersion in liquid nitrogen, and...

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supporting

confidence: 84%

“…5). However, we took greater care to be certain that the hemoglobins were fully saturated with the ligand in the highly concentrated solutions used in those experiments.…”

mentioning

confidence: 99%

Reexamination of the evidence for paramagnetism in oxy- and carbonmonoxyhemoglobins.

Cerdonio¹,

Morante²,

Torresani³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

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“…To maintain reasonable concentrations of uncharged imidazole at low pH requires large concentrations of imidazolium ion and its chloride counter anion. The latter in turn acts as an allosteric effector of hemoglobin and can also interfer with the effects of P6-inositol [15]. For related reasons the aquomet derivative was examined in the presence and absence of P6-inositol at a single pH.…”

Section: Resultsmentioning

confidence: 99%

“…In these data there appears to be an inverse correlation between AAG" and ligand concentration. There are situations in which elevated anion concentrations reduce the allosteric effects of P6-inositol [15], and we wonder if this was occurring in this instance. We have also wondered whether P6-inositol induces a complete conversion to the T state for even those derivatives which do not require high ligand concentrations.…”

Section: Aag" = -R T Ln-= R T L N ( P+ -P?s 2 ) X ( P K -$ )mentioning

confidence: 99%

Studies on the linkage between spin equilibria and protein structure in carp ferric hemoglobin

Noble

Young

Vitale

et al. 1987

European Journal of Biochemistry

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The effects of protein conformation on the spin-state equilibria of several derivatives of carp hemoglobin have been examined. This has been done by measuring the pH dependence of the paramagnetic susceptibilities of these derivatives in the presence and absence of inositol hexakisphosphate, P6-inositol. In all cases the addition of P6-inositol at low pH and the lowering of the pH in the presence of P6-inositol shift the spin-state equilibrium in favor of the high-spin electronic configuration. The P6-inositol and pH dependence of these magnetic properties parallels the pH and P6-inositol dependence of the conformational state of the hemoglobin as determined in earlier studies and further supports a thermodynamic linkage between the electronic state of the iron atoms and the quaternary structure of the hemoglobin molecule.When oxygen binds to hemoglobin there is an alteration in the structure of this protein from the low-affinity, deoxy structure to the high-affinity, ligand-saturated structure. This conformational transition appears to be an essential feature of the cooperative binding of oxygen, carbon monoxide and other heme ligands [l, 21. Such cooperativity is observed as an increase in the ligand affinity of the hemoglobin molecule as the four oxygen-binding or ligand-binding sites become progressively saturated. Under normal circumstances this structural transition in human hemoglobin requires the binding or release of ligand. In contrast to this, the hemoglobin of the carp can be switched between a similar pair of structural states by specific binding or removal of organic phosphate or protons, all of which bind at sites other than the heme group [3]. This remarkable property permits one to examine the effects of protein structure on the ligand-binding site without the need to change the level of saturation or the nature of the ligand. This behavior is found for both ferrous and ferric derivatives of carp hemoglobin [3,4].It is well known that for many ferric derivatives of heme proteins the energy splitting between the low-spin, S = 112, and high-spin, S = 512, states of iron is small enough for a thermal equilibrium to exist between these electronic configurations [5, 61. This spin-state equilibrium depends on the nature of the ligand and varies from one protein to another. In the case of hemoglobin it has also been shown to depend on the structure of the protein. quantify this linkage at room temperature in aqueous solution for carp aquomethemoglobin and azide methemoglobin [lo]. This work has now been extended to several other derivatives of carp hemoglobin and to the determination of the pH dependence of susceptibility in the presence and absence of 1 mM inositol hexakisphosphate (P6-inositol). MATERIALS AND METHODSMagnetic susceptibilities were measured with a superconducting fluxmeter which has been previously described 18, 91. Calibration and procedures for changing samples result in measurements with an overall accuracy of 0.03% of the susceptibility of water in the room temperature range. To co...

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“…Further evidence for an excited state was the observation of temperature-dependent quadrupole splittings and linewidths in the Mossbauer spectra of oxyhemoglobins (6). The diamagnetism of carbonmonoxyhemoglobin came under question with reports of salt-dependent effects (7). Most recently, one researcher has again found oxyhemoglobin to be diamagnetic (8) 1 and 4 in Fig.…”

mentioning

confidence: 99%

Magnetic susceptibility of oxy- and carbonmonoxyhemoglobins.

Savicki¹,

Lang²,

Ikeda‐Saito³

1984

Proc. Natl. Acad. Sci. U.S.A.

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The room temperature magnetic susceptibilities of human and carp oxy-and carbonmonoxyhemoglobin solutions were measured in a 30-gauss (1 G = 10-4 T) field with a superconducting magnetometer. To within experimental uncertainty, the susceptibility was the same for both the oxy and carbonmonoxy forms, and salt concentration did not affect it. A variety of sample preparations were used; the iron chemical state was verified by Mossbauer spectroscopy. A value of -0.580 ± 0.010 x 10-6 centimeter-gram-second (cgs) system was obtained for the mass susceptibility of the protein.We attribute the paramagnetism sometimes observed in oxyhemoglobin solutions to the presence of a small amount of the deoxy form.A controversy has recently grown over the magnetic susceptibility of oxy-and carbonmonoxyhemoglobin under various solution conditions. The data of Pauling and Coryell (1), Taylor and Coryell (2), and Havemann et al. (3) indicate that both forms of the protein are totally diamagnetic at room temperature. Recent work (4, 5) shows spin unpairing in oxyhemoglobin with a singlet-triplet separation of 144 cm'1. Further evidence for an excited state was the observation of temperature-dependent quadrupole splittings and linewidths in the Mossbauer spectra of oxyhemoglobins (6). The diamagnetism of carbonmonoxyhemoglobin came under question with reports of salt-dependent effects (7). Most recently, one researcher has again found oxyhemoglobin to be diamagnetic (8) but in solution conditions vastly different from those in which paramagnetism was seen.The resolution of this conflict is imperative for the development of valid electronic models of reversible oxygen binding to hemes. Therefore, we have constructed a superconducting magnetometer to measure the magnetic susceptibilities of oxy-and carbonmonoxyhemoglobin at room temperature and have attempted to reproduce the conditions of the work which showed paramagnetism (4, 5, 7) as closely as possible. MATERIALS AND METHODSA superconducting magnetometer built in our laboratory was used to make the measurements. Fig. 1). These samples proved to be more paramagnetic than water. A Mossbauer spectrum of one of these, frozen to 77 K immediately after the susceptibility measurement, indicated about 9% deoxyhemoglobin, a spin 2 compound. After this discovery, Mossbauer spectra were taken immediately after all susceptibility measurements. Mossbauer measurements, although less sensitive than optical spectroscopy, avoid dilution and the consequent risk of changing the oxy/deoxy equilibrium.We found that it was difficult to prepare samples free of deoxyhemoglobin by exposing concentrated solutions (15 mM in heme) to air. If a concentrated, mostly oxygenated sample were exposed to CO, all deoxyhemoglobin would be replaced by carbonmonoxyhemoglobin, but a significant fraction of the protein remained in the oxygenated form. By diluting a sample to about 5 mM in heme, we could reliably prepare pure oxy or carbonmonoxy proteins. More concentrated solutions were then obtained through the u...

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Magnetic and Spectral Properties of Carp Carbonmonoxyhemoglobin. Competitive Effects of Chloride Ions and Inositol Hexakisphosphate

Cited by 14 publications

References 23 publications

Reexamination of the evidence for paramagnetism in oxy- and carbonmonoxyhemoglobins.

Reexamination of the evidence for paramagnetism in oxy- and carbonmonoxyhemoglobins.

Studies on the linkage between spin equilibria and protein structure in carp ferric hemoglobin

Magnetic susceptibility of oxy- and carbonmonoxyhemoglobins.

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