Methemoglobin Derivatives Research Articles

Photochemical reduction of methemoglobin and methemoglobin derivatives

Publisher Summary Hemoglobin solutions that must be stored for long periods of time must be kept either at very low temperatures or in the complete absence of oxygen, or both. These are conditions are not easy to achieve. The spontaneous oxidation to methemoglobin can be especially bothersome when solutions of precious hemoglobin variants or hemoglobin derivatives need to be stored for an extended time. One is thus often faced with the problem of having to reduce any methemoglobin that has formed in hemoglobin preparations before valid studies with the hemoglobin can be initiated. There are various procedures for the reduction of methemoglobin present in hemoglobin preparations that use a variety of reducing agents, such as dithionite, ascorbate, 5-hydroxyanthranilic acid, and iron salts. However, the direct reduction of methemoglobin with a reducing agent sometimes presents difficulties. The reduction of methemoglobin with reducing agents, such as ascorbate and cysteine reaches equilibrium when there are still considerable amounts of methemoglobin present in the solution.

Interaction of fully liganded valency hybrid hemoglobin with inositol hexaphosphate. Implication of the IHP-induced T state of human adult methemoglobin in the low-spin state.

To gain further insight into the quaternary structures of methemoglobin derivatives in the low-spin state, the interaction of fully liganded valency hybrid human hemoglobins with IHP was studied by proton NMR spectroscopy. Upon addition of IHP to (alpha CO beta + N3-)2, the same resonances as the previously reported IHP-induced NMR peaks for azidomethemoglobin (alpha + N3-beta +N3-)2 appeared, whereas the binding of IHP did not significantly affect the NMR spectra for (alpha + N3-beta CO)2. The binding of IHP also brought about more pronounced spectral changes for (alpha CO beta + Im)2 and (alpha CO beta + H2O)2 than for (alpha + Im beta CO)2 and (alpha + H2O beta CO)2. Therefore, the IHP-induced NMR peaks for azidomethemoglobin are attributed to the beta heme methyl group. Such IHP-induced beta heme methyl resonances were also observed for (alpha NO beta + N3-)2, which undergoes quaternary structural change, analogously to the R-T transition by the binding of IHP. From the above results, it was suggested that the IHP-induced heme methyl resonances for azidomethemoglobin and (alpha CO beta +N3-)2 may also be associated with the quaternary structure of these Hbs, implying the presence of the IHP-induced "T-like" state in low-spin metHb A.

Spectroscopic studies of protein-heme interactions accompanying the allosteric transition in methemoglobins.

Resonance Raman, optical absorption, and circular dichroism spectroscopic techniques have been used to examine the effect of the addition of inositol hexaphosphate (IHP) to a series of carp and human methemoglobin derivatives. Markers of spin equilibrium in the high-frequency region (1450-1650 cm-1) of the resonance Raman spectrum yield high/low-spin ratios consistent with direct magnetic susceptibility measurements. Changes in the low-frequency region (100-600 cm-1) of the resonance Raman spectrum appear to correlate with the quaternary structure transition. Changes in the ultraviolet absorption spectra and the circular dichroism spectra also appear to be related to the quaternary structure change. By using the resonance Raman spin markers, we find that those derivatives of carp methemoglobin which are in spin equilibrium have a larger ratio of high-spin to low-spin populations than the corresponding derivatives of human methemoglobin. Upon the addition of IHP to the methemoglobins the spin equilibrium is shifted toward a larger high-spin population. This change in equilibrium is larger for the carp protein than for the human protein. We obtain an IHP-induced change in the free energy difference between the high-spin and low-spin states of 300 cal/mol for those human methemoglobins in which a quaternary structure change occurs and 600 cal/mol for carp methemoglobins. Our data are consistent with a quaternary structure change induced by IHP in all the carp methemoglobins studied (F-, H2O, SCN-, NO2-, N3-, and CN-) and in the F-, H2O, and SCN- derivatives of the human protein but not in the NO2-, N3-, and CN- derivatives. The Fe-CN stretching mode has been identified by isotopic substitution and found to be unchanged in frequency in carp CN- metHb when the quaternary structure is changed. On the basis of our results we conclude that the protein forces at the heme due to the addition of IHP do not significantly affect the position of the iron atom with respect to the heme plane. Rather, the changes in spin equilibrium may be caused by protein-induced changes in the orientation of the proximal histidine or tertiary structure changes in the heme pocket which affect the porphyrin macrocycle. Either of these changes, or a combination thereof, leads to changes in the iron d orbital energies and concomitant changes in the spin equilibrium.

Comparison of solid and solution state protein structures. Photoacoustic study of solid state bovine methemoglobin derivatives.

In an effort to examine the consequences of phase state changes on protein structures, optical absorption spectra of several bovine hemoglobin derivatives in lyophilized, ammonium sulfate-precipitated, and crystalline states were examined. Absorption spectra were used to compare protein conformations. Differences between solution and several solid state spectra were apparent. They were greatest for lyophilized preparations, were inhibited by ligand binding, the tightest binding ligand being the most effective inhibitor, and were substantially or totally reversible upon dissolving the solid state protein. Though the magnitude of spectral changes varied, they all indicated characteristically different proportions of the protein were converted to a reversible hemichrome in each type of solid state preparation. The extent of hemichrome formation was correlated with the degree to which the protein tends to be dehydrated in each solid state preparation. This may reflect the role of water in determining the three-dimensional structure of hemoglobin. Absorption spectra of solid state hemoglobin preparations were obtained from photoacoustic spectra using a novel method for spectral analysis. Results presented here demonstrate the utility of this procedure in probing structures of solid state proteins.

Temperature-dependent conformation change in spin-labeled hemoglobin.

Hemoglobin was spin labeled at beta-93(F9)-cysteine with N-oxyl-2,2,6,6-tetramethylpiperidinylmaleimide. The inward shift of the high-field hyperfine line (delta Hx) position in the ESR spectra of the spin label was measured as a function of temperature. One can except that an abrupt change in the microenvironment around the tightly bound spin label will be reflected in the function delta Hx(T) as a discontinuity (break point). This was shown for aquo-, azido-, nitro- and oxyhemoglobin derivatives. The presented results suggest that the microenvironment around the tightly bound spin label in those methemoglobin derivatives that exhibit the mixed-spin state of the heme iron is prone to an abrupt change above a certain ligand-specific temperature. The change in microenvironment of the spin label is probably due to a temperature-dependent change in the teritary structure of the protein.

Resonance Raman studies of methemoglobin derivatives at room temperature and 77 K

Raman spectra in preresonance with the Soret absorption band are reported for the following methemoglobin derivatives: cyanide, cyanate, thiocyanate, hydroxy-, azide, and fluoride methemoglobin at 285 K and 77 K. For the mixed-spin derivatives, Raman intensity is observed to shift from the high-spin marker band (approx. 1480 cm −1) to the low-spin marker band (approx. 1505 cm −1) upon cooling to 77 K. In addition, Raman spectra of cyanate methemoglobin were taken as a function of temperature, and the log of the intensity ratio I 1480/I 1505 was found to be a linear function of 1/T, indicating a thermally activated process. We interpret these results as observations of temperature-induced spin transitions. In the case of cyanate methemoglobin we find the enthalpy and entropy differences between the high-spin and low-spin states to be ΔH = 600 ± 40 cal · mol −1 and ΔS = 4.7 ± 0.7 cal · mol −1 · K −1 . The high-spin to low-spin ratio for cyanate methemoglobin determined by our experiment disagrees with the value reported for magnetic susceptibility measurements.

Influence of quaternary structure of the globin on thermal spin equilibria in different methemoglobin derivatives

We have measured the paramagnetic susceptibilities of sperm whale azide metmyoglobin and of carp azide, thiocyanate, and nitrite methemoglobin in the quaternary oxy (R) and deoxy (T) structures between about 300 and 90 K, using a new sensitive superconducting magnetometer. We have also measured the pressure dependence of the high- and low-spin optical absorption bands of azide metmyoglobin and of carp azide methemoglobin in the R and T structures between 1 and 2000-4000 atmospheres. At low temperatures all the derivatives show normal Curie behavior, but above 200-250 K this is reversed, so that a thermal spin equilibrium is set up and the paramagnetic susceptibilities rise steeply with rising temperature. At all temperatures the effective magnetic moments in the T structure are higher than in the R structure. The magnetic data for azide methemoglobin have been subjected to detailed analysis. Below 250 K the magnetic moment in the R structure is 1.98 microB, characteristic of pure low spin, but that in the T structure is 2.80 microB, suggestive of a random mixture of high- and low-spin centers which have become frozen in by the immobility of the surrounding protein. Comparison of the thermal spin equilibria above 250 K shows that in the T structure the equilibrium is biased toward higher spin by the equivalent of about 1 kcal/mol relative to the R structure. Hydrostatic pressure reduces the optical density of the high-spin band at 630 nm and increases that of the low-spin bands at 541 and 573 nm. We have calibrated the optical density of the band at 630 nm against the measured paramagnetic susceptibilities of sperm whale azide metmyoglobin and carp azide methemoglobin in the R and T structures and have used this calibration to determine the dependence of the spin equilibria on hydrostatic pressure; this has allowed us to calculate the volume contraction associated with the transition from the fully high to the fully low-spin state. This amounts to -6.7 and -13.3 mL/mol heme for carp azide methemoglobins in the R and T structures, respectively, and to -12.5 mL/mol heme for azide metmyoglobin. These volume contractions are larger than those of about -4 mL/mol Fe found in synthetic iron chelates. Apparently stereochemical changes of the globin surrounding the heme also contribute to the volume changes; these must be larger in the T than in the R structure. The significance of these observations for the mechanism of heme-heme interaction is discussed.

Nuclear magnetic resonance studies of hemoprotein Proton hyperfine shifts and structural characterization of the different heme environments in methemoglobin and metmyoglobin

The different features of the nature of heme iron-proximal histidine binding in methemoglobin and metmyoglobin were investigated by high resolution proton nuclear magnetic resonance spectroscopy at 220 MHz. From the comparison of the hyperfine-shifted heme methyl resonances of various methemoglobin and metmyoglobin derivatives it was revealed that the metHb derivatives exhibit smaller hyperfine shifts than the corresponding metMb derivatives. This observation was interpreted in terms of the stronger interaction between proximal histidine and ferric heme iron in methemoglobin than in metmyoglobin. The spectral contributions from the α and β subunits in methemoglobin in high spin state were successfully separated by the analysis of the temperature dependent heme methyl shifts of the methemoglobin azide complex which is in the high and low spin thermal spin equilibrium. It was shown that the β heme methyl resonances have smaller hyperfine shifts than the α heme ones in ferric high spin state. This tendency in methemoglobin derivatives was also interpreted in terms of the stronger iron-histidine binding in the β subunits than in the α subunits within the tetramer. From the comparisons between methemoglobin and metmyoglobin, and between the α and β subunits in methemoglobin, the order of the strength of the iron-histidine bond interaction was deduced as follows: metmyoglobin < α subunits in methemoglobin ⪅ β subunits in methemoglobin

Resonance Raman spectra of methemoglobin derivatives. Selective enhancement of axial ligand vibrations and lack of an effect of inositol hexaphosphate.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTResonance Raman spectra of methemoglobin derivatives. Selective enhancement of axial ligand vibrations and lack of an effect of inositol hexaphosphateSanford A. Asher, Larry E. Vickery, Todd M. Schuster, and Kenneth SauerCite this: Biochemistry 1977, 16, 26, 5849–5856Publication Date (Print):December 1, 1977Publication History Published online1 May 2002Published inissue 1 December 1977https://doi.org/10.1021/bi00645a032RIGHTS & PERMISSIONSArticle Views239Altmetric-Citations77LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (879 KB) Get e-Alerts Get e-Alerts

Circular Dichroism and Magnetic Circular Dichroism Studies on Methemoglobin and Its Derivatives

CD and MCD spectra of human methemoglobin and its derivatives were studied with special reference to the relation of CD and MCD spectra to the magnetic moments. The intensities of MCD peaks of methemoglobin derivatives at 270 nm were inversely proportional to their magnetic susceptibility, and there was a linear relationship between magnetic ellipticity and magnetic susceptibility. The MCD peak intensity of methemoglobin peroxide compound was close to that of methemoglobin cyanide complex, suggesting that the methemoglobin peroxide compound is in a low-spin state.

Temperature dependence of ESR spectra of spin-labelled met-hemoglobin

The maleimide spin-label, firmly fixed to the protein, was used to study conformation changes of various met-hemoglobin derivatives. The temperature dependence of the rotational correlation time shows a distinct conformation change in aquomet-hemoglobin at about 25 degrees C. The other met-hemoglobin derivatives studied (fluoro-, cyano-, aquomet-complexed with inositol hexaphosphate) and carbonmonoxy-hemoglobin exhibit no conformation changes in the temperature range from 0-50 degrees C.

The optically active heme bands of hemoglobin and methemoglobin derivatives. Correlation with absorption and magnetic properties.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTOptically active heme bands of hemoglobin and methemoglobin derivatives. Correlation with absorption and magnetic propertiesTing-Kai Li and Burt P. JohnsonCite this: Biochemistry 1969, 8, 9, 3638–3643Publication Date (Print):September 1, 1969Publication History Published online1 May 2002Published inissue 1 September 1969https://doi.org/10.1021/bi00837a021RIGHTS & PERMISSIONSArticle Views367Altmetric-Citations26LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (650 KB) Get e-Alerts Get e-Alerts

Second spectroscopically abnormal methemoglobin associated with hereditary cyanosis.

Isolation of an abnormal methemoglobin from two families exhibiting dominantly transmitted cyanosis have permitted the recognition of two different pigments of the hemoglobin M type. It is possible that the abnormal properties which characterize the acidic methemoglobin derivatives result from a crevice configuration of the heme, with two Feprotein bonds.