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At least 19 recordsLinked to original sources
PubMed1990

Methemoglobin formation in the blood of Japanese subjects and mice suffering from acatalasemia in response to methemoglobin inducers.

Hemolysates or erythrocytes prepared from Japanese normal and acatalasemic subjects were exposed to nitrogen monoxide or nitrogen dioxide, and methemoglobin formation was determined. Concentrations of methemoglobin in human and mouse acatalasemic hemolysates exposed to nitrogen monoxide or nitrogen dioxide were higher than those in the normal hemolysates. Results similar to Japanese acatalasemic hemolysates or erythrocytes were obtained with mouse hemolysates or erythrocytes. When acatalasemic mice were exposed to nitrogen monoxide, the methemoglobin concentration in the blood was higher than that in the normal mice. Similar results on methemoglobin formation were obtained after exposing mice to nitrogen dioxide, although the rate of methemoglobin formation was lower in the blood of nitrogen dioxide-exposed mice. The methemoglobin concentration in erythrocytes of acatalasemic mice after the addition of sodium nitrite was higher than that in red cells of normal ones. Methemoglobin concentration in the blood of mice receiving sodium nitrites in vivo was determined, and found that the methemoglobin concentration in the blood of acatalasemic mice was higher than that in the blood of normal ones. These results indicated that the formation of methemoglobin from hemoglobin with nitrogen monoxide, nitrogen dioxide and nitrite ion appears to be controlled by the blood catalase.

Animals↗
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↗
PubMed1991

Oxidation of chlorpromazine by methemoglobin in the presence of hydrogen peroxide. Formation of chlorpromazine radical cation and its covalent binding to methemoglobin.

The oxidation of chlorpromazine by methemoglobin plus H2O2 has been studied. The transient formation of the chlorpromazine radical cation in this reaction has been demonstrated by light absorption measurements. Under the experimental conditions complete conversion of chlorpromazine yields approximately 60% chlorpromazine sulfoxide. From studies with 3H-labeled chlorpromazine it appears that the remaining 40% is covalently bound to apohemoglobin. Upon reaction of methemoglobin with H2O2 a stable ferrylhemoglobin is formed. This ferrylhemoglobin is not the reactive species, which accepts the chlorpromazine electron, as its presence is not sufficient to induce chlorpromazine oxidation. For this the presence of H2O2 is a prerequisite. This indicates that a transient species in the formation of the stable ferrylhemoglobin is involved, whether this is a compound I analogue or a ferrylhemoglobin with a free radical on one of the apoprotein residues. Exposition of methemoglobin to H2O2 denatures hemoglobin and induces protein-heme crosslinks, as appears from changes in the visible absorption spectrum and heme retention by the protein after methyl ethyl ketone extraction. Reaction with CPZ partly protects against denaturation and crosslinking.

Chlorpromazine↗
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↗
PubMed1990

Impaired erythrocyte methemoglobin reduction in sickle cell disease: dependence of methemoglobin reduction on reduced nicotinamide adenine dinucleotide content.

We have examined aspects of methemoglobin (metHb) reduction in sickle and in thalassemic red blood cells (RBCs). NADH metHb reductase activity in sickle and thalassemic RBCs was significantly increased compared with normal RBCs. Because in vitro enzyme activity does not necessarily represent in vivo activity, we measured the rate of metHb reduction in intact RBCs. Intact thalassemic RBCs demonstrated a significantly increased rate of metHb reduction compared with normal RBCs. In contrast, intact sickle RBCs had a rate of metHb reduction that was similar to normal RBCs and significantly decreased relative to high reticulocyte RBCs of equivalent cell age. To determine the mechanism for the relative impairment of metHb reduction in sickle RBCs, we measured intraerythrocytic NADH, a cofactor in the metHb reduction reaction. Thalassemic RBCs had a significantly increased NADH content relative to normal RBCs. In contrast, sickle RBCs did not have an increase in NADH content. Furthermore, incubating normal RBCs under conditions that increase the NADH content resulted in an increased rate of metHb reduction. In contrast, conditions that decrease the NADH content in normal RBC resulted in a decreased rate of metHb reduction. These data and other results suggest that metHb reduction in intact RBCs is dependent on NADH content, and that the impaired metHb reduction rate in sickle RBCs may be a result of a lack of increase in NADH content. The dependence of metHb reduction on RBC NADH content and the ability to manipulate NADH content in vitro suggest a new strategy for decreasing oxidant damage to sickle RBCs in vivo.

Anemia, Sickle Cell↗
PubMed1989

Methemoglobin reduction under near physiological conditions.

Pure methemoglobin was prepared from fresh red cells and was used as substrate for methemoglobin reduction reaction. Two sources of methemoglobin reductase were used: (a) red cell hemolysate which was prepared by freezing and thawing of unwashed red cells; (b) purified methemoglobin reductase from bank blood. Methemoglobin reduction rate was measured in a mixture of pure methemoglobin (substrate) and hemolysate (enzyme). In other experiments the rate of methemoglobin reduction was measured in the above mixture with the addition of various other compounds such as NADH, cytochrome b5, and pure methemoglobin reductase. Only the addition of pure enzyme accelerated the rate of methemoglobin reduction. In other experiments, the rate of methemoglobin reduction was measured when the reduction reaction was carried out in the presence of various amounts of deoxyhemoglobin, globin, or albumin. It was shown that all proteins tested here decreased the reduction rate. It is concluded that (a) in the red cell, under normal conditions, only the activity of the methemoglobin reductase controls the speed of methemoglobin reduction, and (b) the inhibition of methemoglobin reduction by reduced hemoglobin is mostly nonspecific suggesting a noncompetitive reaction.

Body Temperature↗
PubMed1978

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 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 alpha and beta 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 thermal spin equilibrium. It was shown that the beta heme methyl resonances have smaller hyperfine shifts than the alpha 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 beta subunits than in the alpha subunits within the tetramer. From the comparisons between methemoglobin and metmyoglobin, and between the alpha and beta subunits in methemoglobin, the order of the strength of the iron-histidine bond interaction was deduced as follows: metmyoglobin less than alpha subunits in methemoglobin less than or equal to beta units in methemoglobin.

Hemeproteins↗
PubMed1979

Differential effects of pH and inositol hexaphosphate on the spectroscopic properties of the alpha and beta subunits in methemoglobins M Milwaukee and A.

The effect of pH and inositol hexaphosphate on the electron spin resonance spectra of the alpha-hemes (g = 6.0) and the beta-hemes (g = 6.7) has been measured in methemoglobin M Milwaukee and compared with that of methemoglobin A (g = 6.0). The beta-hemes are found to be comparatively insensitive to both effectors while the alpha-hemes behave in a manner similar to the heme groups of methemoglobin A. Binding of inositol hexaphosphate enhances the high spin ESR signal of the alpha-hemes in both methemoglobins. Comparison of the optical properties of methemoglobins A and M Milwaukee over the pH range from 5.0 to 8.1 shows that inositol hexaphosphate has a differential effect on the subunit types in these two methemoglobins. At low pH the spectral changes observed upon inositol hexaphosphate binding arise primarily from the beta-hemes, while at neutral and alkaline pH these changes arise from both subunit types. The beta-heme spectral changes appear to be pH independent while those arising from the alpha-hemes are strongly pH dependent. It is concluded that it is the hydroxymet form of the alpha-hemes which undergoes spectral change upon inositol hexaphosphate binding to the beta-subunits. In methemoglobin A the spin state and paramagnetic susceptibility increase only in the neutral and alkaline pH ranges upon inositol hexaphosphate binding (Gupta, R.K. and Mildvan, R.S. (1975) J. Biol. Chem. 250, 246; Perutz, M.F., Sanders, J.K.M., Chenery, D.H., Noble, R.W., Penelly, R.R., Fung, L.W.-M., Ho, C., Giannini, I., Porschke, D. and Winkler, H. (1978) Biochemistry 17, 3640). Therefore the hydroxymet form of the alpha-hemes which is responsible for the observed spectral changes must also be responsible for these increases in the magnetic properties of methemoglobin A. Inositol hexaphosphate can bind to methemoglobin at alkaline pH if the beta-hemes are in the high spin form.

Chemical Phenomena↗
PubMed1987

The uptake of protons by heme-linked ionizable groups on azide binding to methemoglobin.

When azide ion reacts with methemoglobin in unbuffered solution the pH of the solution increases. This phenomenon is associated with increases in the pK values of heme-linked ionizable groups on the protein which give rise to an uptake of protons from solution. We have determined as a functional of pH the proton uptake, delta h+, on azide binding to methemoglobin at 20 degrees C. Data for methemoglobins A (human), guinea pig and pigeon are fitted to a theoretical expression based on the electrostatic effect of these sets of heme-linked ionizable groups on the binding of the ligand. From these fits the pK values of heme-linked ionizable groups are obtained for liganded and unliganded methemoglobins. In unliganded methemoglobin pK1, which is associated with carboxylic acid groups, ranges between 4.0 and 5.5 for the three methemoglobins; pK2, which is associated with histidines and terminal amino groups, ranges from 6.2 to 6.7. In liganded methemoglobin pK1 lies between 5.8 and 6.3 and pK2 varies from 8.1 to 8.5. The pH dependences of the apparent equilibrium constants for azide binding to the three methemoglobins at 20 degrees C are well accounted for with the pK values calculated from the variation of delta h+ with pH.

Azides↗
PubMed1988

The reduction of methemoglobin levels by antioxidants.

Preventing the oxidation of hemoglobin in solution is one of the major requirements for the successful production and long-term storage of hemoglobin-based blood substitutes. To this end we have studied the effects of antioxidants on the rate of methemoglobin formation and disappearance in solutions of human and bovine hemoglobin at 4 degrees C and 37 degrees C. Ascorbate and desferal (5 mM) were observed to act as prooxidants, increasing the rate of methemoglobin formation at 37 degrees C. Trehalose, mannitol, glucose, and EDTA (5 mM) had no significant effect. Glutathione and NADH (10 mM) were the most effective antioxidants tested, causing a significant decrease in the rate of methemoglobin formation at 37 degrees C for periods of up to 50 hours. The combination of these antioxidants in bovine hemoglobin at 4 degrees C resulted in the reduction of methemoglobin levels to nearly undetectable levels in approximately 150 hours. In addition, NADH and glutathione were found to reduce methemoglobin levels to 10% over a period of 100 hours in a sample of human hemoglobin that had been stored at 4 degrees C for one year and had 60% methemoglobin. These results suggest that the prevention and reversal of methemoglobin formation during the long-term storage of hemoglobin solutions and hemoglobin-based blood substitutes may now be possible.

Animals↗
PubMed1976

Effect of organic phosphates on methemoglobin reduction by ascorbic acid.

The rate of methemoglobin reduction by ascorbic acid was accelerated in the presence of ATP,2,3-diphosphoglycerate (2,3-DPG), and inositol hexaphosphate (IHP). The acceleration was as much as three times, four times, and ten times in the presence of ATP, 2.3-DPG, and IHP at pH 7.0, respectively. The changes of the concentrations of methemoglobin and ascorbic acid during the methemoglobin reduction were determined, and the reaction was found to proceed stoichiometrically in the presence of IHP. The reduction rate of methemoglobin by ascorbic acid was compared at different concentrations of organic phosphates (ATP,2,3-DPG, and IHP) at various pH values (6.3, 7.0, 7.7). From the changes in the reduction rate under different concentrations of organic phosphates, the dissociation constants of ATP, 2,3-DPG, and IHP to methemoglobin could be determined and were estimated to be 3.3 X 10(-4) M, 2 X 10(-3) M, and 8 X 10(-6) M at pH 7.0, respectively. On the basis of these results, the acceleration mechanism of methemoglobin reduction by ascorbic acid due to the presence of organic phosphates was described. The physiological role of 2,3-DPG in human red cells was discussed in relation to the reduction of methemoglobin by ascorbic acid.

Adenosine Triphosphate↗
PubMed1975

Conformation and spin state in methemoglobin.

The properties of human methemoglobin have been investigated under a wide variety of conditions to determine its conformation and to test for evidence of the T state conformation which has been proposed by Perutz to exist in the presence of high spin ligands and inositol hexaphosphate (IHP). Subunit dissociation was measured as a criterion for the T state since marked differences in the tetramer-dimer equilibrium exist for oxyhemoglobin (R state) and deoxyhemoglobin (T state). In the absence of IHP, complexes of methemoglobin with both high spin ligands (water, fluoride) or low spin ligands (azide, cyanide) show extensive dissociation in 2,2-bis(hydroxymethyl)-2,2',2"-nitriloethanol buffers, pH 6, 0.1 M NaCl, with values of the tetramer-dimer dissociation constant (K4,2) near 10-5 M. The addition of IHP lowers K4,2 to a value near 10-5 M for all forms of methemoglobin. Combination of IHP with methemoglobin promotes a conformational change, but the change is apparently independence of spin state. The conformation acquired in the presence of IHP is not identical with the T state (K4,2 similar to 10-12 M) and can also occur with hemoglobin in the ferrous form, as revealed by a substantial reduction in K4,2 for CO-hemoglobin upon addition of IHP. Subunit dissociation has also been measured using the haptoglobin reaction, since haptoglobin binds only to hemoglobin dimers. The haptoglobin experiments give results that are qualitatively in agreement with the conclusions reached by ultracentrifuge measurements. Similar results are also obtained by estimating the degree of dissociation on the basis of the material which aggregates following mixing with dithionite. The effect of IHP on azide-binding kinetics with methemoglobin has also been examined. Changes in reactivity is observed upon addition of IHP, but the principal effect is observed upon addition of IHP, but the principal effect is an enhancement of the rate of reaction of the beta chains. Changes in the reactivity of the beta93 sulfhydryl group of methemoglobin also accompany addition of IHP, but in a manner which is largely independent of the spin state of the iron. Similar changes are again found with CO-hemoglobin upon addition of IHP. The rate of binding of bromthymol blue also shows some changes upon addition of IHP, but the changes are more pronounced for deoxyhemoglobin than for methemoglobin. Since the results obtained did not appear to indicate a significant role for spin state in the changes observed, additional studies were undertaken using EPR spectroscopy.

Azides↗
PubMed1978

Mechanism of methemoglobin reduction by ascorbic acid under anaerobic conditions.

The time course of methemoglobin reduction by ascorbic acid under anaerobic conditions was analyzed by using isoelectric focusing on Ampholine plate gel in order to compare results obtained by studies of the changes in absorption during the reaction. The intermediate hemoglobin which appeared all through the reaction was single and identified as the alpha3+beta2+ valency hybrid. In the presence of inositol hexaphosphate, reduction of methemoglobin was considerably accelerated and this acceleration was restricted to the step in which methemoglobin is reduced to the alpha3+beta2+ valency hybrid. The phase containing alpha3+beta2+ valency hybrid reduction to deoxyhemoglobin was not affected by the presence of this organic phosphate. The reaction rate constant of each phase was estimated by the analysis of the changes in three components such as methemoglobin, alpha3+beta2+ valency hybrid, and deoxyhemoglobin. The specific attack of beta-methemoglobin chains in methemoglobin tetramer by ascorbic acid was explained by the functional differences in alpha- and beta-methemoglobin chains in the protein.

Anaerobiosis↗
PubMed1978

Characterization of intermediate hemoglobin produced during methemoglobin reduction by ascorbic acid.

Methemoglobin reduction by ascorbic acid was found apparently to cease halfway without further reduction. Studies by isoelectric focusing on Ampholine plate gel revealed that the solutions of the halfway reduced methemoglobin are composed of about 6% oxyhemoglobin, 59% intermediate hemoglobin, and 35% methemoglobin. The intermediate hemoglobin was isolated by CM Sephadex C-50 column chromatography and identified as alpha3+beta2+ valency hybrid by studies using the pattern of isoelectric focusing of p-chloromercuribenzoate-treated intermediate hemoglobin on Ampholine plate gel, absorption spectra, and difference spectra induced by the addition of inositol hexaphosphate in comparison with the reconstituted valency hybrids, alpha3+beta2+ and alpha2+beta3+. Essentially no alpha2+beta3+ valency hybrid was included in the intermediate hemoglobin solutions. These results suggest that methemoglobin reduction by ascorbic acid is mainly initiated by the attack of beta-methemoglobin chains accompanied by the following scheme. Methemoglobin leads to alpha3+beta2+ valency hybrid leads to oxyhemoglobin. The course of methemoglobin reduction by ascorbic acid through alpha2+beta3+ is likely to be small.

Ascorbic Acid↗
PubMed1990

[Permeability of 5 methemoglobin formers through red cell membrane].

By measuring the methemoglobin formation, the permeabilities of some cyanide antidotes passing through mouse erythrocyte membrane were studied. K3Fe(CN)6(0.1 mol/L) did not permeate the red cell and no methemoglobin formed. To the red cell suspension, adding PAPP 0.07 mmol/L, an useful cyanide antidote, no methemoglobin was found. On the contrary, PHAPP, the metabolite of PAPP, transported into the cell readily and reacted with hemoglobin to form methemoglobin quickly. DMAP and NaNO2 passed through the red cell membrane easily. With comparable amount of methemoglobin formation, the concentration of NaNO2 was about 200 times as much as that of DMAP. A comparison of the anticyanide potency of DMAP and NaNO2, the permeability rate constant, the half time and activation energy were measured as: 0.217 and 0.0506/min; 3.2 and 13.7 min; 17.1 and 50.2 kJ/mol, respectively. Owing to its ready permeability and formation of methemoglobin, DMAP is a better antidote than NaNO2 against cyanide poisoning.

Aminophenols↗
PubMed1977

[On the mechanism of ascorbic acid induced methemoglobin reduction of human erythrocytes (author's transl)].

Ascorbic acid and dehydroascorbic acid penetrate the human erythrocyte membrane. In vitro methemoglobin is reduced nonenzymatically by both substances in concentrations of 10(-2) M to 10(-3) M. Dehydroascorbic acid is reduced nonenzymatically to ascorbic acid by GSH, even with low GSH-content of erythrocytes. Under physiological conditions ascorbic acid induced methemoglobin reduction is far less important than reduction by the NADH dependent methemoglobin reductase system. In methemoglobinemic conditions caused by toxic effects or by congenital methemoglobin reductase deficiency treatment with ascorbic acid is possible. However, critically increased methemoglobin content of the blood higher than 30% makes therapy with methylene blue necessary.

Ascorbic Acid↗