Transformation of hemoglobin a into methemoglobin by sesamol

Sesamol (3,4-methylenedioxyphenol), a monophenolic antioxidant in sesame iol, produced methemoglobin from hemoglobin A (oxyhemoglobin and deoxyhemoglobin) and from red cells. The activity of the compound was more extensive than the polyphenolic compounds. The profiles of the methemoglobin formation by the compound were compared with those by nitrite and hydroxylamine. The formation of methemoglobin from oxyhemoglobin by the compound was rather slowly progressed, but the amount of methemoglobin formed was proportional to the concentration of oxyhemoglobin even when the concentration of the compound was low. The sesamol-induced methemoglobin formation was influenced by inositol hexaphosphate, an allosteric effector of hemoglobin. Thus, the phosphate enhanced the transformation of oxyhemoglobin and inhibited the transformation of deoxyhemoglobin.     

Thermal stability and functional properties of human hemoglobin in the presence of aliphatic alcohols]

Differential scanning microcalorimetry was used to study thermal stability of the ferro- and ferriforms of hemoglobin at pH 7.4 in phosphate buffer and in buffer mixtures of methanol, ethanol, 1-propanol. Denaturation of the human hemoglobin molecule composed of four subunits was cooperative transition. The thermostability of the hemoglobin forms decreased in the order of carboxyhemoglobin (TD = 82.0 degrees C) > oxyhemoglobin (71.0 degrees C) > methemoglobin (67.0 degrees C). The aliphatic alcohols as cosolvents decreased the hemoglobin stability because of loosening the structure of the globin moiety by disturbing its hydrophobic contacts and hydrogen bonds. These alcohols reduced the oxygen affinity for hemoglobin probably due to perturbation of the R<-->T equilibrium by the decreased bulk dielectric constant of the solvent. Oxyhemoglobin and methemoglobin was converted to hemichrome by high alcohol concentrations.     

Interaction of sodium dodecyl sulfate with human native and cross-linked hemoglobins: a transient kinetic study

The interaction of sodium dodecyl sulfate (SDS) at a concentration range (0-515 microM) below the critical micelle concentration (CMC approximately 0.83 mM) with human native and cross-linked oxyhemoglobin (oxyHb) and methemoglobin (metHb) has been investigated by optical spectroscopy and stopped-flow transient kinetic measurements. It is observed that the interaction of SDS with human native and cross-linked oxyHb shows the disappearance of the bands of oxyHb at 541 and 576 nm and the appearance at 537 nm. The resultant spectra are characteristic of low spin (Fe(3+)) hemichrome. Similarly SDS has been found to convert human native and cross-linked high spin (Fe(3+)) metHb to low spin (Fe(3+)) hemichrome. The interaction of SDS with oxyHb suggests a conformational change of the protein in the heme pocket, which may induce the binding of distal histidine to iron leading to the formation of superoxide radical. The formation of hemichrome from metHb is found to be concentration-dependent with SDS. The stopped flow transient kinetic measurements of the interaction of SDS with metHb show that at least four molecules of SDS interact with one molecule of metHb. The interaction of SDS with human cross-linked oxy and met hemoglobin shows results similar to those for human native oxy and met hemoglobin indicating that the covalent modification does not alter the interaction of SDS with cross-linked hemoglobin.     

Conversion of hemoglobin to hemichrome by lysophospholipids.

The interaction of lysophospholipids with human oxy- and methemoglobin was studied. Anionic (acidic) lysophospholipids (lysophosphatidylserine, lysophosphatidylinositol, lysophosphatidylethanol, and lysophosphatidic acid) are potent effectors inducing the conversion of both forms of hemoglobin into hemichrome. Zwitterionic lysophospholipids (lysophosphatidylcholine, lysophosphatidylethanolamine, and lysosphingomyelin) did not influence oxyhemoglobin conversion, whereas methemoglobin conversion into hemichrome required much higher concentrations of these lysophospholipids compared to anionic lysophospholipids. Neutralization of negative charge on phosphate group of acidic lysophospholipids by Ca2+ was accompanied by partial or complete loss of their effector properties. The process of hemoglobin conversion to hemichrome is characterized by two isobestic points in the absorption spectra, indicating lack of stable intermediates. The present results are discussed in terms of the biological sense of the asymmetric distribution of phospholipids in the erythrocyte membrane.     

Hemoglobin conversion to hemichrome under the influence of fatty acids

The effect of free fatty acids on the process of hemoglobin conversion and lipid peroxidation has been studied in model systems and erythrocytes. It has been found that methemoglobin and oxyhemoglobin are converted to the low spin oxidized form, namely, reversible hemichrome under the action of fatty acids. In the case of oxyhemoglobin, an increase in the level of active oxygen forms is observed in the system which initiates the formation of primary and secondary lipid peroxidation products. Incubation of erythrocytes with free fatty acids causes the formation of Heinz bodies and is accompanied by an increase of the lipid peroxidation level.     

Induced hemichrome formation of methemoglobins A,S and F by fatty acids,alkyl ureas and urea

• 1.1. Spectral analysis of the Soret region (450-350 nm) has shown that saturated fatty acids, alkyl ureas and urea induce the conversion of methemoglobins A, S, and F to the hemichrome state.
• 2.2. In the presence of fatty acids (C8-C16), methemoglobin F is converted to the hemichrome state more readily than either methemoglobins A or S.
• 3.3. Using several alkyl ureas (methyl, ethyl, propyl, butyl), the extent of hemichrome formation was as follows: met Hb F > met Hb S > met Hb A. The ability of these compounds to induce hemichrome formation is related to their increasing hydrophobicity.
• 4.4. Conversion to the hemichrome state in the presence of urea (5M) led to the formation of molecular aggregates of hemoglobins S, F and A which may be initated by subunit dissociation and conformational changes, coupled to increased globin-globin interactions.
• 5.5. Similar aggregation occurred for methemoglobin S in the presence of octanoic acid; no significant aggregation was evident for methemoglobin A after 10 hr of exposure to octanoic acid.

     

Effector role of fatty acids in the transformation of methemoglobin into hemichrome

The interaction of fatty acids (FA) with methemoglobin (met-Hb) was investigated. It was found that under the action of FA met-Hb is rapidly converted into a low-spin form (the so-called hemichrome). This process is reversible as proved by the addition of bovine serum albumin into the system. Using differential spectrophotometry, the efficiency of FA action on met-Hb depending on concentration and nature of FA was estimated. Using the circular dichroism (CD) method, it was demonstrated that under the action of FA the concentration of R-conformation in the met-Hb molecule is increased. The stoichiometry of the hemichrome complex with FA at various FA concentrations was assessed. A comparative analysis of the effect of various detergents with known structures on met-Hb was carried out. The mechanism of met-Hb conversion into hemichrome is discussed.     

The stability of the heme-globin linkage in some normal, mutant, and chemically modified hemoglobins.

The rates and equilibria of heme exchange between methemoglobin and serum albumin were measured using a simple new spectrophotometric method. It is based on the large difference between the spectrum of methemoglobin and that of methemealbumin at pH 8-9. The rate of heme exchange was found to be independent of the albumin concentration and inversely proportional to the hemoglobin (Hb) concentration. Taken together with the finding that the rate was 10 times greater for Hb Rothschild, which is completely dissociated into alpha beta dimers and 10 times smaller for two cross-linked hemoglobins, the subunits of which cannot dissociate, this showed that the rate of dissociation of heme from alpha beta dimers is very much greater than from tetramers. Conditions were found for the attainment of an equilibrium distribution of hemes between beta globin and albumin. The equilibrium distribution ratio, R = methemealbumin/albumin/methemoglobin/apohemoglobin, for hemoglobin A was 3.4 with human and 0.005 with bovine serum albumin. Both the rates of exchange and the R values of HbS and HbF were the same as that for HbA. The equilibrium distribution ratio for Hb Rothschild was 7 times greater than that for HbA whereas that of one but not the other of the cross-linked hemoglobins was 10 times smaller. The structural bases for these differences are analyzed.     

Green hemoprotein of erythrocytes: methemoglobin superoxide transferase

Influences of base (pH 10), heat (50 degrees C), microwave radiation (2450 MHz, 103 +/- 4 W/kg), and hydrogen peroxide (5.6 mM) generated by glucose oxidase on oxidation of human oxyhemoglobin to methemoglobin were examined. Conversion of oxyhemoglobin to methemoglobin was followed by the difference in absorbancy of 540 or 542 nm and 576 nm wavelength light versus time. Fresh basic hemolysates auto-oxidized on heating with a zero order rate constant, implying that hemoglobin or another protein saturated with oxyhemoglobin catalyzed the oxidation. Simultaneous microwave irradiation inhibited thermally induced auto-oxidation on the average by 28.6%. However, there was great variability among samples and a decrease in auto-oxidation with aging of individual samples. The auto-oxidation rate was independent of initial oxyhemoglobin concentration. Oxidation of partially purified oxyhemoglobin by hydrogen peroxide was not influenced by microwave irradiation. Adding green hemoprotein isolated from human erythrocytes to the oxyhemoglobin/glucose oxidase reaction mixture yielded absorption spectra (500-600 nm) that were a combination of oxyhemoglobin, deoxyhemoglobin, and methemoglobin spectra. Green hemoprotein was labile in hemolysates but stable in a partially purified ferric form. These results imply that thermally unstable reduced green hemoprotein can reverse oxidation of oxyhemoglobin by hydrogen peroxide and could mediate the thermally induced and microwave inhibited auto-oxidation of oxyhemoglobin.     

Tyrosine residues play an important role in heme detoxification by serum albumin

Background

Serum albumin binds avidly to heme to form heme–serum albumin complex, also called methemalbumin, and this binding is thought to protect against the potentially toxic effects of heme. However, the mechanism of detoxification has not been fully elucidated.

Methods

SDS-PAGE and Western blot were used to determine the efficiency of methemalbumin on catalyzing protein carbonylation and nitration. HPLC was used to test the formation of heme to protein cross-linked methemalbumin.

Results

The peroxidase activity of heme increased upon human serum albumin (HSA) binding. Methemalbumin showed higher efficiency in catalyzing tyrosine oxidation than free heme in the presence of H₂O₂. Methemalbumin catalyzed self-nitration and significantly promoted the nitration of tyrosine in coexistent protein, but decreased the carbonylation of coexistent protein compared with heme. The heme to protein cross-linked form of methemalbumin suggested that HSA trapped the free radical accompanied by the formation of ferryl heme. When tyrosine residues in HSA were modified by iodination, HSA lost of protection effect on protein carbonylation. The low concentration of glutathione could effectively inhibit tyrosine nitration, but had no effect on protein carbonylation.

Conclusion

HSA protects against the toxic effect of heme by transferring the free radical to tyrosine residues in HSA, therefore protecting surrounding proteins from irreversible oxidation, rather than by direct inhibiting the peroxidase activity. The increased tyrosine radicals can be reduced by endogenic antioxidants such as GSH.

General significance

This investigation indicated the important role of tyrosine residues in heme detoxification by HSA and suggested a possible novel mechanism.     

Hydration-dependent conformational states of hemoglobin. Equilibrium and kinetic behavior

The equilibrium and kinetics of methemoglobin conversion to hemichrome induced by dehydration were investigated by visible absorption spectroscopy. Below about 0.20 g water per g hemoglobin only hemichrome was present in the sample; above this value, an increasing proportion of methemoglobin appeared with the increase in hydration. The transition between the two derivatives showed a time-dependent biphasic behavior and was observed to be reversible. The rates obtained for the transition of methemoglobin to hemichrome were 0.31 and 1.93 min-1 and for hemichrome to methemoglobin 0.05 and 0.47 min-1. We suggest that hemichrome is a reversible conformational state of hemoglobin and that the two rates observed for the transition between the two derivatives reflect the alpha- and beta-chains of hemoglobin.     

Stoichiometry of the reaction of oxyhemoglobin with nitrite.

During the reaction of oxyhemoglobin (HbO2) with nitrite, the concentration of residual nitrite, nitrate, oxygen, and methemoglobin (Hb+) was determined successively. The results obtained at various pH values indicate the following stoichiometry for the overall reaction: 4HbO2 + 4NO2- 4H+ leads to 4Hb+ + 4NO3- + O2 + 2H2 O (Hb denotes hemoglobin monomer). NO2- binds with methemoglobin noncooperatively with a binding constant of 340 M-1 at pH 7.4 and 25 degrees C. Thus, the major part of Hb+ produced is aquomethemoglobin, not methemoglobin nitrite, when less than 2 equivalents of nitrite is used for the oxidation.     

Inhibition of human lymphocyte ferrochelatase activity by hemin

Ferrochelatase activity in human lymphocytes was found to be 50% inhibited by 10.5 microM hemin under maximal velocity conditions. The inhibition was not prevented by dithiothreitol or glutathione, suggesting that the hemin was not interacting with the sulphydryl groups of ferrochelatase. Human serum albumin, but not bovine serum albumin was able to prevent the inhibition consistent with the known formation of the tightly bound methemalbumin complex with human albumin. Kinetic studies performed under initial velocity conditions with hemin concentrations ranging from 2 to 8 microM revealed the inhibition to be non-competitive with respect to the metal substrate (zinc) and competitive with respect to the porphyrin substrate (mesoporphyrin). The kinetic analysis indicated that hemin binds to both the enzyme and enzyme-metal complex at a site normally occupied by the porphyrin substrate, and a second molecule of hemin could bind to the enzyme-metal complex but with a much lower affinity than the first molecule. We conclude that the product inhibition of ferrochelatase by hemin should be considered as a possible site of regulation of heme biosynthesis.     

Mechanical stability of hemoglobin subunits: an abnormality in betaS-subunits of sickle hemoglobin

In order to study the mechanism of the ease of precipitation of oxyhemoglobin S by mechanical shaking, the rates of precipitation of α- and β-subunits of oxyhemoglobin A and oxyhemoglobin S were compared. At pH 8.0, the α^A-subunits precipitated rapidly, while the β^A-subunits were very stable, although a part of β^A-bunits converted to the hemichrome form. At pH 6.0, the β^A-subunits precipitated rapidly while the α^A-subunits were stable. Similar studies with β^S-subunits showed that β^S-subunits precipitated rapidly both at acidic and alkaline pHs. The abnormal precipitation of tetrameric oxyhemoglobin S during mechanical shaking may be due to this instability of the β^S-subunits.     

Prevention by antioxidants of the hemolysis of erythrocytes of cattle, pigs and humans treated with t-butyl hydroperoxide

Urate, 3-ribosylurate, ascorbate, glutathione and plasma protected bovine, porcine and human erythrocytes from hemolysis caused by t-butyl hydroperoxide (t-BHP). Urate partially protected porcine erythrocytes from hemolysis by t-BHP when it was added 15 min after the addition of the t-BHP, but it did not protect when added 30 min after the t-BHP. Glutathione and ascorbate protected oxyhemoglobin from oxidation to methemoglobin by t-BHP; 3-ribosylurate gave only slight protection. Urate stimulated the formation of methemoglobin from oxyhemoglobin during treatment with t-BHP.     

Inhibition by superoxide dismutase of methemoglobin formation from oxyhemoglobin.

The formation of methemoglobin from oxyhemoglobin in a solution containing photoreduced riboflavin and oxygen was inhibited by superoxide dismutase. The rate of the reaction was pH-dependent in the range of 6.8 to 7.8, increasing as the pH was reduced. Inhibition by superoxide dismutase was enhanced as the EDTA concentration increased and was dependent on enzymatic activity. Under conditions in which superoxide dismutase inhibition was incomplete, catalase inhibited the reaction but mannitol had no effect. The data support the mediation of methemoglobin formation by superoxide. The hypothesis is offered that superoxide anion reduced the heme-bound oxygen in oxygemoglobin by one electron, permitting the subsequent dissociation of ferrihemoglobin and peroxide. The ability of superoxide dismutase to inhibit the formation of methemoglobin may represent one of its functions in the mature erythrocyte.     

The oxidative effect of bacterial lipopolysaccharide on native and cross-linked human hemoglobin as a function of the structure of the lipopolysaccharide.

The binding of lipopolysaccharide (LPS, also known as bacterial endotoxin) to human hemoglobin is known to result in oxidation of hemoglobin to methemoglobin and hemichrome. We have investigated the effects of the LPSs from smooth and rough Escherichia coli and Salmonella minnesota on the rate of oxidation of native oxyhemoglobin A0 and hemoglobin cross-linked between the alpha-99 lysines. For cross-linked hemoglobin, both smooth LPSs produced a rate of oxidation faster than the corresponding rough LPSs, indicating the importance of the binding of LPS to the hemoglobin. The effect of the LPS appeared to be largely on the initial fast phase of the oxidation reaction, suggesting modification of the heme pocket of the alpha chains. For hemoglobin A0, the rates of oxidation produced by rough and smooth LPSs were very similar, suggesting the possibility that the effect of the LPSs was to cause dissociation of hemoglobin into dimers. The participation of cupric ion in the oxidation process was demonstrated in most cases. In contrast, the rate of oxidation of cross-linked hemoglobin by the LPSs of both the rough and smooth E. coli was not affected by the presence of chelators, suggesting that cupric ion had previously bound to these LPSs. Overall, these data suggest that the physiological effectiveness of hemoglobin solutions now being developed for clinical use may be decreased by the presence of lipopolysaccharide in the circulation of recipients.     

A pH-dependent aquomet-to-hemichrome transition in crystalline horse methemoglobin

In 1947, Perutz and co-workers reported that crystalline horse methemoglobin undergoes a large lattice transition as the pH is decreased from 7.1 to 5.4. We have determined the pH 7.1 and 5.4 crystal structures of horse methemoglobin at 1.6 and 2.1 A resolution, respectively, and find that this lattice transition involves a 23 A translation of adjacent hemoglobin tetramers as well as changes in alpha heme ligation and the tertiary structure of the alpha subunits. Specifically, when the pH is lowered from 7.1 to 5.4, the Fe(3+) alpha heme groups (but not the beta heme groups) are converted from the aquomet form, in which the proximal histidine [His87(F8)alpha] and a water molecule are the axial heme ligands, to the hemichrome (bishistidine) form, in which the proximal histidine and the distal histidine [His58(E7)alpha] are the axial heme ligands. Hemichrome formation is coupled to a large tertiary structure transition in the eight-residue segment Pro44(CD2)alpha-Gly51(D7)alpha that converts from an extended loop structure at pH 7.1 to a pi-like helix at pH 5.4. The formation of the pi helix forces Phe46(CD4)alpha out of the alpha heme pocket and into the interface between adjacent hemoglobin tetramers where it participates in crystal lattice contacts unique to the pH 5.4 structure. In addition, the transition from aquomet alpha subunits to bishistidine alpha subunits is accompanied by an approximately 1.2 A movement of the alpha heme groups to a more solvent-exposed position as well as the creation of a solvent channel from the interior of the alpha heme pocket to the outside of the tetramer. These changes and the extensive rearrangement of the crystal lattice structure allow the alpha heme group of one tetramer to make direct contact with an alpha heme group on an adjacent tetramer. These results suggest possible functional roles for hemichrome formation in vivo.