Hemichrome Formation Research Articles

Hemoglobin degradation in human erythrocytes with long-duration near-infrared laser exposure in Raman optical tweezers

Near-infrared laser (785-nm)-excited Raman spectra from a red blood cell, optically trapped using the same laser beam, show significant changes as a function of trapping duration even at trapping power level of a few milliwatts. These changes in the Raman spectra and the bright-field images of the trapped cell, which show a gradual accumulation of the cell mass at the trap focus, suggest photoinduced aggregation of intracellular heme. The possible role of photoinduced protein denaturation and hemichrome formation in the observed aggregation of heme is discussed.

Plasma Protein Oxidation and Triglyceride Levels in Sickle Cell Anemia.

Abstract 2578Poster Board II-555 Introduction:A mutant and unstable hemoglobin is characteristic in sickle cell anemia (SCA) with an increased formation of methemoglobin, hemichrome and haemin. Increased oxidative stress and altered lipid balance in the plasmas of SCA patients have been documented previously. Lower total plasma cholesterol (TC) and higher-than-avarage triglyceride (TG) levels have also been reported in SCA patients compared to healthy controls. Plasma phospholipids (PL) were normal or increased according to various studies. However, to our knowledge only a few reports documented the plasma protein oxidation in SCA patients. In this study protein oxidation, lipid levels, bilurubin levels and their probable correlation were investigated in the plasmas of SCA patients, carriers and healthy controls. Patient and Methods:15 SCA patients, 10 carriers and 10 healthy controls were included in the study. The patients had not been transfused and hospitalized and had no crisis for the last three months. The oxidation status of plasma and its proteins were detirmined by total sulfhydryl (T-SH) and protein carbonly levels respectively. TC, TG and bilurubin levels were detirmined in the plasma and PL levels were detirmined in the lipid extract from plasma. Results:SCA patients had lower T-SH levels compared to controls (p=0.003) and higher protein carbonyl levels compared to carriers (p=0.001) and healthy children (p=0.032). T-SH values were 417.41±77.90 μM, 457.46±106.26 μM, 522.59±77.18 μM and protein carbonyl levels were 0.93±0.15 nmol/mg protein, 0.74±0.08 nmol/mg protein , 0.82±0.05 nmol/mg protein for patients, carriers and controls respectively. Significantly lower mean TC levels than controls (p<0.0001) and higher TG levels than carriers (p=0.001) were observed in SCA patients. TC levels were 116.67±23.60, 127.53±22.40, 151.62±16.53 and TG levels were 89.31±23.50 mg/dl, 60.94±9.18 mg/dl, 82.96±21.06 mg/dl for patients, carriers and controls respectively. Phospholipid levels were not different between the groups. TG levels were positively correlated to protein carbonyl levels (p=0.002) and to bilirubin levels (p=0.033). Bilurubin levels and protein carbonyl levels are also positively correlated (p<0,0001). Conclusions:Alteration of lipid balance besides protein oxidation and hemolysis were observed in the plasmas of SCA patients compared to the carriers and healthy group. The positive correlation between total bilurubin, TG and carbonyl levels indicates that oxidative stress may affect the SCA plasma extensively, ie. lipids, proteins and erythrocytes are all affected. Disclosures:No relevant conflicts of interest to declare.

Cysteine 93 of Hemoglobin Beta Chain Is the Major Target of Oxidation During Red Blood Cell Storage.

Abstract 4040Poster Board III-976Red blood cells (RBC) stored for prolonged time periods under blood banking conditions are known to accumulate irreversible damage that ultimately affects their viability and function. Among the consequences of this RBC storage lesion are enhanced hemolysis and oxidative stress after transfusion. Stored RBCs accumulate reactive oxygen species (ROS) and surface-associated hemichrome, a partially denatured form of oxidized hemoglobin. In this work, we examined whether the ROS created during RBC storage lead to characteristic oxidative modifications to hemoglobin (Hb) and RBC membrane proteins.To characterize Hb modifications leading to hemichrome formation, we oxidized human hemoglobin A with varied concentrations of hydrogen peroxide (H2O2), an oxidant relevant to RBC storage. The reaction was quenched with excess methionine and the protein digested with trypsin. The resulting peptides were analyzed by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Methionine and cysteine residues are the major targets of oxidation. Oxidation of methionine to methionine sulfoxide and cysteine to cysteine sulfonic/sulfinic acids is accompanied by a mass shift related to the covalent modifications of each residue, which are readily detected by LC-ESI-MS/MS. We found that HBβ-Cys-93 was oxidized to a maximum of approximately 70% after treatment with 200 αM H2O2. This cysteine occurs adjacent to the FeII-coordinating His-92 and is thus exposed to the heme-FeII center. In contrast, HBβ-Cys-112 and HBα-Cys-104, which are buried and located away from the heme-FeII center, were oxidized to only 2% and 0%, respectively. The enhanced oxidation of the heme-FeII proximal HBβ-Cys-93 suggests that ROS are generated through a FeII-dependent mechanism such as Fenton's reaction. Oxidation of each of the three Hb methionines reached 20% upon treatment with 200 μM H2O2. Among these Met residues, HBα-Met-32 is located near the heme-FeII, center, while both Hbα-Met-76 and HBβ-Met-55 are near the protein/solvent interface. Thus, Cys and Met oxidations in Hb appear to be dependent on proximity to either the heme-FeII center or the protein/solvent interface.To test whether these Hb residues are also sensitive to oxidation during storage we acquired 3 RBC units stored for 3 days and 3 RBC units stored for 45 days, from the Puget Sound Blood Center. The cytosolic Hb was extracted by hypotonic lysis and digested with trypsin prior to LC-ESI-MS/MS analysis. We observed that oxidation of HBβ-Cys-93 was dramatically increased from 10% on day 3 to 29% on day 45 (p < 0.02). HBα-Met-32 was also oxidized to a small extent (5% on day 3 to 7.5 % on day 45, p<0.04). The two buried Cys residues were unoxidized, whereas two surface proximal Met residues were oxidized, but not in a significantly storage-time-dependent manner. These data implicate the redox active heme-FeII center in enhanced ROS production and oxidative stress in stored RBCs.Our observations suggest that HBβ-Cys-93 oxidation is a specific and sensitive marker for oxidative stress during storage. Moreover, we have correlated these progressive oxidations with increases in microvesiculation and hemolysis, storage-time-dependent changes that have been well documented. Future studies will evaluate the storage-time-dependence in other oxidative and post-translational modifications to RBC membrane proteins and correlate these findings with functional consequences such as microvesiculation and hemolysis. Disclosures:No relevant conflicts of interest to declare.

Oxidative process in erythrocytes of individuals with hemoglobin S

The understanding of the oxidative stress mechanisms helps to explain many of the processes of cellular lesion and death, especially those related to the hemolytic diseases. Sickle cell anemia, thalassemias and G6-PD deficiency are among the more frequent genetic anomalies accompanied by oxidative stress. In the sickle cells, one of the factors that predisposes to the hemolytic process is the oxidative degradation of the hemoglobin S due to its deoxigenation leading to hemichrome formation and precipitation as Heinz bodies. The oxidative stress contributes to the sickle process and shortening of the erythrocyte survival. Here we analyzed the oxidative process in erythrocytes of patients with two different genotypes for HbS (AS and SS). Units of blood from donors of the Center of Hematology and Hemotherapy of Paraná (HEMEPAR), from normal individuals (AA) and from heterozygote individuals (AS), and venous blood collected from patients with sickle cell anemia (SS) were analyzed. In order to evaluate the protective action of the vitamins C and E in oxidative stress, erythrocytes were treated with antioxidant substances, vitamin C and vitamin E, and then treated with the oxidant tert-butilhydroperoxide (TBHP). The oxidative action induced by TBHP was observed in erythrocytes AA<AS<SS, by the increase in the content of Heinz bodies, methemoglobin, hemolysis, GSH depletion and lowering activities of the enzymes G6-PD and GR. The protective actions of the vitamins C and E for the oxidative stress induced by TBHP were observed for the erythrocytes in the lowering Heinz bodies, methemoglobin, hemolysis, and partial recovery of GSH more efficiently in AS and SS erythrocytes. Recovery was not observed in the levels of the activities of the enzymes G6-PD and GR, under the vitamins actions. The results obtained confirmed the higher susceptibility of the sickle erythrocyte to oxidation which necessitates precaution in the transfusion adequacy of AS erythrocytes. On the other hand, the protective effect of the vitamins C and E over the oxidative stress observed in erythrocytes AS and SS open perspectives for their use for treatment of patients with sickle cell anemias, as well as in the preservation of transfusional erythrocyte bag units.

Structural Characterization of Ferric Hemoglobins from Three Antarctic Fish Species of the Suborder Notothenioidei

Spontaneous autoxidation of tetrameric Hbs leads to the formation of Fe (III) forms, whose physiological role is not fully understood. Here we report structural characterization by EPR of the oxidized states of tetrameric Hbs isolated from the Antarctic fish species Trematomus bernacchii, Trematomus newnesi, and Gymnodraco acuticeps, as well as the x-ray crystal structure of oxidized Trematomus bernacchii Hb, redetermined at high resolution. The oxidation of these Hbs leads to formation of states that were not usually detected in previous analyses of tetrameric Hbs. In addition to the commonly found aquo-met and hydroxy-met species, EPR analyses show that two distinct hemichromes coexist at physiological pH, referred to as hemichromes I and II, respectively. Together with the high-resolution crystal structure (1.5 Å) of T. bernacchii and a survey of data available for other heme proteins, hemichrome I was assigned by x-ray crystallography and by EPR as a bis-His complex with a distorted geometry, whereas hemichrome II is a less constrained (cytochrome b 5-like) bis-His complex. In four of the five Antartic fish Hbs examined, hemichrome I is the major form. EPR shows that for HbCTn, the amount of hemichrome I is substantially reduced. In addition, the concomitant presence of a penta-coordinated high-spin Fe (III) species, to our knowledge never reported before for a wild-type tetrameric Hb, was detected. A molecular modeling investigation demonstrates that the presence of the bulkier Ile in position 67 β in HbCTn in place of Val as in the other four Hbs impairs the formation of hemichrome I, thus favoring the formation of the ferric penta-coordinated species. Altogether the data show that ferric states commonly associated with monomeric and dimeric Hbs are also found in tetrameric Hbs.

The interaction between hemoglobin and two surfactants with different charges

The interactions of hemoglobin (Hb) with sodium dodecyl sulfate (SDS) and dodecyl trimethylammonium bromide (DTAB) are investigated by several methods. We observed the formation of hemichrome below the critical micelle concentration (cmc) of surfactant and the release of heme from Hb above the cmc. When pH value of Hb/surfactant system is lower than isoelectric point (p I) of Hb, the interaction of SDS with Hb is both electrostatic and hydrophobic, while the interaction of DTAB with Hb is hydrophobic mainly. On the contrary, when pH > p I, the interaction of SDS with Hb is hydrophobic mainly, while the interaction of DTAB with Hb is both electrostatic and hydrophobic. In the case where both the electrostatic interaction and hydrophobic interaction exist, the electrostatic interaction plays a more important role. Thus, SDS tends to interact with Hb more obviously than DTAB does when pH < p I and the interaction between DTAB and Hb is stronger when pH > p I.

Sugar-derived Glasses Support Thermal and Photo-initiated Electron Transfer Processes over Macroscopic Distances

Trehalose-derived glasses are shown to support long range electron transfer reactions between spatially well separated donors and protein acceptors. The results indicate that these matrices can be used not only to greatly stabilize protein structures but also to facilitate both thermal and photo-initiated hemeprotein reduction over large macroscopic distances. To date the promise of exciting new protein-based technologies that can harness the exceptional tunability of protein functionality has been significantly thwarted by both intrinsic instability and stringent solvent/environment requirements for the expression of functional properties. The presented results raise the prospect of overcoming these limitations with respect to incorporating redox active proteins into solid state devices such as tunable batteries, switches, and solar cells. The findings also have implications for formulations intended to enhance long term storage of biomaterials, new protein-based synthetic strategies, and biophysical studies of functional intermediates trapped under nonequilibrium conditions. In addition, the study shows that certain sugars such as glucose or tagatose, when added to redox-inactive glassy matrices, can be used as a source of thermal electrons that can be harvested by suitable redox active proteins, raising the prospect of using common sugars as an electron source in solid state thermal fuel cells.

Mechanisms of band 3 oxidation and clustering in the phagocytosis of Plasmodium falciparum-infected erythrocytes

Erythrocytes (RBCs) opsonized by IgG and complement are prevalently recognized and phagocytosed by complement receptor CR1. This mechanism, effective in senescent and damaged RBCs seems to be operative in ring-parasitized RBCs, since infection by Plasmodium falciparum induces stage-dependent binding of auto-antibodies and activated C3 to the RBC membrane. Later, parasite forms are also recognized by non-opsonic receptors, such as scavenger receptor CD36. Malaria parasites induce the oxidative formation of hemichromes which are the trigger for the auto-antigen development. Band 3 protein is the most plausible candidate of the RBC auto-antigen, induced by hemichromes. Auto-antigens isolated from trophozoites were found only in a high-molecular-weight protein aggregates not present in the normal RBC. The immunocomplex was purified by protein-A affinity chromatography, purified proteins digested by trypsin and analyzed by MALDI-TOF. Peptide mapping showed that the main antigen consisted of band 3 protein aggregates that also contained hemichromes, IgGs, complement factor 3 (C3), and traces of spectrin and glycophorin but no parasite proteins. Two cysteines located in the band 3 cytoplasmic domain were found to be particularly reactive to oxidants and mediated band 3 covalent dimerization via disulfide bonds. Thus, parasites promote oxidative alterations in the membrane of the host which lead to exposure of antigenic sites recognized by anti-band 3 auto-antibodies. Formation of band 3 clusters appears to be mediated by cytoplasmic binding of hemichromes and also by direct band 3 oxidation, whereby clustered, oxidized and antigenic band 3 was underglycosylated.

Hemichrome formation observed in human haemoglobin A under various buffer conditions.

To observe hemichrome formation in human haemoglobin A under various buffer conditions. Hemichrome formation of human oxyhaemoglobin A (HbO2) was studied spectrophotometrically in 0.1 m buffer at various temperatures and pH values. Following autoxidation in ferrous HbO2, it was evident that formation of hemichrome, which tends to precipitate, occurred at various stages during the course of the autoxidation reaction namely at initial, intermediate or final stages, depending on temperature and pH of the solution. By varying temperature of the solution from 35 to 55 degrees C and pH from 4.5 to 10.5, it is shown here that HbO2 exhibits high susceptibility for hemichrome formation and its occurrence is a function of pH, temperature and progress of autoxidation of HbO2. Unlike HbO2 and its separated haemoglobin chains, monomeric bovine heart myoglobin (MbO2) did not easily form hemichrome. These findings provide a clue on the crucial role of haemoglobin molecule for senescent cell recognition or homeostasis in the blood circulation.

THE BENEFICIAL EFFECT OF THREE FLAVONOLS AGAINST OXIDATIVE DAMAGE

The ability of three related naturally occurring flavonols in inhibiting Hb oxidation and lipid peroxidation of human erythrocyte membranes was evaluated. The flavonols tested exhibited the following order of potency to inhibit tert-butyl hydroperoxide-induced Hb oxidation: quercetin > rutin > morin. The Hb oxidation was estimated by the extent of metHb and hemichrome formation induced by tert-butyl hydroperoxide. Quercetin or rutin (0.5 mM) increased oxyHb levels about 33% and 10%, respectively. Morin (0.5 mM) was pro-oxidant, decreasing the oxyHb about 7%. Despite the pro-oxidant action on Hb oxidation, morin offered greater protection against lipid peroxidation than rutin, preventing the formation of TBA reactive substances by 33.1%, at 0.2 mM. However, quercetin (0.2 mM) provided approximately 50% protection against TBARS formation. The results show that the flavonoids tested are protective for Hb oxidation and lipid peroxidation on erythrocyte membrane subjected to oxidative stress. Quercetin in particular, may be useful in diminishing oxidative damage to red blood cells

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 μM) below the critical micelle concentration (CMC∼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.

The effect of dodecyl trimethylammonium bromide on the formation of methemoglobins and hemichrome

The interaction of n-dodecyl trimethylammonium bromide (DTAB), a cationic surfactant, with human and greylag goose ( Anser anser) hemoglobins (Hbs) has been studied using various methods, such as a potentiometric technique using a DTAB-selective membrane electrode, UV–Vis spectrophotometry in the absence and the presence of 100 mM NaCl, isothermal titration microcalorimetry at buffer condition of 50 mM Tris, pH 7.2 and 1 mM EDTA. Binding isotherms were obtained from the potentiometric technique and using the Wyman binding potential model, apparent binding constants were calculated. Thermodynamic parameters were obtained for two sets of binding sites from the Hill equation. Binding of DTAB molecules induced the unfolding of hemoglobin accompanied with exposure of the heme pocket and facilitating the oxidation of heme. The concentration of metHb increases and oxyHb and deoxyHb decrease due to the interaction of DTAB–Hb complexes. Increasing the temperature and ionic strength, in the presence of DTAB, increases the autoxidation of oxyhemoglobin. The met process is enhanced by electrostatic contributions induced by the initial ionic binding and subsequently the hemichrome is increased by the hydrophobic effect that is induced by a second set of DTAB binding sites.

Quercetin Diminishes the Binding of Hemoglobin to the Red Blood Cell Membrane

Hemoglobin (Hb) oxidation leads to the formation of hemichrome, which binds to the membrane and causes red blood cell removal by the reticuloendothelial system. In the present investigation, the ef...

Contribution of haemoglobin and membrane constituents modification to human erythrocyte damage promoted by peroxyl radicals of different charge and hydrophobicity

We have investigated the influence of the free radical initiator characteristics on red blood cell lipid peroxidation, membrane protein modification, and haemoglobin oxidation. 2,2′-Azobis(2-amidinopropane) (AAPH) and 4,4′-azobis(4-cyanovaleric acid) (ACV) were employed as free radical sources. Both azo-compounds are water-soluble, although ACV presents a lowed hydrophilicity, as evaluated from octanol/water partition constants. At physiological pH, they are a di-cation and a di-anion, respectively.AAPH and ACV readily oxidise purified oxyhemoglobin in a very efficient free radical-mediated process, particularly for ACV-derived radicals, where nearly one heme moiety was modified per radical introduced into the system, suggesting that negatively charged radicals react preferentially at the heme group. The radicals derived from both azo-compounds lead to different oxidation products. Methemoglobin, hemichromes and choleglobin were produced in AAPH-promoted hemoglobin oxidation, while ACV-derived radicals predominantly form hemichromes, with very low production of choleglobin.Red cell damage was evaluated at the level of hemoglobin and membrane constituents modification, and was expressed in terms of free radical doses. Before the onset of the lytic process, ACV leads to more lipid peroxidation than AAPH, and induces a moderate oxidation of intracellular Hb. This intracellular oxidation is markedly increased if ACV hydrophilicity is decreased by lowering the pH. On the other hand, AAPH-derived radicals are considerable more efficient in promoting protein band 3 modification and cell lysis, without significant intracellular hemoglobin oxidation. These results show that the lytic process is not triggered by lipid peroxidation or hemichrome formation, and suggest that membrane protein modification is the relevant factor leading to red blood cell lysis.

Peroxidation of linoleate at physiological pH: hemichrome formation by substrate binding protects against metmyoglobin activation by hydrogen peroxide

Peroxidation by metmyoglobin, MbFe(III), by metmyoglobin/hydrogen peroxide, MbFe(III)/H 2O 2 , to yield the myoglobin ferryl radical ( MbFe(IV)=O), or by ferrylmyoglobin, MbFe(IV)=O, was investigated at physiological pH (7.4) in oil-in-water linoleate emulsions. Linoleate peroxidation was followed using second derivative ultraviolet (UV)-spectroscopy for monitoring formation of conjugated dienes and quantitative determination of specific linoleate hydroperoxides by liquid chromatography with photodiode absorption detection. Modifications of myoglobins during lipid peroxidation were followed simultaneously by changes in the Soret absorption band (410 or 424 nm), and in the visible absorption region (from 450 to 700 nm), combined with electron spin resonance (ESR) spectroscopy for direct detection of changes in the spin state of the iron center. In contrast to MbFe(IV)=O, MbFe(III) and MbFe(III)/H 2O 2 were not able to initiate linoleate peroxidation in oil-in-water emulsions, and MbFe(III) was converted, by binding of linoleate (but not methyl linoleate), to a low-spin hemichrome derivate, HMbFe(III), with the distal histidine reversibly bound to the iron center. HMbFe(III) is ineffective in initiating lipid peroxidation and cannot be activated to MbFe(IV)=O or MbFe(IV)=O by addition of moderate amounts of H 2O 2. Addition of MbFe(III) to linoleate emulsions containing H 2O 2 results in the competitive formation of MbFe(IV)=O and HMbFe(III) in favor of HMbFe(III), and little linoleate peroxidation is detected, demonstrating the inherent protection, at physiologic pH, against peroxidation by reversible binding of the substrate to the potential myoglobin catalyst.

Surface Modified Methotrexate Loaded Erythrocytes for Enhanced Macrophage Uptake

The unique property of macrophages to distinguish between ‘mature self’ and ‘senescent self’ was applied for the enhanced macrophage uptake upon loading drugs into erythrocytes and provoking environment within which it gets translated into senescent cells. Two different approaches were applied one by depleting the sialic acid from the surface of cells and another by inducing hemichrome formation in the cells. Erythrocytes encapsulation of methotrexate was carried out by preswell dilution technique. Surface modification of erythrocytes was performed using desialation and hemichrome induction by treating with trypsin (Tt) and phenylhydrazine (PhT) respectively. Desialation technique was optimized in terms of concentration of trypsin, incubation temperature and time period. Both of these surface modified erythrocytes were characterized for in vitro macrophage uptake. In vivo organ localization was assessed by recording amount of drug present in different organs. The macrophage uptake of both surface modified erythrocytes was enhanced by almost 3–5 and 5–6 times with Tt and PhT cells, respectively. These new surface modified carriers are found to be excellent candidate for enhanced macrophage uptake and anticipated to be useful for any RES tumors.

Oxidized Heme Proteins in an Animal Model of Hemochromatosis

Hemichrome formation resulting from oxidation of heme proteins has been proposed as sensitive indicator of iron-induced oxidative stress. The Heme Protein Spectra Analysis Program was used to quantitate oxidized heme proteins (OHP) in chronic hepatic iron overload in vivo. Rats were fed a diet containing carbonyl iron for 14 months with or without vitamin E supplementation. A 30-fold increase in hepatic iron concentration was observed in rats fed the iron-containing diet. At baseline, total OHP and TBARS were significantly elevated in iron-loaded livers but no increase in hemichrome was seen. During 3 h of spontaneous oxidation, hemichrome formation increased significantly in iron-loaded livers compared to controls. Although supplemental vitamin E was associated with lower levels of OHP and TBARS in iron-loaded livers at baseline, it did not significantly inhibit hemichrome formation during in vitro oxidation. In conclusion, an increase in hemichrome formation in iron-loaded livers was observed only during oxidation in vitro. These results suggest that total OHP is more sensitive than hemichrome formation as an indicator of oxidative stress in this in vivo model of iron overload.

Fluorescence Studies of Extracellular Hemoglobin of Glossoscolex paulistus in Met Form Obtained from Sephadex Gel Filtration

Abstract Chromatography in Sephadex G-200 of extracellular hemoglobin of the giant worm Glossoscolex paulistus in the met form presents an unique band at pH 7.0 and two bands at pH 9.0 as a result of alkaline dissociation. SDS-PAGE of the intact protein obtained at pH 7.0 is very similar to that for the oxyhemoglobin. Chromatography at pH 9.0 indicates complete dissociation of the oligomeric protein into two low molecular weight fractions corresponding to the trimers and monomers, showing that the oxidized extracellular hemoglobin is less stable than the oxyhemoglobin with respect to alkaline dissociation. Fluorescence quantum yields of different fractions obtained in the chromatography, as well as extinction coefficients at 280 nm and 415 nm, were estimated and compared to human methemoglobin. The fluorescence data are consistent with the high content of aromatic residues in G. paulistus hemoglobin. The increase in the fluorescence quantum yield upon both alkalinization and dissociation was correlated with the reduction of intramolecular quenching but the exposure of tryptophan residues to the solvent did not changed significantly as occurs for the oxy form. The intact native protein has a quantum yield of 0.36% at pH 7.0, increasing to 1.89% at pH 9.0 upon dissociation. The monomer has a fluorescence quantum yield of 1.1% at pH 7.0 increasing to 1.43% at pH 9.0. The maximum emission wavelength of the intact protein (330 nm) is consistent with tryptophan residues being relatively buried; they become more fluorescent upon dissociation into smaller subunits but not more exposed since the wavelength of maximum emission is essentially unchanged at pH 9.0. In the monomer, the tryptophan residues also remain buried inside the protein molecule at pH 9.0 (328 nm). The dependencies of fluorescence quantum yields on the pH show in a clear way the hemichrome transitions observed by optical absorption spectroscopy indicating that the formation of two types of hemichromes accompany the distinct increase in fluorescence quantum yield. One type of hemichrome is irreversibly formed around pH 7.5–8.0 and a second reversible hemichrome is formed above pH 9.5–10.0. They are associated with the bis-imidazole low spin hemichrome and with a high spin hemichrome where the weakening of the iron bond to proximal histidine takes place. Addition of cyanide to the metHb solution produces the cyanomet form of the protein which is considerably more stable towards alkaline dissociation and presents a smaller change in quantum yield as a function of pH. Circular dichroism suggests that the formation of hemichromes is not accompanied by significant protein denaturation.

Effect of Heat Denaturation on the Pro-oxidative Activity of Metmyoglobin in Linoleic Acid Emulsions

To reveal the effect of heat denaturation on the pro-oxidative activity of MMb, the thermal denaturation of metmyoglobin (MMb) was investigated using differential scanning calorimetry (DSC) and UV−vis spectrometry in combination with an investigation of the pro-oxidative activity of heat-denatured MMb in linoleic acid emulsions, as registered by oxygen consumption. Thermograms of MMb solutions obtained using DSC and progress curves of the absorbance changes at 290, 409, and 540 nm during heating showed that temperatures immediately below thermal denaturation of MMb-induced structural changes in the heme protein which subsequently increased the pro-oxidative activity of the molecule. In contrast, temperatures above the denaturation temperature of MMb decreased the pro-oxidative activity of the resulting species compared to native MMb. The decrease in pro-oxidative activity was found to be related to hemichrome formation. Moreover, the amount of free iron released during heat denaturation of MMb was measured and found to be of an order where it would not be expected to play any significant role as an overall pro-oxidant in the initial phase of lipid peroxidation in the presence of other pro-oxidative species formed during heat denaturation of MMb. The latter could be concluded from a comparable study of the catalytic activity of free iron(II), hemin, MMb, and heat-denatured MMb in linoleic acid emulsions. Finally, heat treatment of MMb and the pro-oxidative activity of resulting species are discussed with regard to possible influence on oxidative stability of meat. Keywords: Myoglobin; heat-denatured myoglobin; lipid peroxidation; pro-oxidative species; free iron

The dimer-monomer conversion of Cerithidea myoglobin coupled with the heme iron oxidation

With regard to the distal (E7) residue, gastropod sea mollusc contains both types of myoglobin, one with and the other lacking the distal histidine. We have isolated a myoglobin from the radular muscle of Cerithidea rhizophorarum, a small whelk found on the Japanese coast. Unlike Aplysia myoglobin having a single histidine residue at position 95, Cerithidea myoglobin contains three histidines at positions 48, 66 and 98. Moreover, Cerithidea MbO 2 exists as homodimers and is oxidized, not to the usual form of metMb but to the hemichrome monomers. It was also found that the hemichrome monomers thus produced can easily be converted back to the dimerized oxy-form, if the ferric protein was reduced carefully with a slight excess of sodium hydrosulfite. This dimer-monomer conversion coupled with the heme-iron oxidation in Cerithidea myoglobin is very unique, and the distal histidine at position 66 is probably responsible for its reversible formation of hemichrome from the ferric met-form that occurred transiently in due course of the oxidation reaction of Cerithidea MbO 2