Precise quantification of haemoglobin in erythroid precursors and plasma.

INTRODUCTION: Haemoglobin (Hb) quantification in whole blood is possible by various spectrophotometric methods. However, determination of low-level Hb in erythroid precursors or haemolytic plasma is inaccurate because of contribution from light scatter and/or nonhaemoglobin components with overlapping absorbance. Therefore, this study developed a sole method allowing accurate spectrophotometric quantification of Hb at a low concentration range. METHODS: Advantage was taken of the unique absorption spectra of carbon monoxide-Hb complex (COHb) as compared to oxyHb. The visible absorption spectra of samples were recorded prior and following carbon monoxide exposure. A difference extinction coefficient at the maximal difference absorption was used to calculate Hb concentrations. RESULTS: Known amounts of Hb were added to mouse erythroleukaemia (MEL) cells lysate or plasma to yield known 'theoretical' concentrations. The concentrations were measured by the current and known methods. The current method was found much more accurate compared with previous methods specifically at low concentrations. CONCLUSION: The method is valid for accurate quantification of Hb at a wide concentration range (>0.1 µm/L) in erythroid precursors or plasma and is optional for other biological fluids.

Vascular damage by unstable hemoglobins: the role of heme-depleted globin.

The study compared the damage inflicted to endothelial cells (ECs) by intact hemoglobin (Hb) and isolated chains. To compare optional in vivo contact of acellular Hb with the endothelium, oxy-forms of Hb and its isolated alpha- and beta-chains existing in the thalassemias were added to primary confluent cultures of bovine aorta EC. Cell damage was followed by morphological changes or leakage of lactic dehydrogenase and pre-inserted 51Cr from the cells, followed for 27 h. Under these experimental conditions, Hb did not affect the cells but its chains inflicted damage, beta- more than alpha-chains. Based on the literature and our data, we hypothesized that hemin and/or globin should be responsible for the increased endothelial damage by beta-chains. While hemin hardly affected ECs, globin, unlike the plasma protein hemopexin, was harmful. Since hemin release leaves globin with a large hydrophobic surface, the globin-damage appears to result from adsorptive pinocytosis to endothelial membrane.

Kinetics of hemin distribution in plasma reveals its role in lipoprotein oxidation.

Hemin is a powerful in vitro inducer of low-density lipoprotein (LDL) oxidation, implicated in development of atherosclerosis. To support the proposed role of hemin in atherogenesis, the question of whether hemin has any chance of getting together with LDL in vivo, must be addressed. A stopped-flow technique was employed in order to investigate the fast kinetics of hemin binding to LDL and to other plasma hemin-binding proteins: high-density lipoprotein (HDL), albumin and hemopexin. Based on the measured rate constants of hemin association with and dissociation from each of these proteins, time-dependent hemin distribution in plasma was analyzed. The analysis shows that as much as 80% of total hemin binds initially to LDL and HDL, the plasma components which are most susceptible to oxidation. Only then hemin partially transfers to the antioxidants albumin and hemopexin. The half time of the hemin-LDL complex in plasma, initially comprising 27% of total hemin, was more than 20 s. Not only transient, but also oxidatively active steady-state hemin-lipoprotein complexes in plasma were both predicted from the kinetic analysis and found in experiment. Our data suggest that the hemin-LDL complex may exist in vivo and that its oxidative potential should be considered pro-atherogenic.

Oxidative stress in beta-thalassemia: hemoglobin alpha-chains activate peroxidation of low density lipoproteins.

Under oxidative stress normal hemoglobin (HbA) can trigger oxidation of low density lipoproteins (LDL) in the presence of hydrogen peroxide. Hb variants like isolated HbA chains possess higher peroxidative reactivity than the normal tetramer HbA. Because isolated Hb chains undergo fast autoxidation, a process yielding peroxidants, we studied the relative peroxidative activity of alpha- and beta-chains as well as of HbA without additional peroxidant. The descending order to relative oxidation of LDL protein ApoB (assessed by its crosslinking) and lipids (determined as formation of conjungated diens) was: alpha-chains > beta-chains > HbA. The results of our study indicate that extracellular chains may be the trigger of lipoproteins alterations observed in beta-thalassemia.

A fluorescence method for estimation of toxemia: binding capacity of lipoproteins and albumin in plasma.

The hazard of toxemia, a condition resulting from the spread of toxins by the bloodstream, is regulated by plasma proteins capable of binding with free toxins. As toxin binding results in a reduction of available binding sites, measuring the proteins' binding capacity can be used to estimate toxemia severity. Suggested by this approach, a novel fluorescence method was developed to determine lipoprotein and albumin binding capacities in whole plasma. The method entails two steps: specific binding of N(n-carboxy)phenylimide-4-dimethyl-aminonaphthalic acid with albumin followed by addition of 12-(9-anthroyloxy)stearic acid which, under these conditions, binds mostly with lipoprotein. Reduced fluorescence intensity of the probes in plasma of patients compared to that of healthy donors reflected saturation of binding sites by toxins, thereby estimating toxemia severity. Poor correlation was found between the lipoprotein and albumin binding abilities, suggesting their independent diagnostic values. The simplicity and rapidity of this method are advantageous for its clinical application.

Coordination of nitric oxide by heme-hemopexin.

Hemopexin, which acts as an antioxidant by binding heme (Kd < 1 pM), is synthesized by hepatic parenchymal cells, by neurons of the central and peripheral nervous systems, and by human retinal ganglia. Two key regulatory molecules, nitric oxide (.NO) and carbon monoxide (CO), both bind to heme proteins and since ferroheme-hemopexin binds CO, the possible role of heme-hemopexin in binding .NO was investigated. .NO binds rapidly to hemopexin-bound ferroheme as shown by characteristic changes in the Soret and visible-region absorbance spectra. Circular dichroism spectra of .NO-ferroheme-hemopexin in the Soret region exhibit an unusual bisignate feature with a zero crossover at the absorbance wavelength maximum, showing that exciton coupling is occurring. Notably, the .NO complex of ferroheme-hemopexin is sufficiently avid and stable to allow hemopexin to bind this molecule in vivo and, thus, hemopexin may protect against NO-mediated toxicity especially in conditions of trauma and hemolysis.

Involvement of the oxygen storage protein myoglobin in muscle damage under oxidative stress.

Myoglobin (Mb), the muscular oxygen reservoir, was shown to possess peroxidative reactivity in presence of H2O2 leading to oxidation of isolated cellular proteins like myosin. The objective of this study was to investigate the peroxidative effect of Mb/H2O2 on proteins in intact myofibrils (MF). Incubation of chicken leg MF in isotonic, pH 7.3 buffer at 37 degrees C in the presence of Mb (30 microM) and H2O2 (200 microM), resulted in aggregation of MF material as inspected under light microscope. SDS-PAGE analysis revealed presence of high molecular weight aggregates at the expense of myosin heavy chains, but not actin. This crosslinking was unaffected by S-S reducing agents. Continuous low flow (0.03-3.00 microM/minute), produced by glucose oxidase and glucose, was more active than bolus H2O2 addition in myosin crosslinking in MF material. Hemin which may be released from Mb under oxidative stress, was more active than Mb as a trigger of MF peroxidative aggregation. Calcium-ATPase activity of crosslinked MF was considerably lost. These findings suggest that Mb/H2O2 may lead to oxidation of neighbouring muscular protein thereby jeopardize their functioning thus explaining muscular malfunction under oxidative stress.

Oxidation of low-density lipoprotein by hemoglobin stems from a heme-initiated globin radical: antioxidant role of haptoglobin.

Hemoglobin, known as a poor peroxidase, has been recently found to be a highly reactive catalyzer of low-density lipoprotein (LDL) oxidation resulting in oxidation of LDL lipids and covalent cross-linking of the LDL protein, apo B. We evaluated three possible mechanisms that may account for hemoglobin reactivity: oxidative activation by globin-dissociated hemin following its transfer to LDL; peroxidase-like reactivity of the ferryl iron active state in intact hemoglobin; and oxidation by a globin radical formed in oxidized hemoglobin. The first mechanism was ruled out because only a minor fraction of hemin was actually transferred to LDL in the process of oxidation. The second mechanism was excluded because hemoglobin ferryl, unlike ferryl of horseradish peroxidase, was not consumed in the process of LDL oxidation. Haptoglobin completely inhibited cross-linking of globin in hemoglobin/H2O2 mixtures but not in myglobin/H2O2, as well as cross-linking of apo B and oxidation of LDL lipids. Haptoglobin could not however abolish the hemoglobin ferryl state, a finding that further supported exclusion of the second mechanism. We conclude that the active species in hemoglobin-induced LDL oxidation is the globin radical, as suggested in the third mechanism. The present findings also show that haptoglobin functions as a major antioxidant thus protecting the vascular system.

Oxidative interaction of unpaired hemoglobin chains with lipids and proteins: a key for modified serum lipoproteins in thalassemia.

We searched for a biochemical explanation to the modification of lipoproteins like low-density lipoproteins (LDL) observed in patients with the severe hemolytic anemia beta-thalassemia. Because a large fraction of the LDL surface is composed of phospholipids, we first explored the possible involvement of phospholipids in the oxidative interaction of LDL with hemoglobin (Hb), using brain extract phospholipid liposomes as a model. The relative binding affinity and oxidative interaction of three hemoglobin variants (intact Hb A and isolated beta- and alpha-chains) with LDL and liposome were compared. Studies carried out at low pH/ionic strength and under physiological conditions revealed that association of hemoglobin variants with the phospholipid liposomes is driven by electrostatic forces but their binding is not a prerequisite for oxidative interaction. Unlike phospholipid liposomes, LDL underwent only a negligible association with the Hb variants under all pH/ionic strength conditions. Nevertheless, LDL induced oxidation of Hb variants, mostly alpha-chains. The dissimilar behavior of the liposomes and LDL indicated that LDL protein apo B rather than phospholipids is the actual LDL surface component which interacts with the hemoglobin variants. This agrees with the finding that apo B protein underwent oxidative crosslinking by the hemoglobin variants among which alpha-chains were most active. We concluded from these results that the ability of hemoglobin to undergo autooxidation is the key to its oxidative reactivity toward LDL. The results of the present study indicate that the modified LDL particles observed in beta-thalassemia may reflect lipoprotein oxidation by alpha-chains in circulation.

Role of hemopexin in protection of low-density lipoprotein against hemoglobin-induced oxidation.

Globin-free hemin and certain hemoproteins, predominantly hemoglobin, are active triggers of low-density lipoprotein (LDL) peroxidation, a contributing cause of atherosclerosis. The role of the plasma heme-binding protein, hemopexin, in protecting apolipoprotein B and LDL lipids from oxidation triggered by either hemin or hemoglobin in the presence of low amounts of H2O2, was investigated at physiological pH and temperature. Significantly, hemopexin prevented not only hemin-mediated modification of LDL but also LDL peroxidation induced by hemoglobin, both by met and oxy forms. Analysis of the data revealed that the rate of heme transfer from methemoglobin to hemopexin was highly dependent upon temperature: only minimal heme transfer occurred at 20 degrees C, whereas at the physiological temperature of 37 degrees C, heme transfer was rapid, within the lag phase of LDL oxidation, regardless of the presence or absence of H2O2. Heme did transfer to hemopexin from oxyhemoglobin as well, but only in the presence of H2O2. The proposed mechanism of the inhibition of oxyhemoglobin oxidative reactivity by hemopexin involves peroxidation of oxyhemoglobin (Fe(II)) to ferrylhemoglobin (FeIV), followed by a comproportionation reaction (FeIV+FeII-->2FeIII), yielding methemoglobin (FeIII) from which heme is readily transferred to hemopexin. Taken together, the data demonstrate that hemopexin can act as an extracellular antioxidant against hemoglobin-mediated damage in inflammatory states, which is especially important when haptoglobin is depleted or absent.

Hemoglobin induced apolipoprotein B crosslinking in low-density lipoprotein peroxidation.

Oxidative modification of human low-density lipoprotein (LDL) is thought to play a major role in the development of atherosclerosis. Free hemin, hemoglobin, myoglobin, and horseradish peroxidase (HRP) were reported in different studies as promoters of LDL lipid oxidation. Based on our previous finding that hemin induced oxidative crosslinking of the LDL protein, apolipoprotein B (apo B) (Y. I. Miller and N. Shaklai (1994) Biochem. Mol. Biol. Int. 34, 1121-1129), we compared the ability of free hemin and the above hemoproteins to induce peroxidation modification of apo B using SDS-PAGE. The levels of the final products of lipid peroxidation were determined as thiobarbituric acid-reactive substances. Hemoglobin and myoglobin were found to be as active as free hemin and all these were much more active than the classic peroxidase HRP. Moreover, the products of oxidized apo B differed: hemoglobin, myoglobin, and hemin induced mostly covalent aggregates, while HRP caused fragmentation of apo B. Hemoglobin reactivity was expressed at low H2O2 concentrations even in the absence of molecular oxygen. Desferal, along with other antioxidants, inhibited the hemoglobin-induced LDL oxidation independently of its iron-chelating property. The high peroxidative reactivity of hemoglobin is explained by its ability (unlike HRP) to transfer the oxidative equivalents from the heme active site, through the globin, to LDL. The apo B radicals thus formed are terminated, yielding intermolecular crosslinked protein. It is suggested that small amounts of the highly reactive hemoglobin in plasma, suffice to trigger LDL protein oxidation (along with its lipid oxidation), thereby inflict the atherosclerosis precondition.

Peroxidative interaction of myoglobin and myosin.

Met-myoglobin [Fe(III)] was found to induce myosin cross-linking in the presence of H2O2 [Bhoite-Solomon, V. & Shaklai, N. (1992) Biochem. Int. 26, 181-189]. To assess the relevance of these findings to cellular pathology, peroxidation of myosin was performed with physiological divalent iron [Fe(II)] myoglobins in the oxy and deoxy forms. Both myoglobin forms were capable of mediating cross-linking of myosin. Deoxymyoglobin reactivity was similar to that of met-myoglobin, but the reactivity of oxymyoglobin was retarded compared to deoxymyoglobin. Cross-linking of myosin occurred under a low flow rate of H2O2 (3 microM/min) and in the presence of excess oxymyoglobin over H2O2, known to diminish the steady state of the myoglobin active heme [ferryl, Fe(IV)]state. The adenosinetriphosphatase activity of myosin was reduced to about half due to cross-linking. Addition of myoglobin/H2O2 to high myosin concentrations (> = 20 microM) turned the solutions into gels, a phenomenon explained by the further formation of intermolecular super cross-links of soluble myosin. Thus, at cellular ionic strength in which myosin is insoluble, cross-linking of myosin could still be triggered by myoglobin and H2O2. Based on these data, it is suggested that myoglobin-induced cross-linking of myosin and the consequent loss of adenosinetriphosphatase activity may be involved in muscle malfunction under hypoxia when cellular peroxidants increase and the deoxymyoglobin form prevails.

The involvement of low-density lipoprotein in hemin transport potentiates peroxidative damage.

Hemin binds to isolated low-density lipoprotein (LDL) and thereby triggers LDL oxidation. In this study we investigated whether hemin can get together with LDL under physiological conditions. The relative affinity of three blood components to free hemin was as follows: RBCM < LDL < albumin. At physiological molar ratio of LDL/albumin all the hemin was bound to albumin. In molar excess of albumin over hemin, existing even under pathological conditions, albumin served as an efficient antioxidant for the plasma hemin-induced LDL oxidation. RBCM-embedded hemin, unlike plasma hemin, affected LDL: the mobile hemin was transferred from RBCM to LDL in the absence of albumin, whereas in the presence of albumin most of the mobile hemin finally reached the albumin but partially via LDL. Thus, a transient hemin is built up in LDL. This transient hemin triggered LDL oxidation which was not inhibited but rather promoted by albumin. The involvement of albumin in this oxidation was explained by its acting as a pump thereby increasing the transient hemin in LDL. It is suggested that increased membrane hemin level as in hemoglobinopathies and/or excess LDL in dyslipidemia provide conditions for hemin-induced LDL oxidation.

The role of H2O2-generated myoglobin radical in crosslinking of myosin.

The mechanism of myoglobin/H2O2 derived peroxidation of myosin was studied by comparing the catalytic activity of myoglobin and horseradish peroxidase using O-dianisidine, N-acetyl tyrosine and myosin as substrates. It was found that both hemoproteins induced myosin crosslinking and concomitant tyrosines oxidation to bityrosines, suggesting inter-molecular coupling of tyrosines in the crosslinking. The enzymatic activity of both hemoproteins on myosin was weak compared to small substrates. While horseradish peroxidase was much more active than myoglobin on small substrates, the reverse was true for myosin peroxidation. Since the suicidal interaction of myoglobin with H2O2 forms unstable tyrosine radicals, we suggest that the increased activity of myoglobin on myosin results from an efficient electron transfer between surface tyrosines of myosin and myoglobin but not horseradish peroxidase. These conclusions were supported by evidence that sperm whale myoglobin, which contains two active tyrosines--the heme-adjacent (tyrosine-103) and the surface (tyrosine-151), is more active as a mediator of myosin peroxidation than horse heart myoglobin which is devoid of the surface tyrosine.

Oxidative crosslinking of LDL protein induced by hemin: involvement of tyrosines.

This study investigated free hemin induced modifications in low density lipoprotein (LDL). By use of fluorescent probes hemin was found to associate with LDL thereby inducing peroxidation of both lipids and protein. Upon LDL peroxidation, covalent crosslinking of apolipoprotein B (Apo B) occurred as judged by SDS-PAGE. Concomitantly, a multifluorophore emission developed, which included contribution of bityrosines. The simultaneous formation of protein aggregates and bityrosines was interpreted as the involvement of intermolecular bityrosines in the hemin induced crosslinking of Apo B. Since LDL protein aggregation relates to conversion of macrophages into foam cells, hemin should be considered as an endogenous trigger of atherosclerosis.

Association of erythrocyte glycophorin with protoporphyrin-IX derivatives: a fluorimetric study.

Based on demonstrations that protoporphyrin-IX and its metabolic derivatives bilirubin and hemin bind to the red cell membrane, their association with glycophorin A, the main transmembrane sialoglycoprotein, was assessed. No interaction between bilirubin and glycophorin could be demonstrated but both protoporphyrin-IX and hemin were found to bind to the protein. Interaction of protoporphyrin-IX and glycophorin was demonstrated by changes in both ligand and protein fluorescence characteristics in the presence of the other reactant. Binding of hemin, (Fe+3-protoporphyrin-IX) with glycophorin was revealed by quenching of the proteins' intrinsic fluorescence intensity by hemin and a shifted Soret absorption of hemin in the presence of glycophorin. The association constants of protoporphyrin-IX and hemin with glycophorin at 25 degrees C were calculated as 2.5 +/- 0.5 x 10(6)M-1 and 1.4 +/- 0.4 x 10(6)M-1 respectively.

Myocyte injury by hemin.

Effects of free hemin on myocardium were investigated using a model of neonatal myocyte primary cultures. Cells were subjected to free hemin at concentrations up to 20 microM and equilibrated for 5 h at 37 degrees C. Distribution of hemin in media, cell sarcolemma, and cell interior was evaluated. Time-dependent reduction in beating rate was monitored throughout the entire concentration range of administrated hemin. With time and in a hemin concentration-dependent manner, arrhythmic beatings which were followed by loss of contractility were observed. In parallel, morphologic changes appeared from granulation to complete loss of cell integrity. At the concentration range studied, hemin also induced a biphasic release of cytosolic enzymes. In the first phase, the fraction of enzyme released was dependent of the ratio of hemin:cells and was correlated with the amount of nonviable cells as monitored by a trypan blue test. In the second phase, the fraction of released enzyme was much larger than that of nonviable cells. The data are interpreted as an indication of complete loss of cytosolic content due to sarcolemma damage in first phase and partial damage to cell interior in the prolonged second phase. It is concluded that in similarity with other amphipathic molecules, free hemin is toxic to the myocardium.

Peroxidative crosslinking of myosins.

The effect of myoglobin, free hemin and H2O2 on myosins from heart and skeletal muscle was studied. SDS-gel electrophoresis revealed that each agent caused intermolecular thiol crosslinking of both myosins dissociable by excess of beta-mercaptoethanol. In the simultaneous presence of H2O2 and myoglobin or H2O2 and free hemin, myosin formed covalent aggregates undissociable by beta-mercaptoethanol and therefore assessed to formation of non S-S inter molecular covalent bonds. The latter aggregates are suggested to result from pairing of myosin radicals formed by the H2O2 induced ferryl iron state in myoglobin, free hemin or hemo-myosin.

Long-term intercalation of residual hemin in erythrocyte membranes distorts the cell.

The effect of long-term incubation of residual globin-free hemin on whole red blood cell and isolated cytoskeletal proteins was studied. Hemin at concentrations found in pathological red cells was inserted to fresh erythrocytes. Increased hemolysis developed in the hemin-containing cells after a few days at 37 degrees C and after about four weeks at 4 degrees C. Since lipid and hemoglobin peroxidation did not depend on the presence of hemin, time-dependent effects on the cytoskeleton proteins were studied. Observations were: (1) spectrin and protein 4.1 exhibited a time-dependent increasing tendency to undergo hemin-induced peroxidative crosslinking. (2) The ability of the serum proteins, albumin and hemopexin, to draw hemin from spectrin, actin and protein 4.1 decreased with time of incubation with hemin. These results were attributed to time-dependent hemin-induced denaturation of the cytoskeletal proteins. Albumin taken as a control for physiological hemin trap was unaffected by hemin. Small amounts of hemo-spectrin (2-5%) were analyzed in circulating normal cells, and this in vivo hemo-spectrin also failed to release hemin. It was concluded that slow accumulation of hemin, a phenomenon increased in pathological cells, is a toxic event causing erythrocyte destruction.

Hemin-promoted peroxidation of red cell cytoskeletal proteins.

Hemin-induced crosslinking of the erythrocyte membrane proteins was analyzed at three levels: (i) whole membranes, (ii) integrated or dissociated cytoskeletons, and (iii) isolated forms of the three main cytoskeletal proteins, spectrin, actin, and protein 4.1. Addition of H2O2 and hemoglobin to resealed membranes from without did not affect any of the membrane proteins. Hemin that can transport across the membrane induced, in the presence of H2O2, crosslinking of protein 4.1 and spectrin. Both free hemin and hemoglobin added with H2O2 induced crosslinking of integer cytoskeletons and mixtures of isolated cytoskeletal proteins, but hemin was always more active. Of the three major cytoskeletal proteins, spectrin and protein 4.1 were most active while the participation of actin was only minor. The yield of crosslinked products was increased in all reaction mixtures with pH, with an apparent pK above 9.0. Replacement of H2O2 by phenylhydrazine and tert-butyl hydroperoxide resulted in crosslinking of the same proteins, but with lower activity than H2O2. Bityrosines, which were identified by their specific fluorescence emission characteristics, were formed in reaction mixtures containing hemin and hydrogen peroxide and either spectrin or protein 4.1, but not actin. On the basis of fact that bityrosines were revealed only in reaction mixtures that produced protein adducts, formation of intermolecular bityrosines was analyzed to be involved in crosslinking of the cytoskeletal proteins. Since the levels of membrane-intercalated hemin are correlated with aggregation of membrane proteins, it is suggested that the peroxidative properties of hemin are responsible for its toxicity.