Association of hemoglobin C with erythrocyte ghosts.

The interaction of hemoglobin C (Hb C) with erythrocyte membranes was studied using changes in fluorescence intensity in a membrane-embedded probe. The affinity of Hb C for the membranes at pH 6.0 and pH 6.8 was compared to that of normal hemoglobin (Hb A). Steady-state and kinetic data were delivered. The affinity of Hb C for the erythrocyte membrane at pH 6.8 appeared to be about five times greater than that of Hb A. The associations of Hb C and Hb A with the membrane were reversible to about the same extent. The cytoplasmic portions of band 3 membrane proteins were suggested to be the binding sites for both hemoglobins. The membrane binding of Hb C at pH values of 6.8 to 7.0 indicates that this reaction may occur under physiological circumstances.

Spectroscopic studies on the tyrosine residues of canavalin.

Canavalin is a tetramer with 6 tyrosines per subunit. In the work presented here, we have classified these tyrosines by their spectrophotometric and fluorometric pH titration and their ability to be quenched I-. Of the 6 residues, 2 were found to be exposed to the solvent. One (pK = 10.2) contributes 28% of the total fluorescence intensity; the second has a pK of 11.50, and a lower quantum yield, contributing only 16% of the total intensity. The remaining 4 residues (pK = 12.5, contributing 54% of fluorescence intensity) are buried; their titration is irreversible, requiring protein denaturation.

Association of hemin with protein 4.1 as compared to spectrin and actin.

The interaction of hemin with protein 4.1 isolated from red cell membrane cytoskeleton has been studied. Spectrophotometric titration has shown one strong binding site and additional lower affinity sites for hemin. From fluorescence quenching data an association binding constant of 1.3 . 10(7) M-1 has been calculated for the primary site. The conformation of cytoskeletal proteins after hemin binding was followed by the use of far UV circular dichroism and compared to that of the serum hemin trap, albumin. The secondary structure of albumin was unchanged in the presence of high hemin concentrations. Both spectrin and actin lost their conformation upon hemin binding in a ligand-concentration and time-dependent manner. Unlike spectrin and actin, the secondary structure of protein 4.1 appeared. The findings of this study suggest that protein 4.1 may serve as the cytoskeletal temporary sink for small amounts of membrane-intercalated hemin similarly to the function of albumin in the serum. However, an increased release of hemin under pathological conditions may cause hemin association with the cytoskeletal proteins and as a result the cell membrane is expected to be distorted.

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.

Tyrosyl fluorescence in hemocyanin from the scorpion Leirus quinquestriatus.

The fluorescence properties of hemocyanin from the scorpion Leirus quinquestriatus were studied. Emission and excitation spectra were determined for protein in both its oxygenated and deoxygenated forms. Oxygenation was found to bring about a large blue shift in the position of the fluorescence maximum, in addition to a marked quenching of the fluorescence intensity as noted before for another hemocyanin. An appreciable tyrosyl contribution to the fluorscence of oxyhemocyanin was inferred from the wavelength dependence of its emission and excitation spectra. No tyrosyl fluorescence could be observed in either apo- or deoxyhemocyanin. It was concluded that the inability to observe tyrosyl emmision in deoxyhemocyanin is due to the dominating emission to tryptophan. The implication of the findings to the often noted failure to detect tyrosyl emission in proteins containing both tyrosine and tyrptophan is discussed.

The interaction of hemoglobin with phosphatidylserine vesicles.

The interaction of hemoglobin with phosphatidylserine vesicles at low ionic strength and pH conditions was studied. The fluorescence intensity of a lipid embedded probe was quenched by bound Hb but could not be reversed by an elevation of ionic strength and pH. The irreversibility of the fluorescence quenching is a time-dependent process associated with changes in the heme Soret and visible spectra. The rate of these changes was much faster for methemoglobin than for either cyanomethemoglobin or oxyhemoglobin. Elevation of ionic strength released out of the bound hemoglobin into the water phase most of the globin but only a small fraction of the heme. The data are interpreted as demonstrating the ability of phosphatidylserine vesicles to compete with globin for the heme group. When Hb binds to the liposome, heme is being transferred into the lipid phase and the rate-limiting step is the dissociation of the heme-globin complex. The fact that binding of heme to the lipid vesicles is very strong was demonstrated by the failure of hemin to interact with globin when the two were rapidly mixed in the presence of phosphatidylserine vesicles. A multi-step process is suggested to explain the results of Hb phosphatidylserine interaction.

The interaction of hemin with skeletal muscle actin.

The ability of actin to interact with hemin was studied. It was found that the Soret absorption band of hemin changes in the presence of actin and that hemin is capable of quenching the fluorescence intensity of actin. These findings were indicative of hemin binding to actin. The binding constant for the high affinity site was calculated to be 5.3 X 10(6) M-1. The amounts of native G- and F-actin were estimated by their DNAase I inhibition activity. It was observed that the binding of hemin to G-actin is followed by a slow decrease in the ability of actin to inhibit DNAase I activity and to polymerize upon addition of salts. Binding of hemin to F-actin resulted in a gradual depolymerization of the filaments, to an inactivated form, as expressed by a reduction in the ability of hemin-bound F-actin to inhibit DNAase I activity in the absence as well as in the presence of guanidine-HCl. Electron microscopy studies further corroborated these findings by demonstrating that: (1) hemin-bound G-actin failed to show formation of polymers when salts were added; (2) a marked reduction in the amount of actin polymers was observed in the specimens examined 24 h after mixing with hemin. It is suggested that the elevated amounts of free hemin formed under pathological conditions, might be toxic to cells by interfering with actin polymerization cycles.

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 interaction of hemin and bilirubin with the human red cell membrane.

The incubation of 0.5% suspension of fresh normal erythrocytes with hemin or bilirubin resulted in substantial hemolysis. The amount of hemolysis achieved depended on the concentration of the lytic agents. In each concentration maximum hemolysis was reached within half an hour. The hemolytic effect was somewhat dependent on temperature. Comparison with the hemolytic effect of hemin on mice (Chau, A.C. and Fitch, C.D. (1980) J. Clin. Invest. 66, 856-858) showed that although both cells undergo hemolysis by hemin, the behaviour of each red cell type is different. Centrifugation and fluorescence quenching of membrane embedded probe revealed that both hemin and bilirubin bind to the red cell membrane, hemin having higher affinity. The reaction was found to be hydrophobic and therefore independent of ionic strength. The high affinity of the membrane for hemin was shown by its ability to compete successfully with globin for hemin. Electron microscopy of the red cells which underwent hemolysis indicated cell damage and some membrane destruction. Red cell ghosts were totally disrupted when saturated with hemin. These results suggest an explanation for hemolytic events occurring in cases such as elevation of serum bilirubin or abnormalities leading to hemin release by hemoglobin.

Crosslinking of isolated cytoskeletal proteins with hemoglobin: a possible damage inflicted to the red cell membrane.

Crosslinking of isolated red cell membrane cytoskeletal proteins and hemoglobin mediated by H2O2 was studied. The products of spectrin and hemoglobin interaction were demonstrated electrophoretically to be high-molecular-weight polypeptides crosslinked by nondisulfide covalent bonds. The molecular weight of the protein bands correlated with various combinations of spectrin and hemoglobin chains and the relative amount of the different products was dependent on the molar ratio of the interacting proteins. Free hemin caused spectrin crosslinking as well, but globin in the absence of hemin was inactive. Since the H2O2-mediated reaction resulted in reduction of the spectrin tryptophan fluorescence, the latter was used to monitor the reaction progress under various conditions. Both oxyhemoglobin and methemoglobin were found to be most efficient, whereas cyanmethemoglobin and hemichrome were relatively inactive. Analysis of the data implied that tryptophan oxidation as well as spectrin conformational changes follow an iron-induced crosslinking of the interacting proteins. Actin, the second major protein in the red cell cytoskeleton, behaved similarly to spectrin. The intrinsic fluorescence intensity of both G- and F-actin was decreased upon addition of H2O2 to the mixture of hemoglobin and each of the actin forms. SDS-polyacrylamide gel electrophoresis revealed that G-actin crosslinked one or two hemoglobin chains. F-actin-hemoglobin interaction induced by H2O2 produced very high aggregates that could not penetrate the gel. It is suggested that crosslinking of cytoskeletal proteins in red cells containing membrane-associated hemoglobin provides a rationale for the loss of membrane flexibility.

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.

Accumulation and drainage of hemin in the red cell membrane.

The subject of hemin intercalation in red cell membranes and the correlation of the accumulated hemin level with the membrane pathology was studied. Methods which made use of dioxan and octan-2-ol mixtures to quantitate small amounts of hemin in membranes were developed. Applying these methods, hemin levels were measured in the cytoskeleton and the remaining lipid core of various red cell membranes. The amount of hemin, in both membrane fractions, was higher in pathological cells of sickle cell anemia and beta-thalassemia as compared to normal circulating cells. Correlation exists between the amount of the membrane-accumulated hemin and the severity of the disease. The level of hemin in the membrane was found to be age dependent, old cells in circulation accumulating more hemin than young cells. The level of hemin in all cells tested was much lower than the amount found previously to cause immediate hemolysis when applied externally (Kirschner-Zilber, I., Rabizadeh, E. and Shaklai, N. (1982) Biochim. Biophys. Acta 690, 20-30). This was explained by the differences between the process leading to immediate lysis and membrane changes recognized as pathological by the in-vivo sequestration mechanism. In search of a physiological mechanism which may drain the cell membrane from the hazardeous hemin, albumin, the main serum protein, was found capable of serving as an efficient agent for extracting hemin trapped in red cell membranes. It is suggested that under normal conditions albumin extracts enough hemin to leave the erythrocyte with unharmful hemin amounts, however, under pathological conditions greater amounts accumulate leading to a shorter cell life span.

Association of iron-protoporphyrin-IX (hemin) with myosins.

Addition of myosins isolated from guinea pig heart and rabbit skeletal muscle to hemin solutions resulted in the appearance of new absorption spectra indicating association of hemin and the myosins. Binding stoichiometry based on absorption changes was found to be two hemin sites per myosin molecule. The binding constants calculated from quenching of the intrinsic fluorescence of the myosins by hemin are Ka = 7 (+/- 2) 10(6) M-1 for skeletal muscle myosin, and Ka = 3 (+/- 1) x 10(7) M-1 for heart muscle myosin. Based on these findings, myosins are suggested as potential transporters of free hemin between cell organelles.

Serum proteins as mediators of hemin efflux from red cell membranes: specificity of hemopexin.

The involvement of the serum heme-binding proteins hemopexin and albumin in the clearance of erythrocyte membranes from toxic hemin was compared. In the presence of hemopexin initial rates of hemin efflux from resealed ghosts were faster and the amount of extracted hemin larger. When hemin-containing ghosts were treated with a protein mixture of 1:45 hemopexin to albumin, as present in serum, most of the hemin was extracted in the form of heme-hemopexin. It was concluded that hemopexin is the serum protein responsible for heme extraction from cell membranes.

Glutathione as a scavenger of free hemin. A mechanism of preventing red cell membrane damage.

The interaction of glutathione and hemin was studied at physiological ionic strength and pH conditions. Formation of a glutathione-hemin complex was assessed from the appearance of spectral changes in the visible region. In the presence of excess cyanide and histidine and upon oxidation of the sulfhydryl group, no complex was formed. From these results it was concluded that the thiol group of glutathione serves as a ligand for the heme iron. A binding constant of 3.1(+/- 0.1) x 10(4) M-1 was calculated by use of a Hill plot. The hemolytic effect of hemin on red cell was much reduced in the presence of glutathione. Since hemolysis results from association of hemin with membrane components its binding in the presence and absence of glutathione was studied. It was found that the affinity of hemin for the cytoskeletal membrane proteins as well as for the membrane lipid core was decreased in the presence of glutathione. It was concluded that glutathione competes with the membranes for hemin and by doing so can defend the membrane from injury by hemin.

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.

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.

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.

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.

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.