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.     

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.     

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.     

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.     

The Interplay between Molten Globules and Heme Disassociation Defines Human Hemoglobin Disassembly

Hemoglobin functions as a tetrameric oxygen transport protein, with each subunit containing a heme cofactor. Its denaturation, either in vivo or in vitro, involves autoxidation to methemoglobin, followed by cofactor loss and globin unfolding. We have proposed a global disassembly scheme for human methemoglobin, linking hemin (ferric protoporphyrin IX) disassociation and apoprotein unfolding pathways. The model is based on the evaluation of circular dichroism and visible absorbance measurements of guanidine-hydrochloride-induced disassembly of methemoglobin and previous measurements of apohemoglobin unfolding. The populations of holointermediates and equilibrium disassembly parameters were estimated quantitatively for adult and fetal hemoglobins. The key stages are characterized by hexacoordinated hemichrome intermediates, which are important for preventing hemin disassociation from partially unfolded, molten globular species during early disassembly and late-stage assembly events. Both unfolding experiments and independent small angle x-ray scattering measurements demonstrate that heme disassociation leads to the loss of tetrameric structural integrity. Our model predicts that after autoxidation, dimeric and monomeric hemichrome intermediates occur along the disassembly pathway inside red cells, where the hemoglobin concentration is very high. This prediction suggests why misassembled hemoglobins often get trapped as hemichromes that accumulate into insoluble Heinz bodies in the red cells of patients with unstable hemoglobinopathies. These Heinz bodies become deposited on the cell membranes and can lead to hemolysis. Alternatively, when acellular hemoglobin is diluted into blood plasma after red cell lysis, the disassembly pathway appears to be dominated by early hemin disassociation events, which leads to the generation of higher fractions of unfolded apo subunits and free hemin, which are known to damage the integrity of blood vessel walls. Thus, our model provides explanations of the pathophysiology of hemoglobinopathies and other disease states associated with unstable globins and red cell lysis and also insights into the factors governing hemoglobin assembly during erythropoiesis.     

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.

     

Hemichrome binding to band 3: nucleation of Heinz bodies on the erythrocyte membrane

Hemichromes, the precursors of red cell Heinz bodies, were prepared by treatment of native hemoglobin with phenylhydrazine, and their interaction with the cytoplasmic surface of the human erythrocyte membrane was studied. Binding of hemichromes to leaky red cell ghosts was found to be biphasic, exhibiting both high-affinity and low-affinity sites. The high-affinity sites were shown to be located on the cytoplasmic domain of band 3, since (i) glyceraldehyde-3-phosphate dehydrogenase, a known ligand of band 3, competes with the hemichromes for their binding sites, (ii) removal of the cytoplasmic domain of band 3 by proteolytic cleavage causes loss of the high-affinity sites, and (iii) the isolated cytoplasmic domain of band 3 interacts tightly with hemichromes, rapidly forming a pH-dependent, water-insoluble copolymer upon mixing in aqueous solution. Since the copolymer of hemichromes with the cytoplasmic domain of band 3 was readily isolatable, a partial characterization of its properties was conducted. The copolymer was shown to be of defined stoichiometry, containing approximately 2.5 hemichrome tetramers (or approximately 5 hemichrome dimers) per band 3 dimer, regardless of the ratio of hemichrome:band 3 in the initial reaction solution. The copolymer was found to be of macroscopic dimensions, generating particles which could be easily visualized without use of a microscope. The coprecipitation was also highly selective for hemichromes, since, in mixed solutions with native hemoglobin, only hemichrome was observed in the isolated pellet. Furthermore, no precipitate was ever observed upon mixing the cytoplasmic domain of band 3 with oxyhemoglobin, deoxyhemoglobin, (carbonmonoxy) hemoglobin, or methemoglobin.(ABSTRACT TRUNCATED AT 250 WORDS)     

The use of hemoglobin for determination of phospholipase A2 activity

A spectrophotometric method is proposed for determining phospholipase A2 activity, which is based on the conversion of hemoglobin into hemichrome under the fatty acid action. The spectral difference between hemoglobin and hemichrome was registered by the difference spectrum with a minimum at 405 nm and a maximum at 423 nm. The absorption value determined as the difference between the spectrum maximum and minimum was proportional to the amount of the fatty acid derived from hydrolysis of phospholipids. The method enables the enzyme activity to be determined directly in the spectrophotometric cell.     

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.     

The interaction of hemoglobin with hexadecyltrimethylammonium bromide

The interaction of hemoglobin (Hb) with hexadecyltrimethylammonium bromide (CTAB) is investigated by UV–vis absorption spectra and fluorescence spectra method. CTAB monomer can convert methemoglobin (metHb) to hemichrome, and CTAB molecular assemblies, such as micelle, microemulsion and lamellar liquid crystal, can induce heme monomer to leave the hydrophobic cavity of Hb. TEM results show that Hb maintains the spherical structure in CTAB microemulsions while it is unfolded in CTAB lamellar liquid crystals. The existence of proton in the above systems can increase the stability of metHb.     

Interaction of serum albumin with normal and sickle hemoglobins.

The stability of oxyhemoglobin S during mechanical shaking was enhanced by the addition of human serum albumin. The stabilizing effect was maximum when the concentration of serum albumin approached that of oxyhemoglobin, suggesting a molecular level interaction between them. The effects of serum albumin on oxyhemoglobin A were essentially similar to those on oxyhemoglobin S. Deoxy- and methemoglobins were also stabilized by serum albumin. The addition of human serum albumin to a solution containing sickle cell oxyhemoglobin slowly formed a compound which had an absorbance peak at 620 nm. After purification by Sephadex G-200 column chromatography, this compound was identified as methemalbumin. Comparison of the rates of formation of methemalbumin from hemoglobin with various ligand states and human serum albumin showed that the rate of formation from hemichrome was much faster than from met-, oxy- and deoxyhemoglobin. About 60% of the heme was transferred from hemichrome to albumin when the mixture was kept standing at room temperature for 5 min, in contrast to only 5% from methemoglobin. This result suggests that hemichrome, rather than methemoglobin, is the intermediate in the formation of methemalbumin from oxyhemoglobin and human serum albumin. This hypothesis is supported by the finding that the rate of formation of methemalbumin was faster at alkaline pH values than at acid pH values. Serum albumin from various animal sources showed different stabilizing effects. The formation of methemalbumin from these animal albumins was far less than that from human albumin.     

Oxidation and denaturation of hemoglobin encapsulated in liposomes

A study was made of the in vitro stability of hemoglobin-containing liposomes (‘hemosomes’) prepared from phosphatidylcholines, equimolar cholesterol and red cell lysate by the hand-shaking and ether-injection methods. Absorption spectra indicated hemichrome formation in ‘hemosomes’ prepared by the ether-injection technique, and increased oxidation of hemoglobin in hand-shaken ‘hemosomes’. The denaturation of hemoglobin in ether-injection ‘hemosomes’ was increased if the initial methemoglobin content of the hemolysate, or the temperature of preparation was elevated. It was slower if liposomes were prepared under either N₂ or CO, or if the radical scavenger 1,3-diphenylisobenzofuran was added with the ether. Egg phosphatidylcholine and synthetic saturated phospholipids gave the same results. With hand-shaken ‘hemosomes’ the oxidized product was primarily methemoglobin, and oxidation could be inhibited by using saturated phosphatidylcholines instead of egg phosphatidylcholine. Lysophosphatidylcholine levels were higher and arachidonic acid levels lower in egg phosphatidylcholine ‘hemosomes’ than in equivalent liposomes containing no hemolysate. The ‘hemosome’ seems to be a suitable model for the study of hemoglobin-lipid membrane interactions and the resulting hemoglobin denaturation process.     

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.     

Initiation of lipid peroxidation as a result of hemoglobin transformation into hemichrome induced by free fatty acids

The effects of free fatty acids on hemoglobin conversion and lipid peroxidation were studied in hemoglobin-containing liposomes (hemosomes) formed from an equimolar mixture of phosphatidylcholine (PC) and phosphatidylethanolamine (PE). It was shown that in hemosomes oxyhemoglobin is converted into hemichrome by the interaction of saturated fatty acids (arachidic, stearic, palmitic, myristic and lauric). This is accompanied by accumulation of primary and secondary products of lipid peroxidation. All fatty acids, except for lauric acid, have a stabilizing effect on lipid peroxidation in liposomes prepared from an equimolar mixture of PC and PE. The formation of lipid peroxidation products is inhibited by superoxide dismutase, D-alpha-tocopherol, D-mannitol and thiourea. The relationships between hemoglobin conversion and lipid peroxidation in hemosomes under effects of fatty acids were studied. The mechanisms of these reactions are discussed.     

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.     

Pentacoordinate and hexacoordinate ferric hemes in acid medium: EPR, UV-Vis and CD studies of the giant extracellular hemoglobin of Glossoscolex paulistus

The equilibrium complexity involving different axially coordinated hemes is peculiar to hemoglobins. The pH dependence of the spontaneous exchange of ligands in the extracellular hemoglobin from Glossoscolex paulistus was studied using UV-Vis, EPR, and CD spectroscopies. This protein has a complex oligomeric assembly with molecular weight of 3.1 MDa that presents an important cooperative effect. A complex coexistence of different species was observed in almost all pH values, except pH 7.0, where just aquomet species is present. Four new species were formed and coexist with the aquomethemoglobin upon acidification: (i) a "pure" low-spin hemichrome (Type II), also called hemichrome B, with an usual spin state (d(xy))(2)(d(xz),d(yz))(3); (ii) a strong g(max) hemichrome (Type I), also showing an usual spin state (d(xy))(2)(d(xz),d(yz))(3); (iii) a hemichrome with unusual spin state (d(xz),d(yz))(4)(d(xy))(1) (Type III); (iv) and a high-spin pentacoordinate species. CD measurements suggest that the mechanism of species formation could be related with an initial process of acid denaturation. However, it is worth mentioning that based on EPR the aquomet species remains even at acidic pH, indicating that the transitions are not complete. The "pure" low-spin hemichrome presents a parallel orientation of the imidazole ring planes but the strong g(max) hemichrome is a HALS (highly anisotropic low-spin) species indicating a reciprocally perpendicular orientation of the imidazole ring planes. The hemichromes and pentacoordinate formation mechanisms are discussed in detail.     

Proton NMR study of methemoglobin and its isolated chains. Effect of the subunit association on the structure of the subunits.

In order to investigate the effect of the alpha beta subunit contacts on the subunit structure of human adult methemoglobin, the hyperfine shifted proton NMR spectra of several high spin complexes (water, cyanate, thiocyanate, formate, fluoride, and nitrite) and low spin complexes (imisazole, azide, and cyanide) of hemoglobin and its isolated subunits were characterized at 220 MHz and 22 degrees C. The spectra of ferric low spin derivatives of the isolated subunits were approximately superimposable on the corresponding hemoglobin spectra. On the other hand, the high spin spectra of the isolated subunits were greatly different from each other. The spectral anomaly in the ferric high spin complexes of the isolated beta subunit were interpreted to indicate other structural change than the hemichrome formation in the beta heme pocket. Difference in the subunit association effect between the high and low spin complexes of the isolated beta subunit was interpreted on the basis of a conformational change of the apoprotein dependent on the spin state of the beta heme iron.     

Parameters of interaction of sodium dodecyl sulfate with human hemoglobin

It was shown that sodium dodecyl sulfate at concentrations not exceeding the critical micelle concentrations (0-1.9 mM) induced the conversion of oxy- and methemoglobin but not deoxyhemoglobin to hemichrome. The concentration dependences of hemichrome formation were represented as Hill plots, and the parameters of detergent binding were estimated. OxyHb in 20 mM potassium-phosphate buffer, pH 6.8, has two groups of binding sites: the first group is characterized by the Hill constant n1 = 2 and the concentration of half saturation [SDS]50 = 0.8 mM, and the second group is characterized by the Hill constant n2 = 8 and [SDS]50 = 0.9 mM. In the case of metHb one group of binding sites with the Hill constant n = 2 and half saturation concentration [SDS]50 = 0.2 mM was observed. An increase in environmental pH to 7.9 decreased the affinity of Hb for SDS. It is suggested that primary binding sites for SDS in oxyHb coincide with the anion-binding center of the Hb molecule. The interaction of the detergent with these binding sites induced a structural transition of the hemoprotein molecule. As a result of this transition, secondary binding sites were exposed. In a model system (hemin--imidazole in ethanol solution), the enthalpy of the transition of hemin from a high-spin to a low-spin state was estimated to be 47 +/- 7 kJ/mol.     

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.     

Inhibition of erythrocyte superoxide dismutase by diethyldithiocarbamate also results in oxyhemoglobin-catalyzed glutathione depletion and methemoglobin production

N,N-Diethyldithiocarbamate (DDC), a copper-chelating agent, not only inhibits superoxide dismutase activity in the red cell, but also depletes glutathione and promotes the production of methemoglobin, sulfhemoglobin, and small amounts of lipid peroxidation products. DDC reacts with oxyhemoglobin to yield disulfiram, hydrogen peroxide, and methemoglobin. Disulfiram and hydrogen peroxide both convert GSH to GSSG, while DDC reduces methemoglobin to oxyhemoglobin. Although disulfiram also reacts with the hemoglobin sulfhydryl groups, this reaction does not play a role in the conversion of GSH to GSSG. Other hemoglobin derivatives, ferrous, and ferric ions do not catalyze the oxidation of GSH by DDC. These results support the conclusion that DDC reacts with the super-oxo-ferriheme complex of oxyhemoglobin to generate hydrogen peroxide and disulfiram and that the cyclic conversion of oxyhemoglobin to methemoglobin and DDC and disulfiram results in the net oxidation of GSH. Thus, damage to DDC-treated erythrocytes exposed to a putative superoxide-generating toxin, such as 1,4-naphthoquinone-2-sulfonate, may actually be due to diminished GSH concentration and hemoglobin oxidation rather than to superoxide radicals. Glucose added to the incubation medium of DDC-treated erythrocytes fully prevented glutathione depletion but not the oxidation of oxyhemoglobin to methemoglobin. Several other copper-chelating agents either failed to inhibit the activity of purified superoxide dismutase or when incubated with erythrocytes produced more extensive GSH depletion and hemoglobin oxidation than DDC. It is concluded that the interpretation of results with erythrocytes exposed to copper-chelating agents must consider their effects on GSH and hemoglobin as well as on superoxide dismutase inhibition. Moreover, one must be mindful of the interference by DDC in the analysis of GSH with 5,5'-dithiobis-(2-nitrobenzoic acid) in the absence of sufficient quantities of metaphosphoric acid to destroy DDC and that contamination of DDC with trace quantities of disulfiram may be a significant problem.