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.     

修饰血红素提高血红蛋白类过氧化物酶活性

利用苯酚或对羟基联苯对血红蛋白的血红素辅基进行化学修饰,将修饰后的血红素与脱辅基血红蛋白进行重组得到新的血红蛋白。以光吸收扫描分析修饰血红素和重组血红蛋白,证明新的重组血红蛋白构建成功。酶活力测定表明,修饰血红素得到的重组血红蛋白的类过氧化物酶活性都比天然血红蛋白的酶活力高,用对羟基联苯修饰血红素得到的重组血红蛋白的酶活提高明显,约是天然血红蛋白的1.6倍。     

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)     

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.     

Heme degradation during autoxidation of oxyhemoglobin

Two fluorescent heme degradation compounds are detected during autoxidation of oxyhemoglobin. These fluorescent compounds are similar to fluorescent compounds formed when hydrogen peroxide reacts with hemoglobin [E. Nagababu and J. M. Rifkind, Biochem. Biophys. Res. Commun. 247, 592-596 (1998)]. Low levels of heme degradation in the presence of superoxide and catalase are attributed to a reaction involving the superoxide produced during autoxidation. The inhibition of most of the degradation by catalase suggests that the hydrogen peroxide generated during autoxidation of oxyhemoglobin produces heme degradation by the same mechanism as the direct addition of hydrogen peroxide to hemoglobin. The formation of the fluorescent degradation products was inhibited by the peroxidase substrate, ABTS, which reduces ferrylhemoglobin to methemoglobin, indicating that ferrylhemoglobin is produced during the autoxidation of hemoglobin. It is the transient formation of this highly reactive Fe(IV) hemoglobin, which is responsible for most of the heme degradation during autoxidation.     

Peroxidase activity of hemoglobin, modified at the carboxylic groups of heme and amino acids]

The effects of modification of heme carboxylic groups by omega-aminoenantic acid and L-phenylalamine on the peroxidase activity of hemoglobin were studied. For this purpose the peroxidase activities of the original compounds--hemin, hemin-aminoenantic acid, hemin-phenylalanine and hemoglobins prepared from the hemin and globin compounds--hemoglobin, aminoenantyl-hemoglobin and phenylalanine hemoglobin--were determined. The dependence of the peroxidase activity of these compounds on their concentrations and pH was analyzed. It was shown that 40--50% modification of the heme carboxylic groups by amino acids decreases the peroxidase activity of the modified hemins and that of modified hemoglobins reconstructed from these hemins and globin. A decrease of the catalytic activity of the hemoglobin derivatives is due to a lower peroxidase activity (as compared to hemin) of the modified hemins. It is thus concluded that the amino acid modification of the carboxylic groups of heme does not affect the heme-protein interactions in the hemoglobin molecule.     

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.     

Conversion of hemoglobin to hemichrome by lysophospholipids.

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

The oxidation process of Antarctic fish hemoglobins.

Analysis of the molecular properties of proteins extracted from organisms living under extreme conditions often highlights peculiar features. We investigated by UV-visible spectroscopy and X-ray crystallography the oxidation process, promoted by air or ferricyanide, of five hemoglobins extracted from Antarctic fishes (Notothenioidei). Spectroscopic analysis revealed that these hemoglobins share a common oxidation pathway, which shows striking differences from the oxidation processes of hemoglobins from other vertebrates. Indeed, simple exposure of these hemoglobins to air leads to the formation of a significant amount of the low-spin hexacoordinated form, denoted hemichrome. This hemichrome form, which is detected under a variety of experimental conditions, can be reversibly transformed to either carbomonoxy or deoxygenated forms with reducing agents. Interestingly, the spectra of the fully oxidized species, obtained by treating the protein with ferricyanide, show the simultaneous presence of peaks corresponding to different hexacoordinated states, the aquomet and the hemichrome. In order to assign the heme region state of the alpha and beta chains, the air-oxidized and ferricyanide-oxidized forms of Trematomus bernacchii hemoglobin were crystallized. Crystallographic analysis revealed that these forms correspond to an alpha(aquomet)-beta(bishistidyl-hemichrome) state. This demonstrates that the alpha and beta chains of Antarctic fish hemoglobins follow very different oxidation pathways. As found for Trematomus newnesi hemoglobin in a partial hemichrome state [Riccio, A., Vitagliano, L., di Prisco, G., Zagari, A. & Mazzarella, L. (2002) Proc. Natl Acad. Sci. USA99, 9801-9806], the quaternary structures of these alpha(aquomet)-beta(bishistidyl-hemichrome) forms are intermediate between the physiological R and T hemoglobin states. Together, these structures provide information on the general features of this intermediate state.     

Recombinant hemoglobin(alpha 29leucine --> phenylalanine, alpha 96valine --> tryptophan, beta 108asparagine --> lysine) exhibits low oxygen affinity and high cooperativity combined with resistance to autoxidation.

Using our hemoglobin expression system in Escherichia coli, we have constructed three recombinant hemoglobins (rHbs) with amino acid substitutions located in the alpha(1)beta(1) and alpha(1)beta(2) subunit interfaces and in the distal heme pocket of the alpha-chain: rHb(alphaV96W, betaN108K), rHb(alphaL29F, alphaV96W, betaN108K), and rHb(alphaL29F). rHb(alphaV96W, betaN108K) exhibits low oxygen affinity and high cooperativity and also ease of autoxidation of the heme iron atoms from the Fe2+ state to the Fe3+ state. It has been reported by Olson and co-workers [Carver et al., (1992) J. Biol. Chem. 267, 14443-14450; Brantley et al. (1993) J. Biol. Chem. 268, 6995-7010] that a mutation at position 29 (B10, helix notation), e.g. , Leu --> Phe, can inhibit the autoxidation of the heme iron of myoglobin. We have introduced such a mutation into our rHb having low oxygen affinity and high cooperativity. This triply mutated rHb(alphaL29F, alphaV96W, betaN108K) is stabilized against autoxidation and azide-induced oxidation compared to the double mutant, rHb(alphaV96W, betaN108K), but still exhibits low oxygen affinity and good cooperativity. According to electron paramagnetic resonance results, the oxidized form of the triple mutant shows a high ratio of an anionic form of bishistidine hemichrome. Previous reports have suggested that this form does not have water present at the distal heme pocket. (1)H nuclear magnetic resonance spectra of the triple mutant in the ferric state also exhibit spectral features characteristic of hemichrome-type signals. We have carried out a series of biochemical measurements to characterize these three interesting rHbs and to compare them to human normal adult hemoglobin. These results provide new insights into the structure-function relationship of hemoglobin with amino acid substitutions in the alpha(1)beta(1) and alpha(1)beta(2) interfaces and in the heme pockets.     

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.     

Denaturant dependence of equilibrium unfolding intermediates and denatured state structure of horse ferricytochrome c

Nuclear magnetic resonance spectroscopy is employed to characterize unfolding intermediates and the denatured state of horse ferricytochrome c in guanidine hydrochloride. Unfolded and partially unfolded species with non-native heme ligation are detected by analysis of hyperfine-shifted (1)H resonances. Two equilibrium unfolding intermediates with His-Lys heme axial ligation are detected, as are two unfolded species with bis-His heme ligation. These results are contrasted with previous results on horse ferricytochrome c denaturation by urea, for which only one unfolding intermediate and one unfolded species were detected by NMR spectroscopy. Urea and guanidine hydrochloride are often used interchangeably in protein denaturation studies, but these results and those of others indicate that unfolded and intermediate states in these two denaturants may have substantially different properties. Implications of these results for folding studies and the biological function of mitochondrial cytochromes c are discussed.     

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.     

Clotrimazole enhances lysis of human erythrocytes induced by t-BHP

Clotrimazole (CLT) is an antifungal and antimalarial agent also effective as a Gardos channel inhibitor. In addition, CLT possesses antitumor properties. Recent data provide evidence that CLT forms a complex with heme (hemin), which produces a more potent lytic effect than heme alone. This study addressed the effect of CLT on the lysis of normal human erythrocytes induced by tert-butyl hydroperoxide (t-BHP). For the first time, it was shown that 10 μM CLT significantly enhanced the lytic effect of t-BHP on erythrocytes in both Ca²⁺-containing and Ca²⁺-free media, suggesting that the effect is not related to Gardos channels. CLT did not affect the rate of free radical generation, the kinetics of GSH degradation, methemoglobin formation and TBARS generation; therefore, we concluded that CLT does not cause additional oxidative damage to erythrocytes treated with t-BHP. It is tempted to speculate that CLT enhances t-BHP-induced changes in erythrocyte volume and lysis largely by forming a complex with hemin released during hemoglobin oxidation in erythrocytes: the CLT–hemin complex destabilizes the cell membrane more potently than hemin alone. If so, the effect of CLT on cell membrane damage during free-radical oxidation may be used to increase the efficacy of antitumor therapy.     

Spin label studies on conformational changes of aphohemoglobin due to heme binding.

Human apohemoglobin (globin) was spin-labeled at the beta-93 sulfhydryl groups with 2,2,5,5-tetramethyl-3-aminopyrrolidine-I-oxyl. Spin-labeled globin exhibited an EPR spectra that is less immobilized than that of spin-labeled hemoglobin, indicating the conformational difference in the vicinity of the label between hemoglobin and globin. Spectrophotometric titration of spin-labeled globin with protohemin showed that 1 mol of globin (on the tetramer basis) combines with 4 mol of hemin, producing a holomethemoglobin spectrophotometrically indistinguishable from native methemoglobin. The EPR spectrum was also changed strikingly upon the addition of protohemin. This change, however, was not proportional to the amount of hemin added, but marked changes occurred after 3 to 4 mol of hemin were mixed with 1 mol of spin-labeled globin. The EPR spectrum of spin-labeled hemoglobin thus prepared was identical with that prepared by direct spin labeling to methemoglobin. These results suggest the preferential binding of hemin to alpha-globin chains in the course of heme binding by globin. This assumption was further confirmed by preparing spin-labeled semihemoglobin in which only one kind of chain contained hemin (alpha h betaO SL and alpha O beta h SL). The EPR spectrum of the alpha h beta O SL molecule showed a slightly immobilized EPR spectrum, similar to that of spin-labeled globin mixed with 50% of the stoichiometric amount of hemin. On the other hand, the alpha O beta h SL molecule showed a distinctly different EPR signal from that of globin half-saturated with hemin, and showed an intermediate spectrum between those of beta h SL and alpha h beta h SL. These results indicate that heme binding to globin chains brings about a major conformational change in the protein moiety and that chain-chain association plays a secondary role. We conclude that hemin binds preferentially to alpha-globin chains and that the conformation of globin changes rapidly to that of methemoglobin after all four hemes are attached to globin heme pockets.     

Structural and functional properties of class 1 plant hemoglobins

Nonsymbiotic class 1 plant hemoglobins are induced under hypoxia. Structurally they are protein dimers consisting of two identical subunits, each containing heme iron in a weak hexacoordinate state. The weak hexacoordination of heme-iron binding to the distal histidine results in an extremely high avidity to oxygen, with a dissociation constant in the nanomolar range. This low dissociation constant is due to rapid oxygen binding resulting in protein conformational changes that slow dissociation from the heme site. Class 1 hemoglobins are characterized by an increased rate of Fe3(+) reduction which is likely mediated by cysteine residue. This cysteine can form a reversible covalent bond between two monomers as shown by mass spectrometry analysis and, in addition to its structural role, prevents the molecule from autoxidation. The structural properties of class 1 hemoglobins allow them to serve as soluble electron transport proteins in the enzymatic system scavenging nitric oxide produced in low oxygen via reduction of nitrite. During oxygenation of nitric oxide to nitrate, oxidized ferric hemoglobin is formed (methemoglobin), which can be reduced by an associated reductase. The identified candidate for this reduction is monodehydroascorbate reductase. It is suggested that hemoglobin functions as a terminal electron acceptor during the hypoxic turnover of nitrogen, the process aided by its extremely high affinity for oxygen.     

Occurrence and formation of endogenous histidine hexa-coordination in cold-adapted hemoglobins

Spectroscopic and crystallographic evidence of endogenous (His) ligation at the sixth coordination site of the heme iron has been reported for monomeric, dimeric, and tetrameric hemoglobins (Hbs) in both ferrous (hemochrome) and ferric (hemichrome) oxidation states. In particular, the ferric bis- histidyl adduct represents a common accessible ordered state for the β chains of all tetrameric Hbs isolated from Antarctic and sub-Antarctic fish. Indeed, the crystal structures of known tetrameric Hbs in the bis-His state are characterized by a different binding state of the α and β chains. An overall analysis of the bis-histidyl adduct of globin structures deposited in the Protein Data Bank reveals a marked difference between hemichromes in tetrameric Hbs compared to monomeric/dimeric Hbs. Herein, we review the structural, spectroscopic and stability features of hemichromes in tetrameric Antarctic fish Hbs. The role of bis-histidyl adducts is also addressed in a more evolutionary context alongside the concept of its potential physiological role.     

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

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

Modification of isolated α and β chains of human hemoglobin by the metal tetrasulfonated phthalocyanines. Studies on hybrid phthalocyanine-heme intermediates

α and β chains of hemoglobin have been modified with cobalt(II) tetrasulfonated phthalocyanine in place of heme. They display properties very similar to those of iron(II) phthalocyanine modified α and β chains. Mixed together they form tetrameric cobalt(II) phthalocyanine hemoglobin.Incorporation of Co(II)L into α and β globins results in stabilization of the protein structure, which is shown by a marked increase in its helicity content. Cobalt phthalocyanine substituted α and β chains are able to combine reversibly with oxygen giving more stable oxygenated species than their native analogues. The rate of both processes is lower in the case of the modified α chain. Recombination of the phthalocyanine α and β chains with the alternate heme containing chains give tetrameric hybrid hemoglobins. These comprise two phthalocyanine modified subunits and two heme containing subunits. The helicity content of the tetrameric hybrid hemoglobin calculated for one subunit is lower that the arithmetic mean of helicities for its isolated subunits. This suggests a destabilizing chain-chain interaction within the tetramer. Unlike in the separated subunits, oxygen binding by hybrid hemoglobins is irreversible. Deoxygenation by argon bubbling leads to the formation of inactive species which in oxygen atmosphere undergo irreversible oxidation with destruction of the complex.     

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.