A recombinant polymeric hemoglobin with conformational,functional, and physiological characteristics of an in vivo O2 transporter

With the objective of developing a recombinant oxygen carrier suitable for therapeutic applications, we have employed an Escherichia coli expression system to synthesize in high-yield hemoglobin (Hb) Minotaur, containing alpha-human and beta-bovine chains. Polymerization of Hb Minotaur through S-S intermolecular cross-linking was obtained by introducing a Cys at position beta9 and substituting the naturally occurring Cys. This homogeneous polymer, Hb Polytaur, has a molecular mass of approximately 500 kDa and was resistant toward reducing agents present in blood. In mice, the circulating half-time (3 h) was fivefold greater than adult human Hb (HbA). The half-time of autooxidation measured in blood (46 h) exceeded the circulating retention time. Hypervolemic exchange transfusion resulted in increased arterial blood pressure similar to that with albumin. The increase in pressure was less than that obtained by transfusion of cross-linked tetrameric Hb known to undergo renovascular extravasation. The nitric oxide reactivity of Hb Polytaur was similar to HbA, suggesting that the diminished pressor response to Hb Polytaur was probably related to diminished extravasation. Transfusion of 3% Hb Polytaur during focal cerebral ischemia reduced infarct volume by 22%. Therefore, site-specific Cys insertion on the Hb surface results in uniform size polymers that do not produce the large pressor response seen with tetrameric Hb. Polymerization maintains physiologically relevant oxygen and heme affinity, stability toward denaturation and oxidation, and effective oxygen delivery as indicated by reduced cerebral ischemic damage.     

The hemoglobin of the sea lamprey,Petromyzon marinus

The blood hemoglobin of the sea lamprey presents a curious mixture of primitive and highly specialized properties. Like muscle hemoglobin, it has a molecular weight of about 17,000, and apparently contains a single heme. Its isoelectric point is like that of a typical invertebrate hemoglobin. Its amino acid composition is partly characteristic of invertebrate) partly of vertebrate hemoglobins (Pedersen; Roche and Fontaine). In the present experiments, the oxygen equilibrium curve of this pigment was measured at several pH's. As expected, it is a rectangular hyperbola, the first such function to be observed in a vertebrate blood hemoglobin. Other hemoglobins known to possess this type of oxygen dissociation curve—those of vertebrate muscle, the worm Nippostrongylus, and the bot-fly larva—appear to serve primarily the function of oxygen storage rather than transport. Lamprey hemoglobin on the contrary is an efficient oxygen-transporting agent. It achieves this status by having, unlike muscle hemoglobin, a relatively low oxygen affinity, and a very large Bohr effect. In these properties it rivals the most effective vertebrate blood hemoglobins.     

Intraerythrocytic organic phosphates of fetal and adult seaperch (Embiotoca lateralis): Their role in maternal-fetal oxygen transport

Summary The viviparous seaperch,Embiotoca lateralis, has unique fetal and adult hemoglobins. Stripped fetal hemoglobin has a higher oxygen affinity than stripped adult hemoglobin at pH 6.5–7.1. The oxygen affinities of both adult and fetal hemoglobins are lowered allosterically by ATP at pH 7.1. Both fetal and adult seaperch erythrocytes include approximately 82% ATP and 18% GTP of the total nucleotide triphosphates (NTP) with a trace of AMP. No 2,3-diphosphoglycerate or inositol polyphosphate was detected. Mid- and late-gestation erythrocytes contain less NTP/mole hemoglobin tetramer than do adult cells. The effective NTP concentration in adult cells is higher than that of the fetal erythrocytes even when the intracellular concentration of Mg²⁺, which complexes with NTP, is accounted for. The difference in adult and fetal intraerythrocytic NTP concentration should enhance transfer of oxygen from maternal to fetal blood. Thus, the teleostEmbiotoca lateralis may employ a dual mechanism in maternal-fetal oxygen transfer. A difference in fetal and maternal hemoglobin structure and oxygen affinities is enhanced by a difference in their respective intraerythrocytic organic phosphate concentrations.     

Oxygen affinities of maternal and fetal hemoglobins of the viviparous seaperch,Embiotoca lateralis

Summary The striped seaperch,Embiotoca lateralis, is a viviparous teleost. The hemoglobins of adult and fetal seaperch are both tetrameric proteins which in their native state appear to be indistinguishable from one another by electrophoresis. However, differences in the subunit structure of maternal versus fetal seaperch hemoglobins can be detected by electrophoresis in urea with a reducing agent, amino acid analyses and peptide maps of the respective proteins. Furthermore, stripped adult and fetal hemoglobins have different oxygen binding affinities at all pH's tested between pH 6.8 and 8.0. Mid-gestation fetal hemoglobin has a higher oxygen affinity than late-gestation fetal hemoglobin which in turn has a higher affinity than that of the adult hemoglobin. All three stripped hemoglobins show a similar Bohr effect (=–0.9). These data suggest that a difference in oxygen affinities exists in vivo between the adult and fetal blood of the seaperchEmbiotoca lateralis and that it can be explained in part by the presence of a structurally unique fetal hemoglobin. This report is the first to provide evidence for a mechanism of maternal-fetal oxygen transfer in a teleost fish.Abbreviations A adult - LF late-gestation fetal - MF mid-gestation fetal (hemoglobins)     

Factors in the evolution of hemoglobin function.

The packaging of vertebrate blood hemoglobins within cells places subtle constraints on hemoglobin evolution. Since the concentration of hemoglobin is near the solubility limit a selective advantage should exist for a noncomplementary external topology of amino acid residues. Further, any change in charge on the protein should alter ion distribution across the cell membrane and so modify ion-sensitive oxygen transport. An efficient hemoglobin must not only combine readily with oxygen at prevailing environmental oxygen pressures, but must also release it at metabolically appropriate pressures. These adaptations frequently employ different strategies to achieve the same objective in different animals. Some hemoglobins have evolved special properties unrelated to the transport of oxygen to metabolizing tissues. Thus many teleost fish have hemoglobins that discharge much of their oxygen at low pH even at high oxygen pressures. This property appears to aid in filling the swim bladder with oxygen. The hemoglobins of elasmobranchs have evoked a unique resistance to urea as a consequence of the high urea content of their blood. Sometimes the functional adaptations of hemoglobins are achieved by multiple hemoglobins in the same cells. Often, however, different red cell populations with functionally unique hemoglobins arise sequentially during ontogeny.     

Tendency for oxidation of annelid hemoglobin at alkaline pH and dissociated states probed by redox titration

The redox titration of extracellular hemoglobin of Glossoscolex paulistus (Annelidea) was investigated in different pH conditions and after dissociation induced by pressure. Oxidation increased with increasing pH, as shown by the reduced amount of ferricyanide necessary for the oxidation of hemoglobin. This behavior was the opposite of that of vertebrate hemoglobins. The potential of half oxidation (E_{1 / 2}) changed from − 65.3 to + 146.8 mV when the pH increased from 4.50 to 8.75. The functional properties indicated a reduction in the log P₅₀ from 1.28 to 0.28 in this pH range. The dissociation at alkaline pH or induced by high pressure, confirmed by HPLC gel filtration, suggested that disassembly of the hemoglobin could be involved in the increased potential for oxidation. These results suggest that the high stability and prolonged lifetime common to invertebrate hemoglobins is related to their low tendency to oxidize at acidic pH, in contrast to vertebrate hemoglobins.     

Structure-Function Relationships in Unusual Nonvertebrate Globins

Based on the literature and our own results, this review summarizes the most recent state of nonvertebrate myoglobin (Mb) and hemoglobin (Hb) research, not as a general survey of the subject but as a case study. For this purpose, we have selected here four typical globins to discuss their unique structures and properties in detail. These include Aplysia myoglobin, which served as a prototype for the unusual globins lacking the distal histidine residue; midge larval hemoglobin showing a high degree of polymorphism; Tetrahymena hemoglobin evolved with a truncated structure; and yeast flavohemoglobin carrying an enigmatic two-domain structure. These proteins are not grouped by any common features other than the fact they have globin domains and heme groups. As a matter of course, various biochemical functions other than the conventional oxygen transport or storage have been proposed so far to these primitive or ancient hemoglobins or myoglobins, but the precise in vivo activity is still unclear.

In this review, special emphasis is placed on the stability properties of the heme-bound O₂. Whatever the possible roles of nonvertebrate myoglobins and hemoglobins may be (or might have been), the binding of molecular oxygen to iron(II) must be the primary event to manifest their physiological functions in vivo. However, the reversible and stable binding of O₂ to iron(II) is not a simple process, since the oxygenated form of Mb or Hb is oxidized easily to its ferric met-form with the generation of superoxide anion. The metmyoglobin or methemoglobin thus produced cannot bind molecular oxygen and is therefore physiologically inactive. In this respect, protozoan ciliate myoglobin and yeast flavohemoglobin are of particular interest in their very unique structures. Indeed, both proteins have been found to have completely different strategies for overcoming many difficulties in the reversible and stable binding of molecular oxygen, as opposed to the irreversible oxidation of heme iron(II). Such comparative studies of the stability of MbO₂ or HbO₂ are of primary importance, not only for a full understanding of the globin evolution, but also for planning new molecular designs for synthetic oxygen carriers that may be able to function in aqueous solution and at physiological temperature.     

Multiple hemoglobins of the cutthroat trout, Salmo clarki

Nine hemoglobins were purified from blood of Salmo clarki by ion-exchange chromatography and preparative isoelectric focusing. The subunit structures of eight of the purified hemoglobins were studied by electrophoresis of globins in the presence of urea. Six are alpha 2 beta 2 tetramers while two appear to be heterotetramers of the type alpha alpha' beta 2 and alpha alpha' beta beta'. The effects of pH, nucleotides, and temperature on the oxygen equilibria of the purified hemoglobins were studied. Five hemoglobins with isoelectric points from 9.1 to 7.1 and one minor hemoglobin with an isoelectric point of 5.9 appear to have essentially identical oxygen binding properties. All have similar oxygen equilibria which are independent of pH and temperature and not affected by saturating amounts of ATP. Another minor hemoglobin with an isoelectric point below 5.9 has similar oxygen equilibria except for a possible pH dependence. Two hemoglobins, with isoelectric points of 6.5 and 6.4, have oxygen binding properties which are strongly pH and temperature dependent. Addition of ATP or GTP causes a large decrease in the oxygen affinity without affecting the cooperativity of oxygen binding. The effect of GTP is slightly greater than that of ATP. No significant differences were observed in the oxygen equilibria of these two hemoglobins. The red blood cells of S. clarki were found to contain large amounts of both ATP and GTP, with an ATP:GTP ratio of 3:1. Both nucleotides may be important modulators of hemoglobin oxygen affinity in S. clarki, in contrast to the situation in S. gairdneri, in which red blood cell GTP concentrations are considerably lower. The presence of six or possibly seven hemoglobins with identical oxygen binding properties in S. clarki suggests that, to a large extent, the physiological role of multiple hemoglobins in this species involves phenomena not directly related to the oxygen binding properties of the hemoglobins.     

Studies on avian erythrocyte metabolism. Effect of organic phosphates on oxygen affinity of embryonic and adult-type hemoglobins of the chick embryo.

The effects of 2,3 diphosphoglyceric acid (2,3-DPG), adenosine triphosphate (ATP), and inositol hexaphosphate (IHP) on the oxygen affinity of whole “stripped” hemoglobin (WSH), hemoglobin H (Hb-H), hemoglobin A (Hb-A) and hemoglobin D (Hb-D) isolated from 18-day chick embryo blood have been determined. The effect of the three organic phosphates upon the oxygen dissociation curves is similar and the following order of decreasing oxygen affinity of the organic phosphates was observed for each hemoglobin: 2,3-DPG < ATP < IHP. 2,3-DPG appears to have a slightly greater effect upon the P₅₀ of Hb-H than upon that of either of the two adult-type hemoglobins. However, this effect seems insufficient to suggest a preferential interaction of 2,3-DPG with Hb-H which would account for either the large amounts of 2,3-DPG in the erythrocytes of embryos or the higher oxygen affinity of the whole blood. The effects of the organic phosphates upon the Hill constant of the purified hemoglobins are variable. It is concluded that since the distribution of hemoglobins H, A, and D in the erythrocytes during the developmental period from 18-day embryos to 6-day chicks remains fairly constant, the previously described progressive decrease in oxygen affinity of the whole blood during this period results from changes in the total amount and distribution of the intraerythrocytic organic phosphates.²     

Purification of hemoglobin from red blood cells using tangential flow filtration and immobilized metal ion affinity chromatography

Two methods for purifying hemoglobin (Hb) from red blood cells (RBCs) are compared. In the first method, red blood cell lysate is clarified with a 50 nm tangential flow filter and hemoglobin is purified using immobilized metal ion affinity chromatography (IMAC). In the second method, RBC lysate is processed with 50 nm, 500 kDa, and 50-100 kDa tangential flow filters, then hemoglobin is purified with IMAC. Our results show that the hemoglobins from both processes produce identical Hb products that are ultrapure and retain their biophysical properties (except for chicken hemoglobin, which shows erratic oxygen binding behavior after purification). Therefore, the most efficient method for Hb purification appears to be clarification with a 50 nm tangential flow filter, followed by purification with IMAC, and sample concentration/polishing on a 10-50 kDa tangential flow filter.     

Primary structure of chain I of the heterodimeric hemoglobin from the blood clamBarbatia virescens

The blood clamBarbatia virescens has a heterodimeric hemoglobin in erythrocytes. Interestingly, the congeneric clamsB. reeveana andB. lima contain quite different hemoglobins: tetramer and polymeric hemoglobin consisting of unusual didomain chain. The complete amino acid sequence of chain I ofB. virescens has been determined. The sequence was mainly determined from CNBr peptides and their subpeptides, and the alignment of the peptides was confirmed by sequencing of PCR-amplified cDNA forB. virescens chain I. The cDNA-derived amino acid sequence matched completely with the sequence proposed from protein sequencing.B. virescens chain I is composed of 156 amino acid residues, and the molecular mass was calculated to be 18,387 D, including a heme group. The sequence ofB. virescens chain I showed 35–42% sequence identity with those of the related clamAnadara trapezia and the congeneric clamB. reeveana. An evolutionary tree forAnadara andBarbatia chains clearly indicates that all of the chains are evolved from one ancestral globin gene, and that the divergence of chains has occurred in each clam after the speciation. The evolutionary rate for clam hemoglobins was estimated to be about four times faster than that of vertebrate hemoglobin. We suggest that blood clam hemoglobin is a physiologically less important molecule when compared with vertebrate hemoglobins, and so it evolved rapidly and resulted in a remarkable diversity in quaternary and subunit structure within a relatively short period.     

Variable Subunit Contact and Cooperativity of Hemoglobins

Tertiary structures of proteins are conserved better than their primary structures during evolution. Quaternary structures or subunit organizations, however, are not always conserved. A typical case is found in hemoglobin family. Although human, Scapharca, and Urechis have tetrameric hemoglobins, their subunit contacts are completely different from each other. We report here that only one or two amino acid replacements are enough to create a new contact between subunits. Such a small number of chance replacements is expected during the evolution of hemoglobins. This result explains why different modes of subunit interaction evolved in animal hemoglobins. In contrast, certain interactions between subunits are necessary for cooperative oxygen binding. Cooperative oxygen binding is observed often in dimeric and tetrameric hemoglobins. Conformational change of a subunit induced by the first oxygen binding to the heme group is transmitted through the subunit contacts and increases the affinity of the second oxygen. The tetrameric hemoglobins from humans and Scapharca have cooperativity in spite of their different modes of subunit contact, but the one from Urechis does not. The relationship between cooperativity and the mode of subunit contacts is not clear. We compared the atomic interactions at the subunit contact surface of cooperative and non-cooperative tetrameric hemoglobins. We show that heme-contact modules M3–M6 play a key role in the subunit contacts responsible for cooperativity. A module was defined as a contiguous peptide segment having compact conformation and its average length is about 15 amino acid residues. We show that the cooperative hemoglobins have interactins involving at least two pairs of modules among the four heme-contact modules at subunit contact. Received: 12 January 2001 / Accepted: 3 April 2001     

Bacterial hemoglobins and flavohemoglobins: versatile proteins and their impact on microbiology and biotechnology

In response to oxygen limitation or oxidative and nitrosative stress, bacteria express three kinds of hemoglobin proteins: truncated hemoglobins (tr Hbs), hemoglobins (Hbs) and flavohemoglobins (flavo Hbs). The two latter groups share a high sequence homology and structural similarity in their globin domain. Flavohemoglobin proteins contain an additional reductase domain at their C-terminus and their expression is induced in the presence of reactive nitrogen and oxygen species. Flavohemoglobins detoxify NO in an aerobic process, termed nitric oxide dioxygenase reaction, which protects the host from various noxious nitrogen compounds. Only a small number of bacteria express hemoglobin proteins and the best studied of these is from Vitreoscilla sp. Vitreoscilla hemoglobin (VHb) has been expressed in various heterologous hosts under oxygen-limited conditions and has been shown to improve growth and productivity, rendering the protein interesting for biotechnology industry. The close interaction of VHb with the terminal oxidases has been shown and this interplay has been proposed to enhance respiratory activity and energy production by delivering oxygen, the ultimate result being an improvement in growth properties.     

Conservation and diversity of ancient hemoglobins in Bacteria

A group of single-domain proteins in Bacteria similar to thermoglobin, an oxygen-avid hemoglobin representative of the ancestral form, reveals the primordial structure, function, and evolvability of the family. Conserved residues at specific positions function to bind ligand or participate in hydrophobic packing of the protein core during protein folding. A potential hydrogen bond network consisting of a tyrosine and glutamine residue in the distal ligand-binding site of most hemoglobins suggests that the ancestral protein bound oxygen avidly. Two divergent hemoglobins with mutations at generally conserved positions contain non-canonical ligand-binding sites, illustrating plasticity of the fold. One binds heme in a manner similar to cytochromes and may represent an evolutionary link to the precursor of the hemoglobin fold. Conservation suggests specific biochemical properties of the ancestral protein; diversity suggests an evolvability of this group of hemoglobins tolerant of mutations that perturb conserved biochemical properties for adaptation to novel functions.     

Peculiarities of structural organization of hemoglobin of <Emphasis Type="Italic">Chiromonus plumosus</Emphasis> L. (Diptera: Chironomidae)

In Ch. plumosus and Ch. riparius there were revealed various structural hemoglobin variants including monomers, dimers, trimers, tetramers, hexamers, and octamers. The multitude of the protein organization ways seems to be based on diversity of monomers with the approximately equal molecular mass from 12 to 16 kDa in Ch. plumosus and from 11.8 to 15.2 kDa in C. riparus. In PAAG with 8 M urea, the hemoglobins were aggregated into high molecular complexes with mol. masses about 260 and 180 kDa in Ch. plumosus and about 523 and 174 kDa in C. riparus.     

Anionic Control of Function in Vertebrate Hemoglobins

Point mutations in the amino acid sequence of normal human hemoglobinhave provided a powerful means of probing structure-functionrelationships in this respiratory protein. Through studies ofspecific hemoglobin variants it has been possible to gain abetter understanding of how electrostatic interactions exercisecontrol over the functional properties of hemoglobin. Humanhemoglobin variants of particular interest in this respect arethose with alterations of amino or carboxyl terminal residuesand alterations at or near the binding site for the physiologicallyimportant cofactor 2,3-diphosphoglycerate. In deoxy hemoglobinit has been established that salt bridges formed by the terminalresidues constrain the tetramer in a low affinity conformation.From the information presently available, it appears that thecharge cluster of the 2,3 diphosphoglycerate binding site isan important part of the innerspring mechanism that tends todestabilize the deoxy conformation. When anions bind to theseresidues the repulsive interactions between the positively chargedresidues of this region are decreased. This provides a directmeans by which anionic interactions with hemoglobin can shiftthe conformational equilibrium toward the low affinity state.Accordingly anion and pH effects are decreased in a number ofhemoglobin variants whose substitutions reduce the positivecharge density in the region of the binding site for polyphosphates.The presence of the charge cluster provides for a fine tuningof hemoglobin s functional properties that is responsive tothe concentration of metabolic effectors in vivo. The degreeto which this is possible varies in the vertebrate hemoglobinswhich have been examined. In human hemoglobin eight positivelycharged residues contribute to the charge cluster and anionicmodulation of oxygen affinity is effective. Susceptibility toanionic modulation is decreased in hemoglobins where the chargecluster is less developed and is completely absent in some vertebratehemoglobins. Anionic modulation, which occurs via an effecton the equilibrium between conformational states of high andlow oxygen affinity is possible even in systems which do notshow cooperative interactions in oxygen binding. This is wellestablished by studies on isolated chains of human hemoglobinand by studies on enzymatically modified tetramers of Amphiumahemoglobin.     

Oxygen equilibria and structural characteristics of the tetrameric and polymeric intracellular hemoglobins from the bivalve molluscBarbatia reeveana

Summary The arcid bivalveBarbatia reeveana contains within its erythrocytes two hemoglobins with remarkably different structures and oxygen equilibrium properties. A tetrameric hemoglobin (M _r about 60,000) with non-identical subunits (₂₂) constitutes about 60% of the erythrocytic heme protein. This hemoglobin has a relatively low oxygen affinity (P ₅₀=19 Torr at 20°C, pH 7.2), shows cooperativityn _H=2.2, shows no Bohr effect between pH 6.8 and 7.6 and a heat of oxygenation (H) of –5.4 kcal/mole between 15 and 35°C. Its oxygen affinity appears to be insensitive to ATP.B. reeveana erythrocytes also contain another hemoglobin withM _r=430,000, the largest intracellular hemoglobin known in any organism. The subunit of this hemoglobin is unusual, having aM _r of 32–34,000 and two heme oxygen binding sites per polypeptide chain. The large hemoglobin has a very low oxygen affinity (P ₅₀=33 Torr at 20°C, pH 7.2), shows slight cooperativity,n _H=1.8, and no Bohr effect (Grinich and Terwilliger 1980). The H at pH 7.2 equals –2.9 kcal/mole, a low value for most hemoglobins, and its O₂ affinity appears to be insensitive to ATP. The two hemoglobins ofB. reeveana, so different in their structure, are also different in their functional properties.