Unique features of the hemoglobin system of the Antarctic notothenioid fish Gobionotothen gibberifrons.

The hemolysate of the Antarctic teleost Gobionotothen gibberifrons (family Nototheniidae) contains two hemoglobins (Hb 1 and Hb 2). The concentration of Hb 2 (15-20% of the total hemoglobin content) is higher than that found in most cold-adapted Notothenioidei. Unlike the other Antarctic species so far examined having two hemoglobins, Hb 1 and Hb 2 do not have globin chains in common. Therefore this hemoglobin system is made of four globins (two alpha- and two beta-chains). The complete amino-acid sequence of the two hemoglobins (Hb 1, alpha2(1)beta2(1); Hb 2, alpha2(2)beta2(2)) has been established. The two hemoglobins have different functional properties. Hb 2 has lower oxygen affinity than Hb 1, and higher sensitivity to the modulatory effect of organophosphates. They also differ thermodynamically, as shown by the effects on the oxygen-binding properties brought about by temperature variations. The oxygen-transport system of G. gibberifrons, with two functionally distinct hemoglobins, suggests that the two components may have distinct physiological roles, in relation with life style and the environmental conditions which the fish may have to face. The unique features of the oxygen-transport system of this species are reflected in the phylogeny of the hemoglobin amino-acid sequences, which are intermediate between those of other fish of the family Nototheniidae and of species of the more advanced family Bathydraconidae.     

The hemoglobins of the cold-adapted Antarctic teleost Cygnodraco mawsoni

The blood of the teleost Cygnodraco mawsoni, of the endemic Antarctic family Bathydraconidae, contains a major hemoglobin (Hb 1), accompanied by a minor component (Hb 2, about 5% of total). The two hemoglobins have identical alpha chains and differ by the beta chain. The complete amino acid sequence of the three chains has been elucidated, thus establishing the primary structure of both hemoglobins. The sequences show a 53-65% identity with non-Antarctic poikilotherm fish species; on the other hand, a very high degree of similarity (83-88%) has been found between Hb 1 and the major component of another Antarctic species of a different family. The hemoglobin functional properties relative to oxygen binding have been investigated in intact erythrocytes, 'stripped' hemolysate and purified components of C. mawsoni. The hemoglobins display the Bohr and Root effects, indicating fine regulation of oxygen binding by pH and by the physiological effectors organic phosphates.     

The hemoglobin system of Antarctic and non-Antarctic notothenioid fishes

Studies of the hemoglobin system of fish of the suborder Notothenioidei have been extended to non-Antarctic species of Pseudaphritis urvillii and Notothenia angustata. The two species belong to families that were the first to diverge within the suborder. The degree of amino acid sequence identity with Antarctic notothenioids and other non-Antarctic fish species is analyzed with respect to phyletic and ecological diverence.     

Hemoglobin from the Antarctic fish Notothenia coriiceps neglecta. 1. Purification and characterisation

Antarctic fishes live at a constant temperature of -1.8 degrees C, in an oxygen-rich environment. In comparison with fishes that live in temperate or tropical waters, their blood contains less erythrocytes and hemoglobin. A study was initiated on the structure and function of Antarctic fish hemoglobin. The erythrocytes of the Antarctic benthic teleost Notothenia coriiceps neglecta, of the family Nototheniidae, have been shown to contain two hemoglobins, accounting for about 90% and 5% of the total content. These hemoglobins have been isolated, and obtained in crystalline form. They are tetramers and contain two pairs of globin chains. The globin chains of each hemoglobin have been purified and characterised. The two hemoglobins appear to have one of the two globin chains in common. The Root and Bohr effects have been investigated in erythrocytes, 'stripped' hemolysates and pure hemoglobins, indicating that the functional properties are finely regulated by pH and allosteric effectors.     

Structure and function of the Gondwanian hemoglobin of Pseudaphritis urvillii, a primitive notothenioid fish of temperate latitudes

The suborder Notothenioidei dominates the Antarctic ichthyofauna. The non-Antarctic monotypic family Pseudaphritidae is one of the most primitive families. The characterization of the oxygen-transport system of euryhaline Pseudaphritis urvillii is herewith reported. Similar to most Antarctic notothenioids, this temperate species has a single major hemoglobin (Hb 1, over 95% of the total). Hb 1 has strong Bohr and Root effects. It shows two very uncommon features in oxygen binding: At high pH values, the oxygen affinity is exceptionally high compared to other notothenioids, and subunit cooperativity is modulated by pH in an unusual way, namely the curve of the Hill coefficient is bell-shaped, with values approaching 1 at both extremes of pH. Molecular modeling, electronic absorption and resonance Raman spectra have been used to characterize the heme environment of Hb 1 in an attempt to explain these features, particularly in view of some potentially important nonconservative replacements found in the primary structure. Compared to human HbA, no major changes were found in the structure of the proximal cavity of the alpha-chain of Hb 1, although an altered distal histidyl and heme position was identified in the models of the beta-chain, possibly facilitated by a more open heme pocket due to reduced steric constraints on the vinyl substituent groups. This conformation may lead to the hemichrome form identified by spectroscopy in the Met state, which likely fulfils a potentially important physiological role.     

Hemoglobin from the antarctic fish Notothenia coriiceps neglecta. Amino acid sequence of the beta chain

1. Notothenia coriiceps neglecta is a cold-adapted notothenioid teleost, widely distributed in the Antarctic waters. 2. In comparison with fishes from temperate waters, the blood of this teleost contains a reduced number of erythrocytes and concentration of hemoglobin; the erythrocytes contain two hemoglobins, Hb1 and Hb2, respectively accounting for approximately 90, and 5% of the total. 3. The two components differ by the alpha chain; the amino acid sequence of the beta chain in common to the two hemoglobins has been established, thus completing the elucidation of the primary structure of the major component Hb 1.     

Hemoglobin of the Antarctic fishes Trematomus bernacchii and Trematomus newnesi: structural basis for the increased stability of the liganded tetramer relative to human hemoglobin

Hemoglobins extracted from fishes that live in temperate waters show little or no dissociation even in the liganded form, unlike human hemoglobin (HbA). To establish whether cold adaptation influences the tendency to dissociate, the dimer-tetramer association constants (L(2,4)) of the carbonmonoxy derivatives of representative hemoglobins from two Antarctic fishes, Trematomus newnesi (Hb1Tn) and Trematomus bernacchii (Hb1Tb), were determined by analytical ultracentrifugation as a function of pH in the range 6.0-8.6 and compared to HbA. HbA is more dissociated than fish hemoglobins at all pH values and in particular at pH 6.0. In contrast, both fish hemoglobins are mostly tetrameric over the whole pH range studied. The extent of hydrophobic surface area buried at the alpha(1)beta(2) interface upon association of dimers into tetramers and the number of hydrogen bonds formed are currently thought to play a major role in the stabilization of the hemoglobin tetramer. These contributions were derived from the X-ray structures of the three hemoglobins under study and found to be in good agreement with the experimentally determined L(2,4) values. pH affects oxygen binding of T. bernacchii and T. newnesi hemoglobins in a different fashion. The lack of a pH effect on the dissociation of the liganded proteins supports the proposal that the structural basis of such effects resides in the T (unliganded) structure rather than in the R (liganded) one.     

Hemoglobin structure/function and globin-gene evolution in the Arctic fish Liparis tunicatus

The importance of the Arctic, in contributing to the knowledge of the overall ensemble of adaptive processes influencing the evolution of marine organisms, calls for investigations on molecular adaptations in Arctic fish. Unlike the vast majority of Antarctic Notothenioidei, several Arctic species display high hemoglobin multiplicity. The blood of four species, the spotted wolffish of the family Anarhichadidae and three Gadidae, contains three functionally distinct major components. Similar to many Antarctic notothenioids, Arctic Liparis tunicatus (suborder Cottoidei, family Liparidae) has one major hemoglobin (Hb 1) accompanied by a minor component (Hb 2). This paper reports the structural and functional characterisation of Hb 1 of L. tunicatus. This hemoglobin shows low oxygen affinity, and pronounced Bohr and Root effects. The amino-acid sequence of the beta chain displays an unusual substitution in NA2 (beta2) at the phosphate-binding site, and the replacement of Val E11 (beta67) with Ile. Similar to some Antarctic fish Hbs, electron paramagnetic resonance spectra reveal the formation of a ferric penta-coordinated species even at physiological pH. The amino-acid sequences have also been used to gain insight into the evolutionary history of globins of polar fish. L. tunicatus globins appear close to the notothenioid clades as predicted by teleostean phylogenies. Close phylogenetic relationships between Cottoidei and Notothenioidei, together with their life style, seem to be the main factor driving the globin-sequence evolution.     

The functionally distinct hemoglobins of the Arctic spotted wolffish Anarhichas minor

The Arctic fish Anarhichas minor, a benthic sedentary species, displays high hemoglobin multiplicity. The three major hemoglobins (Hb 1, Hb 2, and Hb 3) show important functional differences in pH and organophosphate regulation, subunit cooperativity, and response of oxygen binding to temperature. Hb 1 and Hb 2 display a low, effector-enhanced Bohr effect and no Root effect. In contrast, Hb 3 displays pronounced Bohr and Root effects, accompanied by strong organophosphate regulation. Hb 1 has the beta (beta(1)) chain in common with Hb 2; Hb 3 and Hb 2 share the alpha (alpha(2)) chain. The amino acid sequences have been established. Several substitutions in crucial positions were observed, such as Cys in place of C-terminal His in the beta(1) chain of Hb 1 and Hb 2. In Hb 3, Val E11 of the beta(2) chain is replaced by Ile. Homology modeling revealed an unusual structure of the Hb 3 binding site of inositol hexakisphoshate. Phylogenetic analysis indicated that only Hb 2 displays higher overall similarity with the major Antarctic hemoglobins. The oxygen transport system of A. minor differs remarkably from those of Antarctic Notothenioidei, indicating distinct evolutionary pathways in the regulatory mechanisms of the fish respiratory system in the two polar environments.     

Structure and function of hemoglobin in antarctic fishes and evolutionary implications

Summary The hematological features of cold-adapted, red-blooded Antarctic teleosts has prompted this study on the relationship between hemoglobin molecular structure and oxygen-binding properties. The hemolysates from 21 species of 5 families contained one component (Hb 1), often accompanied by an additional, minor one (Hb 2, 5%–10% of total). On the other hand, 3 species of Zoarcidae, a non-endemic family, had 4–5 components. All purified hemoglobins from the former group, but only 1–2 of the 4–5 hemoglobins of Zoarcidae, showed a strong Root effect (pH regulation of oxygen binding). Globins from each hemoglobin have been purified and characterised with respect to molecular structure in several species. The similarity between the complete amino acid sequence of one -chain and those of non-Antarctic -chains is lower than that among the latter sequences, suggesting independent pathways of evolution.Presented at the 5th SCAR Symposium on Antarctic Biology, Hobart, Australia (August 29th-September 3rd, 1988)     

Temperature differentially affects adenosine triphosphatase activity in Hsc70 orthologs from Antarctic and New Zealand notothenioid fishes

To test the temperature sensitivity of molecular chaperones in poikilothermic animals, we purified the molecular chaperone Hsc70 from 2 closely related notothenioid fishes--the Antarctic species Trematomus bernacchii and the temperate New Zealand species Notothenia angustata--and characterized the effect of temperature on Hsc70 adenosine triphosphatase (ATPase) activity. Hsc70 ATPase activity was measured using [alpha-32P]-adenosine triphosphate (ATP)-based in vitro assays followed by separation of adenylates by thin-layer chromatography. For both species, a significant increase in Hsc70 ATPase activity was observed across a range of temperatures that was ecologically relevant for each respective species. Hsc70 from T bernacchii hydrolyzed 2-fold more ATP than did N angustata Hsc70 at 0 degrees C, suggesting that the Antarctic molecular chaperone may be adapted to function more efficiently at extreme cold temperatures. In addition, Q10 measurements indicate differential temperature sensitivity of the ATPase activity of Hsc70 from these differentially adapted fish that correlates with the temperature niche inhabited by each species. Hsc70 from T bernacchii was relatively temperature insensitive, as indicated by Q10 values calculated near 1.0 across each temperature range measured. In the case of Hsc70 purified from N angustata, Q10 values indicated thermal sensitivity across the temperature range of 0 degrees C to 10 degrees C, with a Q10 of 2.714. However, Hsc70 from both T bernacchii and N angustata exhibited unusually high thermal stabilities with ATPase activity at temperatures that far exceeded temperatures encountered by these fish in nature. Overall, as evidenced by in vitro ATP hydrolysis, Hsc70 from T bernacchii and N angustata displayed biochemical characteristics that were supportive of molecular chaperone function at ecologically relevant temperatures.     

The amino acid sequence of the alpha chain of HB 2 completes the primary structure of the hemoglobins of the Antarctic fish Notothenia coriiceps neglecta

1. The blood of Notothenia coriiceps neglecta (a cold-adapted notothenioid fish, widely distributed in Antarctic waters, and characterized by a relatively low content of erythrocytes and hemoglobin), contains two hemoglobin components, Hb 1 and Hb 2; the amino acid sequences of the beta chain of Hb 1 and Hb 2 are identical. 2. The amino acid sequence of the alpha chain of Hb 2 has been established, thus completing the elucidation of the primary structure of the two hemoglobins.     

Axolotl hemoglobin: cDNA-derived amino acid sequences of two alpha globins and a beta globin from an adult Ambystoma mexicanum

Erythrocytes of the adult axolotl, Ambystoma mexicanum, have multiple hemoglobins. We separated and purified two kinds of hemoglobin, termed major hemoglobin (Hb M) and minor hemoglobin (Hb m), from a five-year-old male by hydrophobic interaction column chromatography on Alkyl Superose. The hemoglobins have two distinct alpha type globin polypeptides (alphaM and alpham) and a common beta globin polypeptide, all of which were purified in FPLC on a reversed-phase column after S-pyridylethylation. The complete amino acid sequences of the three globin chains were determined separately using nucleotide sequencing with the assistance of protein sequencing. The mature globin molecules were composed of 141 amino acid residues for alphaM globin, 143 for alpham globin and 146 for beta globin. Comparing primary structures of the five kinds of axolotl globins, including two previously established alpha type globins from the same species, with other known globins of amphibians and representatives of other vertebrates, we constructed phylogenetic trees for amphibian hemoglobins and tetrapod hemoglobins. The molecular trees indicated that alphaM, alpham, beta and the previously known alpha major globin were adult types of globins and the other known alpha globin was a larval type. The existence of two to four more globins in the axolotl erythrocyte is predicted.     

Some like it hot, some like it cold: the heat shock response is found in New Zealand but not Antarctic notothenioid fishes

Previous research on Antarctic notothenioids has demonstrated that cells of cold-adapted Antarctic notothenioids lack a common cellular defense mechanism called the heat shock response (HSR), the induction of a family of heat shock proteins (Hsps) in response to elevated temperatures. The goal of this study was to address how widespread the loss of the HSR is within the Notothenioidei suborder and, specifically, to ask whether cold temperate non-Antarctic notothenioids possess the HSR. In general, Antarctic fish have provided an important opportunity for physiologists to examine responses to selection in the environment and to ask whether traits of the notothenioids represent cold adaptation, or whether the traits are related to history and are characteristics of the notothenioid lineage. Using in vivo metabolic labeling, results indicate that one of the two New Zealand notothenioids possess an HSR. The thornfish, Bovichtus variegatus Richardson, 1846, expressed heat shock proteins (Hsp) in response to heat stress, whereas the black cod, Notothenia angustata Hutton, 1875, did not display robust stress-inducible Hsp synthesis at the protein-level. However, further analysis using Northern blotting clearly demonstrated that mRNA for a common Hsp gene, hsp70, was present in cells of both New Zealand species following exposure to elevated temperatures. Overall, combined evidence on the HSR in notothenioid fishes from temperate New Zealand waters indicate that the loss of the HSR in Antarctic notothenioid fishes occurred after the separation of Bovichtidae from the other Antarctic notothenioid families, and that the HSR was most likely lost during evolution at cold and constant environmental temperatures.     

High-altitude respiration of birds. Structural adaptations in the major and minor hemoglobin components of adult Rüppell's Griffon (Gyps rueppellii, Aegypiinae): a new molecular pattern for hypoxic tolerance

The primary structures of the hemoglobins Hb A, Hb A', Hb D and Hb D' of Rüppell's Griffon (Gyps rueppellii), which can fly as high as 11,300 m, are presented. The globin chains were separated on CM-Cellulose in 8M urea buffers, the four hemoglobin components by FPLC in phosphate buffers. The amino-acid sequences of five globin chains were established by automatic Edman degradation of the globin chains and of the tryptic peptides in liquid-phase and gas-phase sequenators. The sequences are compared with those of other Falconiformes. A new molecular pattern for survival at extreme altitudes is presented. For the first time four hemoglobins are found in blood of a bird; they show identical beta-chains and differ in the alpha A- and alpha D-chains by only one replacement. These four hemoglobins cause a gradient in oxygen affinities. The two main components Hb A and Hb A' differ at position alpha 34 Thr/Ile. In case of Ile as found in Hb A' an alpha 1 beta 1-interface is interrupted raising oxygen affinity compared to Hb A. In addition the hemoglobins of the A- and D-groups differ at position alpha 38 Pro or Gln/Thr (alpha 1 beta 2-interface). Expression of Gln in Hb D/D' raises the oxygen affinity of these components compared to Hb A/A' by destabilization of the deoxy-structure. The physiological advantage lies in the functional interplay of four hemoglobin components. Three levels of affinity are predicted: low affinity Hb A, Hb A' of intermediate affinity, and high affinity Hb D/D'. This cascade tallies exactly with oxygen affinities measured in the isolated components and predicts oxygen transport by the composite hemoglobins over an extended range of oxygen affinities. It is contended that the mechanisms of duplication of the alpha-genome (creating four hemoglobins) and of nucleotide replacements (creating different functional properties) are responsible for this remarkable hypoxic tolerance to 11,300 m. Based on this pattern the hypoxic tolerances of other vultures are predicted.     

Biochemical adaptations of notothenioid fishes: comparisons between cold temperate South American and New Zealand species and Antarctic species

Fishes of the perciform suborder Notothenioidei afford an excellent opportunity for studying the evolution and functional importance of diverse types of biochemical adaptation to temperature. Antarctic notothenioids have evolved numerous biochemical adaptations to stably cold waters, including antifreeze glycoproteins, which inhibit growth of ice crystals, and enzymatic proteins with cold-adapted specific activities (k(cat) values) and substrate binding abilities (K(m) values), which support metabolism at low temperatures. Antarctic notothenioids also exhibit the loss of certain biochemical traits that are ubiquitous in other fishes, including the heat-shock response (HSR) and, in members of the family Channichthyidae, hemoglobins and myoglobins. Tolerance of warm temperatures is also truncated in stenothermal Antarctic notothenioids. In contrast to Antarctic notothenioids, notothenioid species found in South American and New Zealand waters have biochemistries more reflective of cold-temperate environments. Some of the contemporary non-Antarctic notothenioids likely derive from ancestral species that evolved in the Antarctic and later "escaped" to lower latitude waters when the Antarctic Polar Front temporarily shifted northward during the late Miocene. Studies of cold-temperate notothenioids may enable the timing of critical events in the evolution of Antarctic notothenioids to be determined, notably the chronology of acquisition and amplification of antifreeze glycoprotein genes and the loss of the HSR. Genomic studies may reveal how the gene regulatory networks involved in acclimation to temperature differ between stenotherms like the Antarctic notothenioids and more eurythermal species like cold-temperate notothenioids. Comparative studies of Antarctic and cold-temperate notothenioids thus have high promise for revealing the mechanisms by which temperature-adaptive biochemical traits are acquired - or through which traits that cease to be of advantage under conditions of stable, near-freezing temperatures are lost - during evolution.     

Functional antifreeze glycoprotein genes in temperate-water New Zealand nototheniid fish infer an Antarctic evolutionary origin

The fish fauna of the Antarctic Ocean is dominated by five endemic families of the Perciform suborder Notothenioidei, thought to have arisen in situ within the Antarctic through adaptive radiation of an ancestral stock that evolved antifreeze glycoproteins (AFGPs) enabling survival as the ocean chilled to subzero temperatures. The endemism results from geographic confinement imposed by a massive oceanographic barrier, the Antarctic Circumpolar Current, which also thermally isolated Antarctica over geologic time, leading to its current frigid condition. Despite this voluminous barrier to fish dispersal, a number of species from the Antarctic family Nototheniidae now inhabit the nonfreezing cool temperate coasts of the southern continents. The origin of these temperate-water nototheniids is not completely understood. Since the AFGP gene apparently evolved only once, before the Antarctic notothenioid radiation, the presence of AFGP genes in extant temperate-water nototheniids can be used to infer an Antarctic evolutionary origin. Genomic Southern analysis, PCR amplification of AFGP genes, and sequencing showed that Notothenia angustata and Notothenia microlepidota endemic to southern New Zealand have two to three AFGP genes, structurally the same as those of the Antarctic nototheniids. At least one of these genes is still functional, as AFGP cDNAs were obtained and low levels of mature AFGPs were detected in the blood. A phylogenetic tree based on complete ND2 coding sequences showed monophyly of these two New Zealand nototheniids and their inclusion in the monophyletic Nototheniidae consisted of mostly AFGP-bearing taxa. These analyses support an Antarctic ancestry for the New Zealand nototheniids. A divergence time of approximately 11 Myr was estimated for the two New Zealand nototheniids, approximating the upper Miocene northern advance of the Antarctic Convergence over New Zealand, which might have served as the vicariant event that lead to the northward dispersal of their most recent common ancestor. Similar secondary northward dispersal likely applies to the South American nototheniid Paranotothenia magellanica, which has four AFGP genes in its DNA, but not to the sympatric nototheniid Patagonotothen tessellata, which does not appear to have any AFGP sequences in its genome at all.     

Glutathione mixed disulfides and heterogeneity of chicken hemoglobins.

1. Adult chicken hemoglobins Hb A and Hb D interact with glutathione disulfide, GSSG. The major hemoglobin, Hb A, forms at least two new components, termed GHb AI and GHb AII, and Hb D forms at least one, GHb DI. 2. At pH 8.0 and 5 degrees C, glutathione disulfide (GSSG) in a molar excess of 50 x took 6 days to complete the reaction, although at pH 8.6 and 41 degrees C only 1 hr was needed, where the hemoglobins Hb A and Hb D were converted to their most mobile forms GHb AII and GHb DI. 3. Slight molar excess (2.7 GSSG/Hb, pH 7.4, 41 degrees C), reacting for 1 hr, showed extensive formation of GHb AI and some GHb AII. 4. Electrophoretic patterns, from the reaction products of 54 GSSG/Hb excess at different times, showed a marked pH dependence. 5. Titration with pCMB (p-chloromercuribezoic acid) of DTE (dithioerythrytol)-reduced samples showed 8.0 +/- 0.4 (N = 5) -SH (sulfhydryl) per tetramer. In hemolysates not reacted with DTE, 6.0 +/- 0.4 (N = 3) -SH were detected. 6. DTE-reduced and GSSG-reacted hemoglobins showed 4.6 +/- 0.5 (N = 7) -SH and 1.5 +/- 0.4 (N = 6) -SH, respectively, as titrated by DTNB, pH 8.0. DTE-reduced hemoglobins showed four fast-reacting -SH groups, no longer present in GSSG-reacted hemoglobins. 7. Our data indicate that chicken GHb AI and GHb DI probably have two glutathionyl residues per tetramer whereas GHb AII has four.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proton nuclear magnetic resonance studies of hemoglobin Malm?: implications of mutations at homologous positions of the alpha and beta chains.

The abnormal human hemoglobin Malm? (beta97FG4 His leads to Gln) has been studied and its properties are compared with those of normal adult hemoglobin A. The data presented here show that the ring-current shifted proton resonances of both HbCO and HbO2 Malm? are very different from the corresponding forms of Hb A. The hyperfine shifted proton resonances of deoxy-Hb Malm? do not differ drastically from those of deoxy-Hb A. This result, together with the finding that the exchangeable proton resonances of the deoxy form of the two hemoglobins are similar, suggests that unliganded Hb Malm? can assume a deoxy-like quaternary structure both in the absence and presence of organic phosphates We have also compared the properties of Hb Malm? with those of Hb Chesapeake (alpha92FG4 Arg leads to Leu). This allows us to study the properties of two abnormal human hemoglobins with mutations at homologous positions of the alpha and beta chains in the three-dimenstional structure of the hemoglobin molecule. Our present results suggest that the mutaion at betaFG4 has its greatest effect on the teritiary structure of the heme pocket of the liganded forms of the hemoglobin while the mutation at alphaFG4 alters the deoxy structure of the hemoglogin molecule but does not alter the teriary structure of the heme pockets of the liganded form of the hemoglobin molecule. Both hemoglobins undergo a transition from the deoxy (T) to the oxy (R) quaternary structure upon ligation. The abnormally high oxygen affinities and low cooperativities of these two hemoglobins must therefore be due to either the structural differences which we have observed and/or to an altered transition between the T and R structures.     

Effects of substitutions of lysine and aspartic acid for asparagine at beta 108 and of tryptophan for valine at alpha 96 on the structural and functional properties of human normal adult hemoglobin: roles of alpha 1 beta 1 and alpha 1 beta 2 subunit interfaces in the cooperative oxygenation process.

Using our Escherichia coli expression system, we have produced five mutant recombinant (r) hemoglobins (Hbs): r Hb (alpha V96 W), r Hb Presbyterian (beta N108K), r Hb Yoshizuka (beta N108D), r Hb (alpha V96W, beta N108K), and r Hb (alpha V96W, beta N108D). These r Hbs allow us to investigate the effect on the structure-function relationship of Hb of replacing beta 108Asn by either a positively charged Lys or a negatively charged Asp as well as the effect of replacing alpha 96Val by a bulky, nonpolar Trp. We have conducted oxygen-binding studies to investigate the effect of several allosteric effectors on the oxygenation properties and the Bohr effects of these r Hbs. The oxygen affinity of these mutants is lower than that of human normal adult hemoglobin (Hb A) under various experimental conditions. The oxygen affinity of r Hb Yoshizuka is insensitive to changes in chloride concentration, whereas the oxygen affinity of r Hb Presbyterian exhibits a pronounced chloride effect. r Hb Presbyterian has the largest Bohr effect, followed by Hb A, r Hb (alpha V96W), and r Hb Yoshizuka. Thus, the amino acid substitution in the central cavity that increases the net positive charge enhances the Bohr effect. Proton nuclear magnetic resonance studies demonstrate that these r Hbs can switch from the R quaternary structure to the T quaternary structure without changing their ligation states upon the addition of an allosteric effector, inositol hexaphosphate, and/or by reducing the temperature. r Hb (alpha V96W, beta N108K), which has the lowest oxygen affinity among the hemoglobins studied, has the greatest tendency to switch to the T quaternary structure. The following conclusions can be derived from our results: First, if we can stabilize the deoxy (T) quaternary structure of a hemoglobin molecule without perturbing its oxy (R) quaternary structure, we will have a hemoglobin with low oxygen affinity and high cooperativity. Second, an alteration of the charge distribution by amino acid substitutions in the alpha 1 beta 1 subunit interface and in the central cavity of the hemoglobin molecule can influence the Bohr effect. Third, an amino acid substitution in the alpha 1 beta 1 subunit interface can affect both the oxygen affinity and cooperativity of the oxygenation process. There is communication between the alpha 1 beta 1 and alpha 1 beta 2 subunit interfaces during the oxygenation process. Fourth, there is considerable cooperativity in the oxygenation process in the T-state of the hemoglobin molecule.