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In the absence of effectors, log p 50 remained below zero in the pH range 8. In addition to this remarkable difference, the oxygen affinity and cooperativity in the presence of chloride and ATP at pH 6. At alkaline pH the oxygen affinity was very high 0. This unusual modulation of cooperativity by pH is clearly depicted by the bell-shaped curve of the Hill coefficient Fig. The stripped hemolysate had oxygen-binding features reflecting those of the major component Hb 1 Fig.

Whole blood and intact erythrocytes were assayed in the absence of effectors, and also showed strong Bohr and Root effects not shown. Under all experimental conditions, that is, in the absence and presence either of chloride alone, or of both chloride and organophosphates, the highest value in absolute terms of the enthalpy change of oxygenation was at pH 8. At lower pH values, the overall enthalpy change progressively decreased as a function of increasing proton concentration, due to the endothermic contribution of the heterotropic effectors, released upon oxygen binding, and of the allosteric transition.

The value at pH 6. In this case, the enthalpy change was positive i. On the grounds of the primary structure analysis, a hypothesis on the binding of chloride will be discussed below.


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As expected, the values were much less negative than those of mammalian Hbs. Spectroscopy was the first approach aimed at finding a structural explanation to the striking oxygen-binding features. Electronic absorption spectra of Hb 1. The Met-F complex at pH 5. The visible region is expanded sixfold. Resonance Raman spectra of Hb 1. The spectra of deoxy Hb 1 at pH 6.

In oxy Hb 1, the 2-nm blue-shift of the electronic absorption maxima at pH 7. The formation of the Met form at the expense of the oxy upon laser exposure is confirmed by a slight Soret-band blue-shift after the RR experiment. As the frequencies of the Fe—CO complex are more sensitive to the properties of the heme cavity than those of the oxy form, carbomonoxy Hb 1 was also studied.

The electronic absorption spectrum of the CO complex is characterized by bands at , , and nm not shown , very similar to human HbA—CO Antonini and Brunori Hence, again any variation in the properties of the heme cavities of the two proteins must be of limited extent. The electronic absorption spectrum of human Met-HbA at pH 6. In contrast, Met-Hb 1 at pH 6. In agreement with the principal features of the electronic absorption spectrum, the high-frequency RR spectrum of Met-Hb 1 Fig.

As observed for the other P. This is consistent with an increased proportion of LS species compared to pH 6. Slight differences are evident particularly in the visible region compared to the spectrum of HbA at alkaline pH George et al. The transition to the hydroxide complex could not be followed to completion as the protein was denatured at pH values higher than 9. The complex formed between Met-Hb 1 and fluoride at pH 5. The absorption spectrum is similar to that of HbA Perutz et al. It is worth stressing that only one vinyl band is present in the corresponding spectrum of the HbA-F complex Choi et al.

Thus, the spectroscopic analysis of Met-Hb 1 provides evidence for hemichrome formation and supports the presence of two vinyl stretching modes. Another approach aimed at correlating the structure of the heme pocket with the oxygen-binding properties was molecular modeling. De-oxy and carbomonoxy models were based on the X-ray coordinates of T. Detailed analysis of the final models was performed in comparison to human HbA and T.

Identical and similar residues are in dark and in white boxes, respectively. Modeling indicates that the methyl group CD1 atom, according to the standard nomenclature for amino acids and heme group used by the Protein Data Bank of Leu FG3 is at only 3. The distal side of the heme in Hb 1 was compared to that of HbA. However, a slight conformational modification of His E7, with respect of HbA, was detected only in the carbomonoxy form.


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The models of the carbomonoxy and oxy forms of Hb 1 suggest that Thr E10 does not interact with ligands, as its hydroxyl group is oriented away from distal His. It is worth noting that Thr is also present in E10 of sperm-whale myoglobin; the side chain of Thr E10 is always directed toward the solvent in deoxy, carbomonoxy, and oxy forms of myoglobin PDB codes 1a6n, 1a6g and 1a6m, respectively; Vojtechovski et al. The side chains of E10, E7, and E11, by means of feasible movements, have been indicated Case and Karplus as a possible pathway for ligand escape in sperm-whale myoglobin.

Replacement of Leu B13 with Cys in P. The SG atom of Cys B13 is 6. In each protein, selected residues are labeled in the color of the backbone trace. Multiplicity of Hb components in fish is usually linked to the need to respond to a mutable environment or different habitats. For this reason, a single Hb in lower amounts is regarded as the consequence of a lesser role for oxygen carriers in Antarctic notothenioids, possibly due to their sluggish mode of life, slower metabolism, and to the peculiarity of the environmental conditions high stability and constancy of physicochemical features.

Hb is a direct link between environment and body requirements, and must fulfill its primary function under a wide variety of conditions; consequently, it has experienced major evolutionary pressures to adapt and modify its functional features, while largely retaining its molecular structure. Similar to a large number of notothenioid species, the hemolysate of P. The low amount of Hb 2 can be considered a synapomorphy, connecting P. Three notothenioid families, that is, Bovichtidae only one out of 10 species is Antarctic , Pseudaphritidae, and Eleginopidae, presumably diverged and became established in waters around areas corresponding to New Zealand, Australia, and South America before Antarctica became isolated.

The absence of any detectable antifreeze glycoprotein coding sequence in Bovichtus variegatus , P. However, the three families carry the trypsinogen-like protease gene, in keeping with occurrence of diversification before the evolution of the antifreeze glycoproteins gene from this ancestral gene.

From the evolutionary standpoint, the analysis of the oxygen-transport system of non-Antarcvatic notothenioids is of great interest. The characterization of the Hb system of P. Taking advantage of the wealth of information available on a high number of Antarctic notothenioid species e. The trees are in agreement with those obtained by morphological analysis and sequence studies on mitochondrial RNA Ritchie et al. However, the identity between Hb 1 of P. This argues in favor of a common origin within notothenioids, but also suggests that the primary structure of the major Hb has undergone modifications only to a limited extent.

If sequence mutations in Antarctic fish are indeed related to the development of cold adaptation, this may imply divergence during the first stages of the cooling process, and in any case before the event that gave rise to antifreeze glycoproteins. The relevance of studying Hbs of non-Antarctic species in the evolution of Notothenioidei is highlighted by the analysis of the features of the Hb system of another temperate notothenioid.

Recent studies indicate that the genome of the more recent Notothenia angustata family Nototheniidae , common near the coast of southern New Zealand, does have the antifreeze glycoproteins genes Cheng et al. The amino acid sequence identity between Hbs of noncold-adapted N. The presence of antifreeze genes suggests that, unlike P. The very high similarity of Hb sequence of N. On the other hand, both species have high hematocrit, erythrocyte number, Hb content, and cellular concentration.

Unlike primary structure, these features might be considered a short-term response to environmental changes. In evolutionary processes, in the same time span changes in amino acid sequence would occur at a much slower overall rate, thus producing fewer variations.

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We suggest that the common phylogenetic origin remains the prevailing factor in primary structure in the course of readaptation of a notothenioid to a temperate environment. Changes in factors other than sequence, such as for instance regulatory processes or enhanced protein synthesis, may well be additional mechanisms of temperature compensation.

The Root effect is largely ATP-induced.

In the temperature dependence of oxygen binding, the positive endothermic enthalpy change observed around pH 6. At pH values where the T state becomes preferentially populated, but still in the presence of a significant degree of heterotropic interactions, the contribution of the effectors and of the conformational transition may even overcome that of the T state, thereby producing a change of sign in the overall enthalpy change, which, in fact, becomes endothermic, favoring oxygen release when the organism is challenged by lower temperatures.

It has been shown De Rosa et al. Similar to human fetal, horse, and bear Hbs, P. Such conclusion, supported by crystallographic data, may apply to P. The affinity and cooperativity are modulated by pH and allosteric effectors in a remarkable way. The oxygen affinity of Hb 1 is exceptionally high compared to other notothenioids.

Although the Hb multiplicity closely resembles that of sedentary Antarctic bottom dwellers, this high affinity possibly related to the peculiar habitat constraints clearly differentiates this species from other notothenioids. As discussed in detail below, the spectroscopic and modeling studies indicate, however, that each of these replacements leave the conformation and electrostatic field surrounding His E7 essentially unmodified with respect to both HbA and T. In fact, although T.

This nonconservative substitution, although uncommon in fish Hbs, is found also in T. Although this replacement, therefore, cannot directly explain the high affinity of P. The subunit cooperativity, mostly induced by allosteric effectors, is regulated by pH in a very unusual way, in that it not only disappears at low physiological pH as in all Root-effect Hbs , but is absent also at high pH. Thus, the curve of the Hill coefficient as a function of pH is bell-shaped, and its maximum identifies the pH value where the ligand-induced conformational transition is fully active.

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In the common sting ray, Dasyatis sabina Mumm et al. The spectroscopic measurements reveal two characteristics which distinguish Hb 1 from HbA, namely the presence of a hemichrome species in the Met form and reduced steric constraints on vinyl group 4. It is noteworthy that the proportion and nature of the LS species observed in P.

At both pH 6. Hemichromes have been generally observed in association with Hb-denaturation processes but, unlike hemichromes of other heme proteins that are reduced to hemo-chrome, the oxidized form of P. Although the formation of hemichrome in a native Hb structure is rare, it has recently been reported in the monomeric Hb of the sea cucumber Caudina arenicola Mitchell et al. Moreover, it has recently been found that in five Hbs from four Antarctic notothenioids the oxidation process, promoted by air or ferricyanide, leads to the reversible formation of hemi-chrome Vitagliano et al.

These findings provide new information on tetrameric Hbs in the hemichrome state, and suggest a physiological role of in vivo hemichrome formation in fish, which appears to occur regardless of whether a species is cold adapted or not. It is worth noting that fish are highly sensitive to a number of environmental stress factors. Although fish erythrocytes may contain low levels of Met-Hb, stress often leads to rapid increase of this form Graham and Fletcher It is feasible that fish might use hemichrome formation as a protective response to otherwise irreversible stress-induced Hb oxidation.

In other words, spontaneous autoxidation, occurring at higher rates than in mammalian Hbs, might be balanced by hemichrome formation. This in vivo mechanism has been suggested also in other organisms Robinson et al. The formation of a hemichrome bisHis in P. A slightly modified distal cavity, compared to HbA, is also suggested by retention of an aquo HS species in the Met form at alkaline pH, which indicates that the pKa of the alkaline transition in P. Although the plant, invertebrate, and vertebrate Hbs mentioned above differ significantly in their primary structures, all exhibit two hemichrome-induced structural characteristics, namely a shift of the heme group to a more exposed position and a narrowing of the heme pocket.

These differences found in P. In fact, in accordance with the modeling studies, which suggest a slightly modified distal His disposition in the ligand-bound forms but a largely unchanged electrostatic field, the Fe—CO and CO frequencies of the CO complex are not significantly modified. These frequencies are sensitive indicators of modified heme interactions H-bonding, polarity and steric hindrance and hence confirm the similarity of the heme cavities of P. The absence of any variation in the Raman frequency of the Fe—O 2 stretching mode of P. The presence of two vinyl modes in the RR spectra of P.

In particular, it was found that, when the protein matrix exerts no contraints on the vinyl groups, the latter take the tortional conformation usually found in model compounds in solution Kalsbeck et al. Hence, it is suggested that the presence of two distinct vinyl stretches in the RR spectra of P.

This spectroscopic finding is consistent with the reduced steric constraints of the vinyl 4 substituent group of P. It can be anticipated that the reduced steric hindrance upon replacement of the residue interacting with heme vinyl 4 contributes to the increased mobility of the heme, allowing it to shift to a more exposed position, as required for hemichrome formation.

In conclusion, spectroscopy and molecular modeling were used to investigate correlations between the peculiar oxygen-binding features of P. On the other hand, two closely related and unique features distinguish P. Although their correlation with the oxygen-binding features of P. All other reagents were of the highest purity commercially available. Blood samples were drawn from the caudal vein of anesthetized fish with heparinized syringes.

Oxyhemoglobin Dissociation Curve Right Left Shift Explain (Hb vid 2)

Purification of globins, fractionation of tryptic and cyanogen-bromide-cleaved peptides, and subsequent amino acid sequencing were carried out as previously described Verde et al. The following strategy was applied for inferring the trees. First, a distance matrix and the corresponding neighbor-joining NJ tree were estimated.

An exhaustive topology search of top-ranking trees was performed on a partially constrained starting tree where the taxa of some undisputed clades were clustered together. The constrained topology contained the major and minor Antarctic globin sequences as fixed groups because their topology was well supported by bootstrap analysis and was congruent with previous assessments Verde et al. Finally, the user tree option was used to pick up the best tree among the trees inferred by exhaustive search.

Bootstrap values for internal tree branches were calculated as the sum of the bootstrap probabilities of all the trees showing the node being analyzed among the alternatives. Hemolysate stripping was carried out as described Tamburrini et al.

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Sensitivity to chloride was assessed by adding NaCl to a final concentration of mM. As the crystal structure of P. Crystallographic structures of deoxy and carbomonoxy T. As the PDB coordinates of oxy T. This program is based on a distance-restraint algorithm using spatial restraints extracted from a multiple alignment of the target sequences with the template structures and from the CHARMM force field Brooks et al. The model with the lowest value of MODELER objective function F, molecular probability density function violation was selected as the representative model for each chain in each structure.

The Met-Hb derivatives were prepared by oxidation of the oxy form using excess potassium ferricyanide followed by gel filtration on a Biogel P-6DG column equilibrated with the appropriate buffer to remove the oxidant. The carbomonoxy complex was prepared by flushing Met-Hb with nitrogen, then with CO and reducing the protein as described above.

Electronic absorption spectra were measured with a Cary 5 spectrophotometer. Tris-HCl at pH 7. RR spectra were obtained at room temperature with excitation from the To minimize local heating by the laser beam, all samples except deoxy Hb were cooled by a gentle flow of nitrogen run through liquid nitrogen. The publication costs of this article were defrayed in part by payment of page charges.

Article published online ahead of print. Article and publication date are at http: National Center for Biotechnology Information , U. Journal List Protein Sci v. Cinzia Verde , 1 Barry D. Howes , 2 M. This article has been cited by other articles in PMC. Abstract The suborder Notothenioidei dominates the Antarctic ichthyofauna. Results Purification of Hbs and separation of globins Similar to most Antarctic notothenioids di Prisco et al. Open in a separate window. Phylogenetic positions of P. Oxygen-binding properties The oxygen-binding properties were investigated Fig. Spectroscopic characterization Spectroscopy was the first approach aimed at finding a structural explanation to the striking oxygen-binding features.

Molecular modeling Another approach aimed at correlating the structure of the heme pocket with the oxygen-binding properties was molecular modeling. Discussion Evolution of notothenioidei Multiplicity of Hb components in fish is usually linked to the need to respond to a mutable environment or different habitats. Oxygen affinity and subunit cooperativity P. Molecular Adaptation in Extreme and Temperate Environments. Be the first to review this item.

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