The Hemoglobins of the Antarctic Fishes Artedidraco orianae and Pogonophryne scotti
1998; Elsevier BV; Volume: 273; Issue: 49 Linguagem: Inglês
10.1074/jbc.273.49.32452
ISSN1083-351X
AutoresMaurizio Tamburrini, Mario Romano, Vito Carratore, Andreas Kunzmann, Massimo Coletta, Guido di Prisco,
Tópico(s)Neonatal Health and Biochemistry
ResumoThe oxygen-transport system of two species of Antarctic fishes belonging to the family Artedidraconidae,Artedidraco orianae and Pogonophryne scotti, was thoroughly investigated. The complete amino acid sequence of the α and β chains of the single hemoglobins of the two species was established. The oxygen-binding properties were also investigated, and were found not to differ significantly from those shown by blood, intact erythrocytes, and unstripped hemolysates. Both hemoglobins have unusually high oxygen affinity and display a relatively small Bohr effect; the Root effect is elicited only by organophosphates and is also reduced. Remarkably, the Hill coefficient is close to one in the whole pH range, indicating absence of cooperative oxygen binding which, in A. orianae hemoglobin, could be ascribed to the subunit heterogeneity shown upon oxygen dissociation. In comparison with the other families of the suborder Notothenioidei, the oxygen-transport system of these two species of Artedidraconidae has unique characteristics, which raise interesting questions on the mode of function of a multisubunit molecule and the relationship with cold adaptation. The oxygen-transport system of two species of Antarctic fishes belonging to the family Artedidraconidae,Artedidraco orianae and Pogonophryne scotti, was thoroughly investigated. The complete amino acid sequence of the α and β chains of the single hemoglobins of the two species was established. The oxygen-binding properties were also investigated, and were found not to differ significantly from those shown by blood, intact erythrocytes, and unstripped hemolysates. Both hemoglobins have unusually high oxygen affinity and display a relatively small Bohr effect; the Root effect is elicited only by organophosphates and is also reduced. Remarkably, the Hill coefficient is close to one in the whole pH range, indicating absence of cooperative oxygen binding which, in A. orianae hemoglobin, could be ascribed to the subunit heterogeneity shown upon oxygen dissociation. In comparison with the other families of the suborder Notothenioidei, the oxygen-transport system of these two species of Artedidraconidae has unique characteristics, which raise interesting questions on the mode of function of a multisubunit molecule and the relationship with cold adaptation. hemoglobin high performance liquid chromatography 2-[bis(2-hydroxyethyl)amino]- 2-(hydroxymethyl)-propane-1,3-diol. During adaptation to low temperatures, Antarctic fishes have acquired special hematological features which clearly differentiate them from fishes of temperate and tropical climates. The hematocrit and hemoglobin (Hb)1concentration are highly reduced in the blood of Antarctic fishes (1Everson I. Ralph R. Bull. Br. Antarct. Surv. 1968; 15: 59-62Google Scholar, 2Hureau J.-C. Petit D. Fine J.M. Marneux M. Llano G.A. Adaptations within Antarctic Ecosystems. Smithsonian Institution, Washington, D. C.1977: 459-477Google Scholar, 3Wells R.M.G. Ashby M.D. Duncan S.J. Macdonald J.A. J. Fish Biol. 1980; 17: 517-527Crossref Scopus (98) Google Scholar), as well as the number of Hb components. At the extreme of such evolution, the 15 species of the family Channichthyidae are characterized by lack of Hb (4Ruud J.T. Nature. 1954; 173: 848-850Crossref PubMed Scopus (281) Google Scholar). The hematological peculiarities of Antarctic teleosts prompted an investigation on the molecular structure and ligand binding properties of hemoglobins (Hbs) from these organisms, in order to characterize the adaptation of the oxygen-transport mechanism at the molecular level. We focused our interest on the suborder Notothenioidei, largely endemic and confined south of the Antarctic Polar Front. The suborder includes six families with 120 species, 95 of which are Antarctic (5Gon O. Heemstra P.C. Fishes of the Southern Ocean. JLB Smith Institute of Ichthyology, Grahamstown, South Africa1990Crossref Google Scholar, 6Eastman J.T. Antarctic Fish Biology: Evolution in a Unique Environment. Academic Press, San Diego1993Google Scholar): Bovichtidae, Nototheniidae, Harpagiferidae, Artedidraconidae, Bathydraconidae, and Channichthyidae (in fact, the families might be seven, since recent evidence (7Balushkin A.V. J. Ichthyol. 1992; 30: 132-147Google Scholar) suggests that Bovichtidae should be grouped into Pseudaphritidae and Bovichtidae). Notothenioids generally have a single major Hb (Hb 1), often accompanied by a minor component (Hb 2), accounting for approximately 95 and 5% of the total, respectively (8di Prisco G. D'Avino R. Antarct. Sci. 1989; 1: 119-124Crossref Scopus (16) Google Scholar, 9di Prisco G. D'Avino R. Camardella L. Caruso C. Romano R. Rutigliano B. Polar Biol. 1990; 10: 269-274Crossref Scopus (17) Google Scholar, 10di Prisco G. D'Avino R. Caruso C. Tamburrini M. Camardella L. Rutigliano B. Carratore V. Romano M. di Prisco G. Maresca B. Tota B. Biology of Antarctic Fish. Springer-Verlag, Berlin1991: 263-281Crossref Google Scholar). A cathodal Hb (Hb C) is present in trace amounts, except in Trematomus newnesi, of the family Nototheniidae, in which Hb C is approximately 25–30% of the total (11D'Avino R. Caruso C. Tamburrini M. Romano M. Rutigliano B. Polverino de Laureto P. Camardella L. Carratore V. di Prisco G. J. Biol. Chem. 1994; 269: 9675-9681Abstract Full Text PDF PubMed Google Scholar). In this study, the oxygen-transport system of species of the family Artedidraconidae, which comprises 24 of the 80 red-blooded Antarctic species of the suborder Notothenioidei so far identified (5Gon O. Heemstra P.C. Fishes of the Southern Ocean. JLB Smith Institute of Ichthyology, Grahamstown, South Africa1990Crossref Google Scholar, 6Eastman J.T. Antarctic Fish Biology: Evolution in a Unique Environment. Academic Press, San Diego1993Google Scholar), was thoroughly investigated for the first time. Artedidraconids are benthic fish, have a wide depth distribution, and are largely confined in the Antarctic continental shelf and slope (12Andriashev A.P. Monogr. Biol. 1965; 25: 491-550Google Scholar). This paper reports the complete amino acid sequence of the α and β chains of the single Hbs of the artedidraconids Artedidraco orianae and Pogonophryne scotti, along with kinetic and thermodynamic characterization of ligand binding. The functional features of Hbs were very similar to those measured in whole blood, intact erythrocytes, and unstripped hemolysates. DEAE-cellulose (DE52) was from Whatman; trypsin (EC 3.4.21.4), treated with l-1-tosylamide-2-phenylethylchloromethyl ketone, from Cooper Biomedical; Tris and bisTris from Sigma; dithiothreitol from Fluka; all sequanal-grade reagents from Applied Biosystems; HPLC-grade acetonitrile from Lab-Scan Analytical. All other reagents were of the highest purity commercially available. A. orianae was caught by means of Agassiz Trawl in the northeastern Weddell Sea, Antarctica, and P. scotti by gill nets in the vicinity of Terra Nova Bay Station, Ross Sea, Antarctica. Immediately after catch, fish were transferred to aquariums supplied with running, aerated seawater at approximately −1.0 °C. Blood samples were drawn from the caudal vein of living animals by means of heparinized syringes; the red blood cells were washed three times in isotonic saline solution (1.7% NaCl, in 1 mmTris-HCl, pH 8.1). Hemolysates were prepared as described (13D'Avino R. di Prisco G. Comp. Biochem. Physiol. B Comp. Biochem. 1988; 90: 579-584Crossref Scopus (41) Google Scholar). Hb concentration and purification from minor contaminants was carried out by ion-exchange chromatography on a DE52 column (1 × 3 cm), equilibrated with 10 mm Tris-HCl, pH 8.1, and eluted with 100 mm Tris-HCl, pH 7.1. Gel filtration was performed by fast protein liquid chromatography (Pharmacia) on a ProteinPak 300 SW column (Waters), equilibrated with 50 mm Tris-HCl, pH 8.0, containing 100 mm NaCl, at a flow rate of 0.2 ml/min. Globins were precipitated with 10 volumes of acetone containing 5 mm HCl at −20 °C; α and β chains were separated by reverse-phase HPLC on a μBondapak C18 column (Waters, 0.39 × 30 cm). In P. scotti, the eluents were (A) 45% acetonitrile containing 0.3% trifluoroacetic acid and (B) 55% acetonitrile; a linear gradient from 0 to 100% of eluent B in 15 min was used. In A. orianae, the eluents were (A) 0.1% trifluoroacetic acid and (B) acetonitrile containing 0.08% trifluoroacetic acid; the elution was carried out with a multistep gradient of eluent B. Flow rate was 1 ml/min. Cellulose acetate electrophoresis of hemolysates and purified Hbs and SDS-polyacrylamide gel electrophoresis of the purified globins were carried out as described (14Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207538) Google Scholar, 15D'Avino R. di Prisco G. Eur. J. Biochem. 1989; 179: 699-705Crossref PubMed Google Scholar). Globin denaturation, alkylation of the sulfhydryl groups with 4-vinylpyridine, tryptic digestion, and cleavage of Asp-Pro bonds were carried out according to described procedures (16D'Avino R. Caruso C. Romano M. Camardella L. Rutigliano B. di Prisco G. Eur. J. Biochem. 1989; 179: 707-713Crossref PubMed Scopus (24) Google Scholar, 17Tamburrini M. Brancaccio A. Ippoliti R. di Prisco G. Arch. Biochem. Biophys. 1992; 292: 295-302Crossref PubMed Scopus (53) Google Scholar). Tryptic peptides were purified by reverse-phase HPLC on a μBondapak C18 column (Waters, 0.39 × 30 cm), equilibrated with eluent A (0.1% trifluoroacetic acid); peptides were eluted with a multistep gradient of eluent B (acetonitrile, containing 0.08% trifluoroacetic acid). Amino acid analyses were performed with an Applied Biosystems automatic derivatizer model 420 A, equipped with the hydrolysis option and with on-line detection of phenylthiocarbamyl amino acids. Alternatively, gas-phase manual hydrolysis was carried out for 1 h at 155 °C in 6 n HCl containing 1% 2-mercaptoethanol and 1% phenol. Amino acid sequencing was carried out with an Applied Biosystems automatic sequencer model 477A, equipped with on-line detection of phenylthiohydantoin-derivatives. Sequencing of Asp-Pro-cleaved globins was performed after treatment with o-phthalaldehyde (18Brauer A.W. Oman C.L. Margolies M.N Anal. Biochem. 1984; 137: 134-142Crossref PubMed Scopus (106) Google Scholar), to reduce the background due to sequencing from the N terminus. Molecular mass measurements were carried out with a Hewlett-Packard mass spectrometer model 5989B, equipped with the electrospray source model 59987A. Oxygen saturation experiments were carried out at 2 °C, as described previously (15D'Avino R. di Prisco G. Eur. J. Biochem. 1989; 179: 699-705Crossref PubMed Google Scholar). Oxygen-equilibrium curves were obtained by the tonometric method (19Giardina B. Amiconi G. Methods Enzymol. 1981; 76: 417-427Crossref PubMed Scopus (139) Google Scholar) at 2, 10, and 20 °C, in the pH range 6.5–8.0. The heat of oxygenation was calculated from the integrated van't Hoff equation, ΔH=−4.574·[(T1·T2)/(T1−T2)]·ΔlogP50/1000(Eq. 1) and the values obtained were corrected for the heat of oxygen solubilization (−3 kcal/mol; 1 kcal = 4.184 kJ). Kinetic measurements were carried out with a Gibson-Durrum stopped-flow system equipped with a 2-cm pathlength observation cell and interfaced to a computer for data acquisition (On Line System, Jefferson, GA). Oxygen dissociation measurements were undertaken mixing oxy-Hb of A. orianae (5 μm heme before mixing, in low-ionic strength HEPES buffer, pH 7.0) with sodium dithionite (20 mg/ml after mixing) in buffer at the desired pH at higher ionic strength. For carbon monoxide binding kinetics, deoxy-Hb of A. orianae or of P. scotti in sodium dithionite at low ionic strength and pH 7.0 (3 μm heme before mixing, in HEPES buffer) were mixed with a higher ionic strength buffer at the desired pH containing a given concentration of dissolved carbon monoxide. Both measurements were performed at 20 °C and monitored at 430 or 419 nm, following the appearance or disappearance of unliganded Hb. Electrophoretic analysis on cellulose acetate showed that the hemolysates of A. orianae and P. scotti have a single Hb. The α and β chains of each Hb were purified by HPLC (Fig. 1). The N terminus of the two α chains was unavailable to automated Edman degradation, indicating the presence of a blocking group. An internal region became accessible in both chains after cleavage of an Asp-Pro bond. After treatment with o-phthalaldehyde, sequencing proceeded from Pro96 to Thr119 in A. orianae, and from Pro96 to Ala122in P. scotti. Tryptic peptides were purified by reverse-phase HPLC. Fig. 2 shows the patterns of the α chains of A. orianae (panel A) and P. scotti(panel B). All peptides were identified and sequenced. The sequence of the blocked N-terminal peptides (residues 1–7) of the two chains was established after incubation in 30% trifluoroacetic acid at 55 °C for 2.5 h. The molecular mass of these peptides, obtained by electrospray mass spectrometry, was 834.6 Da, a value compatible with the presence of an acetyl group at the N terminus. In the α chain of A. orianae, mass spectrometry analysis also revealed a minor component having a molecular mass of 592.5 Da, corresponding to the sequence from acetyl-Ser1 to Lys5. In the α chain of A. orianae Hb, trypsin partially failed to cleave at Lys5 and Lys62. Thus, it was possible to recover two forms of peptide T10 (T10A and T10B), having Lys62 and Val63 at the N terminus, respectively. The sequence obtained after Asp-Pro cleavage provided overlap from T14 to T16. In the α chain of P. scotti Hb, trypsin failed to cleave at Lys5 and at Arg93; the alignment of T12 and T13 was thus obtained. The sequence obtained after Asp-Pro cleavage provided overlap from T13 to T15. Tryptic peptides were purified by reverse-phase HPLC. Fig. 2 shows the patterns of the β chains of A. orianae (panel C) and P. scotti (panel D) Hbs. All peptides were identified and sequenced. Direct sequencing from the N terminus proceeded for 30 (A. orianae) and 31 (P. scotti) residues, providing overlap from T1 to T3 and from T1 to T4, respectively. After cleavage of the internal Asp-Pro bond and treatment with o-phthalaldehyde, sequencing proceeded from Pro100 to Val114(A. orianae) and from Pro100 to Phe133 (P. scotti), providing overlap from T9 to T10 and from T8 to T11, respectively. Fig. 3 shows the complete sequence of the α (142 residues) and β (146 residues) chains of the two Hbs. The amino acid compositions are reported in TableI. Alignment of the tryptic peptides was established by the described overlaps and by homology with other known sequences. The calculated molecular masses were: (i) 15,499 and 15,566 (α chains); (ii) 16,203 and 16,129 (β chains), for A. orianae and P. scotti Hbs, respectively.Table IAmino acid composition of the α (A and B) and β (C and D) chains of A. orianae and P. scotti, respectivelyAmino acidABCDAsp/Asn12.6 (15)15.2 (15)13.8 (19)16.2 (16)Glu/Gln6.4 (5)5.2 (5)11.9 (11)11.7 (10)Ser10.1 (13)14.3 (14)8.0 (10)10.1 (11)Gly6.7 (6)6.8 (6)10.7 (11)10.7 (10)His8.6 (5)6.1 (6)7.2 (5)8.2 (9)Arg3.7 (3)3.0 (3)4.1 (4)3.0 (3)Thr5.4 (5)5.3 (5)5.0 (5)4.6 (4)Ala14.7 (18)16.9 (16)12.8 (13)15.1 (16)Pro6.7 (6)6.4 (6)4.0 (4)3.8 (4)Tyr3.7 (3)3.2 (3)5.6 (5)5.2 (5)Val9.9 (11)10.7 (12)10.1 (11)7.7 (9)Met1.6 (4)4.5 (4)1.2 (4)3.6 (3)CysND1-aND, not determined. (1)ND (1)ND (2)1.6 (2)Ile10.1 (10)7.9 (9)8.7 (9)7.8 (10)Leu14.2 (13)13.0 (13)14.2 (14)15.0 (16)Phe6.7 (6)6.2 (6)8.5 (9)8.1 (8)Lys15.2 (16)15.4 (16)8.7 (8)8.1 (8)TrpND (2)ND (2)ND (2)ND (2)No. of residues142142146146Molecular mass (Da)15,49915,56616,20316,129The number of residues from the sequence are indicated in parentheses.1-a ND, not determined. Open table in a new tab The number of residues from the sequence are indicated in parentheses. At 2 °C (close to physiological conditions) the affinity for oxygen of the two Hbs was high: at pH 8.0,P 50 was 2.45 and 4.6 mm Hg in the absence, and 3.63 and 5.5 mm Hg in the presence, of chloride and organophosphates, for A. orianae and P. scotti Hbs, respectively. In comparison with other notothenioids (10di Prisco G. D'Avino R. Caruso C. Tamburrini M. Camardella L. Rutigliano B. Carratore V. Romano M. di Prisco G. Maresca B. Tota B. Biology of Antarctic Fish. Springer-Verlag, Berlin1991: 263-281Crossref Google Scholar), the Hbs showed a modest, effector-enhanced alkaline Bohr effect (becoming virtually absent at 10 °C in A. orianae Hb, not shown) in the pH range 6.5–8.0 (Fig. 4, panels A and B); the Bohr coefficient ΔlogP 50/ΔpH was, respectively, −0.35 and −0.31 in the absence, and −0.51 and −0.44 in the presence, of the physiological effectors. Although the overall oxygen affinity of whole blood (as well as erythrocytes and unstripped hemolysate) of A. orianae and P. scotti (Fig. 4, panels A and B, respectively) was slightly lower than that measured in Hb in the presence of chloride and phosphates, the slopes of the oxygen-equilibrium curves were very similar in the pH range 7.0–7.5, where the Bohr effect is active. In the whole pH range, the Hill coefficient of the Hbs of both species was close to one (Fig. 4, panels C and D), regardless of the presence of chloride and organophosphates, with only slightly higher values observed in P. scotti. These results were taken as strong evidence of the absence of cooperative oxygen binding. In order to ascertain whether the low Hill coefficient values were due to subunit dissociation, A. orianae Hb was analyzed by fast protein liquid chromatography gel filtration at pH 8.0 (see “Experimental Procedures” for details). The hemoprotein was eluted from the column at the retention time of tetrameric Hb (not shown), without any apparent dissociation. Therefore, the absence of subunit cooperativity is due to intrinsic properties of the tetrameric molecule; further work is required to establish correlations with molecular structure. The Root effect (20Root R.W. Biol. Bull. (Woods Hole). 1931; 61: 427-456Crossref Google Scholar, 21Brittain T. Comp. Biochem. Physiol. B Comp. Biochem. 1987; 86: 473-481Crossref PubMed Scopus (85) Google Scholar) often displayed by fish Hbs, which leads to incomplete saturation of Hb in air, was not found, at 2 °C, in A. orianae and P. scotti Hbs in the absence of organophosphates, but it was induced to a limited extent by ATP or inositol hexakisphosphate (Fig. 5). The Root effect of A. orianae and P. scotti blood, erythrocytes, and unstripped hemolysate was very similar to that shown by the isolated Hb (Fig. 5, panels A and B). The regulation of the oxygen affinity by temperature was investigated in the range 2–20 °C. A very strong ΔH, larger than that of mammalian Hbs (22Coletta M. Ascenzi P. Smulevich G. Mantini A.R. Del Gaudio R. Piscopo M. Geraci G. FEBS Lett. 1992; 296: 184-186Crossref PubMed Scopus (11) Google Scholar), was measured in A. orianae Hb at pH 8.0, in the absence and presence of chloride and ATP. However, lowering the pH to 7.0 brought about a dramatic decrease of the oxygen-binding enthalpy, which became almost zero in the presence of effectors (Table II).Table IIApparent heat of oxygenation of A. orianae and P. scotti HbsΔHpH 7.0pH 7.5pH 8.0kcal mol−1 oxygenWithout effectors A. orianae−6.75−19.6 P. scotti−9.96369With effectors A. orianae−1.55−15.1 P. scotti−12.06369 Open table in a new tab Fig. 6 shows the progress curve for oxygen dissociation of Hb of A. orianae at different pH values and at 21 °C, displaying a biphasic process characterized by two exponentials, which can be attributed to a different ligand dissociation behavior for the two subunits α and β of the tetramer, even though the kinetic heterogeneity is not as marked as in the Root effect Hb of the temperate fish Chelidonichthys kumu(23Fago A. Romano M. Tamburrini M. Coletta M. D'Avino R. di Prisco G. Eur. J. Biochem. 1993; 218: 829-835Crossref PubMed Scopus (28) Google Scholar). The amplitude of the process decreased as the pH was lowered (Fig. 6), indicating that A. orianae Hb was already partially deoxygenated at atmospheric oxygen pressure and at 21 °C, before mixing with sodium dithionite. However, this effect appeared markedly temperature-dependent, since at lower, physiological temperatures, no Root effect, i.e. no partial deoxygenation, was observed upon pH lowering at atmospheric pressure (Fig. 5). Both deoxygenation rate constants displayed a pH-dependent behavior in the observed pH range (Fig. 7); the data were analyzed according to, kobs=kalk/(1+Ka[H+])+kacKa[H+]/(1+Ka[H+])(Eq. 2) where k obs is the observed oxygen dissociation rate constant, k alk and k ac are the oxygen dissociation rate constants for the unprotonated and protonated Hb, respectively, and K a (=10pK a) is the proton binding constant to oxygenated Hb. The pH dependence described by the continuous lines indicated a single protonation event with a pK a = 7.1 ± 0.15, closely similar for both phases and essentially unaffected by 3 mm ATP (Fig. 7,panels A and B, and TableIII). This behavior suggests that the two subunits of the tetramer, although showing different values of the oxygen dissociation rate constants (as from the biphasic progress curves in Fig. 6), are functionally regulated by the same protonation process, indicating that differences in the observed rates should be related to variations in the conformation of the distal side of the heme pocket.Figure 7pH dependence of oxygen dissociation rate constant (k; s−1) of A. orianae Hb, in the absence (panel A) and presence (panel B) of 3 mm ATP. Wavelength, 430 nm; temperature, 21 °C.Continuous lines are the best fitting according to Equation2, with parameters reported in Table IV. Open circles, fast phase; filled squares, slow phase. Details are given in the text.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IIIParameters obtained from the analysis according to Equation 2 of the pH dependence of oxygen dissociation of A. orianae Hb in the absence and presence of 3 mm ATP, at 20 °Ck ack alkpK as−1Without ATP Fast phase62.6 ± 2.4214.4 ± 5.76.97 ± 0.12 Slow phase26.2 ± 0.774.4 ± 3.37.06 ± 0.10With ATP Fast phase73.0 ± 2.9198.3 ± 5.97.04 ± 0.11 Slow phase28.6 ± 1.065.2 ± 2.87.20 ± 0.12 Open table in a new tab Fig. 8 shows the pH dependence of the rate constant for the monophasic carbon monoxide binding process to both Hbs. The behavior, substantially unaffected by 3 mm ATP (data not shown), was investigated over a very wide pH range, spanning from pH 2.5 to 9.0. This range has been shown to be suitable in order to follow the effect of the protonation of the proximal and distal histidine on the CO binding process, since over this pH interval both events are taking place (24Coletta M. Ascenzi P. Brunori M. J. Biol. Chem. 1988; 263: 18286-18289Abstract Full Text PDF PubMed Google Scholar, 25Coletta M. Ascenzi P. Traylor T.G. Brunori M. J. Biol. Chem. 1985; 260: 4151-4155Abstract Full Text PDF PubMed Google Scholar). The pH dependence was analyzed according to Equation 2 modified only in that k values of Equation 2 are substituted by l' since the rate constants are referring to the bimolecular carbon monoxide binding rate constants. Furthermore, it must be outlined that theK a value measured in CO binding kinetics refers to the proton binding constant to unliganded Hb. The continuous lines refer to the fitting of the experimental data from the Hbs of A. orianae and P. scotti according to Equation 2, employing the set of parameters reported in TableIV.Table IVKinetic parameters of carbon monoxide bindingl′ acl′ alkpK am−1 s−1A. orianae2.2 (±0.3) × 1064.1 (±0.4) × 1043.52 ± 0.27P. scotti1.3 (±0.2) × 1064.7 (±0.4) × 1043.04 ± 0.17Parameters obtained from the analysis according to Equation 2 of the pH dependence of carbon monoxide binding to A. orianae and P. scotti Hbs, at 20 °C. l′, second order carbon monoxide association rate constant. Open table in a new tab Parameters obtained from the analysis according to Equation 2 of the pH dependence of carbon monoxide binding to A. orianae and P. scotti Hbs, at 20 °C. l′, second order carbon monoxide association rate constant. Species of the Antarctic family Artedidraconidae have only one Hb (26di Prisco G. Camardella L. Carratore V. Caruso C. Ciardiello M.A. D'Avino R. Fago A. Riccio A. Romano M. Rutigliano B. Tamburrini M. Battaglia B. Bisol P.M. Varotto V. Proceedings of the 2nd Meeting on Antarctic Biology. Edizioni Universitarie Patavine, Padova1994: 157-177Google Scholar, 27di Prisco G. Giardina B. Soc. Exp. Biol. Semin. Ser. 1996; 59: 23-51Google Scholar, 28di Prisco G. Battaglia B. Valencia J. Walton D.W.H. Proceedings of the SCAR 6th Biological Symposium, Venice (Antarctic Communities: Species, Structure and Survival). Cambridge University Press, Cambridge1997: 251-260Google Scholar, 29di Prisco G. Tamburrini M. D'Avino R. Soc. Exp. Biol. Semin. Ser. 1997; 66: 143-165Google Scholar) in the adult stage. This is the first report of the complete primary structure of the single Hbs of two artedidraconid species,A. orianae and P. scotti. A very high degree of sequence identity (96% for the α chains, and 90% for the β) was found between the Hbs of A. orianaeand P. scotti (Table V), higher than the identity with the major Hbs of species belonging to other Antarctic families (82–91 and 77–83%, respectively). As usual (10di Prisco G. D'Avino R. Caruso C. Tamburrini M. Camardella L. Rutigliano B. Carratore V. Romano M. di Prisco G. Maresca B. Tota B. Biology of Antarctic Fish. Springer-Verlag, Berlin1991: 263-281Crossref Google Scholar), the identity with minor Hbs (Hb 2 and Hb C) of Antarctic fish and with Hbs of non-Antarctic species was substantially lower (51–68%).Table VSequence identity (%) in α and β chains of some fish hemoglobinsSpeciesC. carpio 5-aNon-Antarctic species.S. irid 5-aNon-Antarctic species. Hb IVS. irid 5-aNon-Antarctic species. Hb IT. newnesi Hb 2N. coriiceps Hb 2A. orianaeP. scottiG. acuticepsC. mawsoni Hb 1, Hb 2T. bernacchii Hb 1T. newnesi Hb 1, Hb Cα Chains N. coriiceps Hb 15957556163919182838987 T. newnesi Hb 1, Hb C58625263668685929097 T. bernacchii Hb 1646257657087889191 C. mawsoni Hb 1, Hb 26062536469848593 G. acuticeps58625365678282 P. scotti666060656896 A. orianae6761606468 N. coriiceps Hb 263636293 T. newnesiHb 2615862 Salmo irideus 5-aNon-Antarctic species. Hb I6660 S. irideus 5-aNon-Antarctic species. Hb IV63C. carpio 5-aNon-Antarctic species.S. irid 5-aNon-Antarctic species. Hb IVS. irid 5-aNon-Antarctic species. Hb IC. mawsoni Hb 2T. newnesi Hb CA. orianaeP. scottiG. acuticepsC. mawsoni Hb 1T. bernacchii Hb 1T. newnesi Hb 1, Hb 2β Chains N. coriiceps Hb 1, Hb 25763536570838280889086 T. newnesi Hb 1, Hb 257625364687977808493 T. bernacchii Hb 1616658667080798387 C. mawsoni Hb 15662536770818385 G. acuticeps56595565677778 P. scotti566051626590 A. orianae5962546468 T. newnesi Hb C57625789 C. mawsoniHb 2556054 S. irideus 5-aNon-Antarctic species. Hb I6459 S. irideus 5-aNon-Antarctic species. Hb IV735-a Non-Antarctic species. Open table in a new tab Although cladograms (6Eastman J.T. Antarctic Fish Biology: Evolution in a Unique Environment. Academic Press, San Diego1993Google Scholar, 30Iwami T. Mem. Natl. Inst. Polar Res. Ser. E Biol Med. Sci. 1985; 36: 1-69Google Scholar) indicate the family Bathydraconidae to be evolutionarily farther apart from Nototheniidae than Artedidraconidae, the artedidraconid sequences show lesser identity with those of two nototheniid species (T. newnesi and Trematomus bernacchii) than the identity between the nototheniids and two bathydraconids (Gymnodraco acuticeps and Cygnodraco mawsoni). Sequences of Hbs of other notothenioids, together with evidence from phylogenetic analysis based on partial sequences of 12 S and 16 S mitochondrial ribosomal RNA (31Bargelloni L. Ritchie P.A. Patarnello T. Battaglia B. Lambert D.M. Meyer A. Mol. Biol. Evol. 1994; 11: 854-863PubMed Google Scholar), will hopefully contribute to understanding the evolutionary history. Among the functionally important amino acid residues (32Perutz M.F. Brunori M. Nature. 1982; 299: 421-426Crossref PubMed Scopus (148) Google Scholar), in the β chain of both Hbs, Ser F9, Glu FG1, Gln HC1, and His HC3 are conserved, Arg H21 is conservatively replaced by Lys, whereas Asp NA2 and Lys EF6 are replaced by Gln and Met, respectively. At the α1β2 interface, the residues forming the flexible joint between the α1 FG corner and the β2 C helix (Arg βC6, Trp βC3, Arg αFG4, Asp αG1, and Pro αG2) are conserved; among the residues forming the switch region between the α1 C helix and the β2 FG corner, His βFG4 and Thr αC6 are conserved, whereas Thr αC3 and Pro αCD2 are replaced by Gln (as in all fish hemoglobins) and Ala (as in Cyprinus carpio and Catostomus clarkii), respectively. The Hbs of A. orianae and P. scotti (as well as those of other species of Artedidraconidae, such as Artedidraco shackletoni, D. longedorsalis, Pogonophrynesp. 1, sp. 2 and sp. 3) 2G. di Prisco, unpublished data., 3M. Tamburrini and G. di Prisco, unpublished data. are characterized by a modest Bohr effect, very weak or no Root effect, and very low cooperativity of oxygen binding. Similar results were obtained with blood, intact erythrocytes, or unstripped hemolysates. ATP slightly enhances the Bohr effect, and induces the Root effect to a limited extent. The Root effect is further induced upon addition of ATP to blood, intact erythrocytes, or unst
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