The primary structure of hemoglobin from reindeer (Rangifer tarandus tarandus) and its functional implications

Biochimica et BiophysicaActa, 1076(1991)221-224 © 1991ElsevierSciencePublishersB.V.(BiomedicalDivision)0167-4838/91/$03.50 ADONIS 0167483891000512

221

BBAPRO33823

The primary structure of hemoglobin from reindeer ( Rangifer tarandus tarandus) and its functional implications Raffaete Petmzzel!i i, Donatella Barra 2 F r a n c e s c o Bossa 2, Saverio G. C o n d 6 1 Ole Brix s, Matti N u u t i n e n 4 a n d Bruno G i a r d i n a i t Oipartimentodi Biologia e di Medicina Sperimentalee Scien2e Biochimiche Universitd di Roma Tot Vergata, Roma (italy), : Dipartimento di Scienze Biochimicheand CNR Centro di Biologia Molecolare Universitd di Roma La Sapienza, Rema (Italy), J Zoological Laboratory, Unieersityof Bergen, Bergen (Norway) and 4 Departmentof Pediatrics, Universityof Oalu, Oalu, (Finland)

(Received25 July1990)

Keywords: Aminoacidsequence:Hemoglobin;(Reindeer) The primary stmctmes of a- and ,8-eheins of hemoglobin from reindeer (Raagi]er tarandus t a n m d m ) were determined. Comparison of the re'mdeer hemoglobin sequence with those of human and bovine hemoglobins showed 50 and 29 substitutions per aj8 dimer, respectively. The influence oi replacements on the modulation of hemoglobin oxygen affinity by heterothopic ligands and temperature, as well as their importance on the strueture-function relationships in hemoglobin are discussed.

Introduction The functional properties of hemoglobin from the reindeer were recently reported [1-3]. Compared to human HbA, the protein shows an intrinsically low oxygen affinity and displays a very unusual response to changes in temperature. In fact, while AH of oxygen binding to the T state of reindeer Hb is strongly exothermic, oxygen binding to the R state is very close to zero. This marked difference in the thermodynamics of the two conformational states of the molecule results in a unique dependence of the ~emperature effect on the degree of oxygen saturation, which could be regarded as a very interesting case of molecular adaptation to extreme environmental conditions. The very minor enthalpy change of the ~'~ction with oxygen of reindeeer Hb (about 3-times lower than that of HbA under the same experimental conditions) slaould in fact be considered in connection with the very low environmental temperatures that this species experi-

* Data supplementaryto this article are depositedin the BBA Data Bank under accessionnumberDD/452//33823/1076 (1991)221. They can be obtained free of charge from: BBA Editorial Secretariat, p.o. Box 1345.1000BH Amsterdam,The Netherlands. Abbreviation:2,3-DPG,2,3-diphosphoglycerate. Correspondence: R. Petmr2.elfi,Dipartimentodi Biologia,Universit~t di RomaTor Vergata, Via Orazio Raimondo,00173Roma,Italy.

ences in its living habitat throughout the year (down to -40°C). Hence, because o; the unusual thermodynamics of the oxygen binding, hemoglobin from reindeer does not require much energy input during its oxygenation-deoxygenation cycle and oxygen delivery in this animal species is not drastically impaired at the level of peripheral tissues, where temperature may be significantly lower than that of the lungs. Moreover, the functional properties of reindeer Hh, as for other ruminants hemoglobins [4-7], are not modulated, in the presence of chloride anions, by 2,3-diphosphoglycerate (2,3-DPG) [1], this observation being in accord with the finding of the very low intraerythrocitic level of this organic phosphate (0.4 raM). In order to elucidate the molecular basis of this unique functional behaviour, we have undertaken an investigation on the amino acid sequence of both a- and fl-chalns of reindeer Hb. From the results obtained, and from a comparison with other available mammalian hemoglobin sequences, many of the peculiar properties shown by reindeer Hb can be satisfactorily explained at the molecular level. Materials and Methods Blood samples were collected as already described [1]. Trypsin-TPCK was from Cooper Biomedical; 6, vinyipyridine from Aldrich Chemic; HPLC grade acetonitrile from Farmitalia Carlo Erba; sequence-grade re-

222 Amino acid analyses were carried out with an LKB 4151 Alpha plus instrument equipped with an LKB 2221 integrator after the hydrolysis of samples (0.5-2 nmol) in 6 M HCI at ll0°C for 24 h. Automated Edman degradation was carried out using an Applied Biosystems model 470 A gas-phase sequencer equipped with an Applied Biosystems model 120A PTH-analyzer for the on-line detection of PTHamino acids. 0.1-2 nmol of peptides were loaded onto prewashed, polybrene-coated TFA-treated glass fiber filters. CNBr fragments of the fl-chain were directly loaded onto the gas phase sequencer for collection of sequence data without purification of the peptide mixture, according to the procedure of Shlyapnikov [10]. Similarly the fragments derived from acid cleavage were subjected to automated Edman degradation after in situ treatment with o-phtahalaldehyde [11].

agents from Applied Biosystems; all other reagents were of highest purity commercially available. The a- and p-chains were purified by high-performance liquid chromatography; the hemoglobin was appiied in several aliquots on a reverse-phase column (Aquapore RP 300, 7 × 250 ram, Brownlee Laboratories), developed in 30 rain with a linear gradient from 35 to 505 acetonitrile in 0.2% trifluoroacetic acid (TFA), generated in a Beckman model 340 instrument at a flow rate ely 3.0 ml/min. The absorbance of the effluent was monitored at 220 nm with a Beckman model I65 spectrophotometer. The a- and/]-chains (2 mg each) were alkylated with 4-vinylpyridine essentially as described [8] and then digested with trypsin (enzyme to protein ratio 1/50 (w/w) in 0.1 M ammonium bicarbonate (pH 7.8) for 4 h at 37°C). Cleavage of the//-chain (2 nmol) with CNBr was performed in 70% formic acid for 15 h at room temperature. Another aliquot of 2 nine! of the a- and fl-chains was incubated in 70% formic acid at 37°C for 48 h, to cleave ti~e polypeptide chains at the unique Asp-Pro bond [3]. Tryptic peptide mixture was purified on a reversephase column (Acquapore RP 300, 4.6 x250 ram, Brownlee Laboratories), developed in 30 min with a linear gradient from 0 to 50~ of acetonitrile in 0.2% TFA generated in a Beckman model 340 instrument at a flow rate of 1 ml/min. The elution of the peptides was monitored both at 220 and 280 run.

Results and Disemsion

The amino acid sequence of the a- and fl-chains of reindeer hemoglobin is presented in Figs. 1 and 2, respectively, along with the corresponding sequences of other mammalian hemoglobins, namely human, bovine and elk. Direct sequence analysis of the purified chains allowed collection of structural information for the first 48 and 64 residues of the a- and fl-globins, respectively. The sequence of the two globins was reconstructed following the purification and analysis of the complete

20 Human Bovine Elk Reindeer

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Fig I. Comp~son of the amino acid sequence of the a-chain of reindeer Hb ~th the corresponding chains of human, bovine and elk hemo~obin. Boxes in~cate identical residues. Arrows above the human sequence indicate the helical re~ons as established for mamm~ian hemoglobim.

• extentof the variousfragmentsusedto reconstructthe sequence.I", trypticpeptides;P, add cleavagepepfide.~ denoteresiduesidentified after automatedsequencingof the intactchain.

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set of tr~ptic peptides; in Figs. 1 and 2 only the tryptic peptides, which were necessary,for the reconstruction of the sequence, are reported. Overlaps were obtained by means of CNBr fragments and those derived after acid cleavage. At any rate, the high similarity with the corresponding chains of elk and ox renders the need for overlapping sequences less stringent. Details on amino acid composition, yield and sequence determination for these peptides are deposited with the supplementary data. Comparison of the amino acid sequence of the aand/]-chains from reindeer hemoglobin with those previously reported for the human, ox and elk proteins gives the number of amino acid replacement shown in Table L The data reported are in agreement with the phylogenetic relatedness of reindeer, elk and ox which belong to different fatuities (reindeer and elk to Cervidae; ox to Bovidae) of the same sub-order (Ruminants) of Artiodactyls and clearly indicates that in these animals the e-chains evolved more slowly than the /i-chains. With respect to haman hemoglobin the amino acid sequence of reindeer Hb differs at 21 positions along the a-chain and at 28 along the/l-chain. These differences include several residues which have been shown by X-ray crystallography [12] to be involved in al/il contacts. In contrast, all the residues that are located at the al/i2 interface in human Hb and are involved in the oxygenation-linked shift of the quaternary structure are

all conserved in reindeer Hb. On this basis, the differences in the functional properties of reindeer and HbA, i.e., the intrinsic low oxygen affinity of reinder Hb and its insensitivity to the presence of 2,3-DPG, have to be ascribed to other regions of the molecule. Inspection of the sequence data of the/i-chain shows that, in comparison with the human r-chain, in the reindeer Va~/il (NA1) is deleted and His/i2 (NA2) is replaced by a methionyl residue, as already observed in all the other ruminant hemoglobins. The presence of a hydrophobic residue at position NA2 fl has been proposed to be at the basis of the low intrinsic oxygen affinity displayed by hemoglobins of ruminants [5]. Thus, the hydrophobic methionine would have to point into the interior of the protein, thereby mimicking 2,3DPG action in stabilizing the tertiary deoxy-structure of the/i subunits.

TABLEI Number of amino add subslimtions between a (above the diagona# and /] (below the diagonal) human, bovine, elk and reindeer hemoglobin chains Human Human Bovine Elk Reindeer

Bovine 17

23 24 28

15 ]8

Elk 2t 13 10

Reindeer 21 11 2

224 However, these substitutions account only in part for the observed properties, since insensitivity of reindeer hemoglobin to 2,3-DPG is not only an intrinsic property of the molecule, but is due also to the interplay between the binding of chloride and that of 2,3-DPG. In fact, in comparison to human HbA, reindeer Hb is characterized by a lower affinity for 2,3-DPG and by an higher affinity for chloride, As a result, chloride at physiological concentrations (= 100 mM) competes efficiently with 2,3-DPG and the latter anion is not able to modulate the functional properties of the hemoglobin molecule. The low-affinity constant of 2,3-DPG can be explained in part by the deletion of Vai tOm and the replacement of His ,02 by Met. In addition reindeer Hb, as all other low oxygen affinity hemoglobins, does not contain a proline residue at position /35 (/~4 if the deletion of Val ,or is taken into consideration). This residue seems to be a characteristic of all the hemoglobins with a functional behaviour similar to that of HbA, Recently, it has been suggested [13] that this position may have a role in determining the stereochemistry of the anion binding pocket and therefore the sensitivity toward a specific anion. All the,;e considerations are in full agreement with the data reported on bovine ,hemoglobin where, chloride ions mask the functional effect of 2,3-DPG [14,151. In fact, in the absence of chloride, bovine Hb does respond to 2,3-DPGjust as reindeer Hb I151. In the case of bovine Hb, the molecule possesses high-alfinity sites that are able to bind both 2,3-DPG and chloride as well as low-affinity sites that bind only chloride anions [15]. Recently Hueno et al. [16] reported that the high-affinity chloride binding sites in bovine Hb are formed by Met .01 and Lys .081 for the liganded form and by Val am and Lys .08m for the unliganded form. It may be envisaged that the same sites occur also in reindeer hemoglobin, since all these residues are present in the a- and *0-chains, Concerning the nature of the low-affinity chloride binding sites the inspection of the hydropathy profiles of/~-¢hains from both reindeer and man is most revealing. Thus, the E helix of the chain from reindeer appears more hydrophilic than that of the ,0-chain of human HbA due to the substitutions Gly 69 (El3) --, Asp and Ala 76 (E20) --, Lys which are present in all the sequences of ruminant hemoglobins so far investigated. Such substitutions, by increasing the hydrophilicity of the E helix, may contribute to modulate the solvent phobicity of these hemoglobins being good candidates for the low-affinity chloride binding sites in view of the proximity of Lys ,076 to Lys ,0slIt may be proposed that, this additional low-affinity chloride binding site could be responsible, at least in

part, for the unusual thermodynamic properties of reindeer Hb with respect to oxygen binding. Thus oxygenation of the hemoglobin molecule would be accompanied by a concomitant endothermic release of chloride ions that would cancd some of the heat released upon oxygen bi'~,ling, thereby contributing to lower the overall enthalp~ change of the reaction. In this perspective, since bovine Hb displays the same structural characteristics, it should be characterized by a small AH of oxygen binding. Although more functional data on ruminant hemoglobins are needed to fully support this hypothe, sis, it is very suggestive that preliminary data, obtained by measuring the oxygen affinity of bovine Hb at different temperatures (in 0.1 M Hepes buffer at pH 7.4 plus 0.1 M NaCI) give a a H value (after correction for the heat contribution of oxygen in solution) of - 2 . 6 Kcal/mol, comparable with that of reindeer Hb under the same experimental conditions ( - 3.3 Keal/mol). Acknowledgements This work was supported in part by grant from Ministero della Pubblica lstruzione (Italy). Dr. Ole Brix was supported by the Norwegian Research Council (NAVF) grant No. 425.90/002.

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