The amino acid sequence and oxygen-binding properties of the single hemoglobin of the cold-adapted Antarctic teleost Gymnodraco acuticeps

ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

Vol. 292, No. 1, January, pp. 295-302, 1992

The Amino Acid Sequence and Oxygen-Binding Properties of the Single Hemoglobin of the ColdAdapted Antarctic Teleost Gymnodraco acuticeps Maurizio

Tamburrini,*

Andrea

Brancaccio,?

Rodolfo

Ippoliti,t

and Guido

di Prisco*”

*Institute of Protein Biochemistry and Enzymology, C.N.R., Via Marconi 10, 80125 Naples, Italy; and fC.N.R. Center of Molecular Biology and Department of Biochemical Sciences, University ‘La Sapienza,” Piazza A. Moro 5, 00185 Rome, Italy

Received July 26, 1991, and in revised form September 20, 1991

The complete amino acid sequence of the single hemoglobin of the Antarctic teleost Gymnodraco acuticeps has been determined. The (Y chain contains 142 amino acid residues; an acetylated seryl residue is at the amino terminal. The fl chain contains 146 residues. A very high degree of sequence identity has been found with hemoglobins of other Antarctic fishes. Oxygen binding is not modulated by pH and allolsteric effecters. The Bohr and Root effects are absent, alt.hough specific amino acid residues, considered responsible of most of these functions, are conserved in the seque:nce, thus posing new questions about the molecular basis of these mechanisms. The low heat of oxygenation may be interpreted as one of the mechanisms involved in the process of cold adaptation. Q 19%

Academic

Press,

Inc.

The temperature of the oxygen-rich coastal Antarctic ocean is constantly at -1.87”C, the equilibrium temperature of seawater and ice. Antarctic fishes can survive in this environment, since they have developed mechanisms of cold adaptation. For example, they synthesize “antifreeze” (glyco)peptides, which lower the freezing temperature of blood and other fluids in a noncolligative way (1). A further aspect of fish cold adaptation is the modification of hematological cha.racteristics. In general, studies on several families of Antarctic teleosts indicate a decrease in the number of erythrocytes and hemoglobin concentration (2-4). This feature compensates for the increase in blood viscosity at low temperatures. The total lack of hemoglobin and of cells containing this protein in the blood of fishes of the fami1.y Channichthyidae (5) represents the extreme stage of such evolution.

’ To whom correspondence

should be addressed.

0003-9861/92 $3.00 Copyright 0 1992 hy Academic Press, Inc. All rights of reproduction in any form reserved.

The hematological peculiarities of Antarctic teleosts prompted an investigation on the molecular structure and oxygen-binding properties of hemoglobins from these organisms. Fish species of the suborder Notothenioidei, largely endemic and confined within the Antarctic ocean, have only one major hemoglobin, often accompanied by a minor (approximately 5% of total) component (6, 7). The amino acid sequence of the two hemoglobins of Notothenia coriiceps neglecta (family Nototheniidae) and of Cygnodraco mawsoni (family Bathydraconidae) have recently been reported (8-ll), and the functional properties of several hemoglobins of Antarctic teleosts have been described. These hemoglobins are generally characterized by a strong alkaline Bohr effect and also display the Root effect (12-14). Gymnodraco acuticeps, of the endemic family Bathydraconidae, is a benthic sedentary fish living in shallow waters close to the anchor ice. In this paper we report the structural and functional characterization of its single hemoglobin. The complete amino acid sequence of CYand p chains has been elucidated. Studies on the oxygen- and carbon monoxide-binding properties have shown the absence of the Bohr and Root effects. Thus, in the single hemoglobin of G. acuticeps, oxygen binding is not regulated by pH or by allosteric effecters, such as endogenous organic phosphates. This is a unique feature, not only among species of extreme latitudes, but also (to our knowledge) among fishes in general. Moreover, thermodynamic analysis of oxygen equilibria suggests that the unusually low enthalpy change in the oxygenation of hemoglobin may reflect a mechanism of molecular adaptation to extreme life conditions. EXPERIMENTAL

PROCEDURES

Specimens of G. acuticeps were collected by means of gill nets, set 100 m deep on the bottom, in the vicinity of Terra Nova Bay Station, Ross Sea (74’42’S, 164O07’E), Antarctica. Fishes were kept in aquaria supplied with running, aerated seawater at approximately -1.O”C. 295

296

TAMBURRINI

DEAE-cellulose (DE 52) was from Whatman; trypsin (EC 3.4.21.4), treated with L-1-tosylamide-2-phenylethylchloromethylketone, from Cooper Biomedical; carboxypeptidases A (EC 3.4.17.1) and B (EC 3.4.17.2), Tris, and bistris’ buffers from Sigma Chemical Co.; dithiothreitol from Fluka; and all sequanal-grade reagents from Applied Biosystems; HPLC-grade acetonitrile from Carlo Erba. All other reagents were of the highest purity commercially available. Preparation of hemolysates, cellulose acetate electrophoresis, “stripping” for organic phosphates, and preparation of globins were carried out as previously described (15). The hemolysate was ion-exchange chromatographed on a DE 52 column (2.2 X 20 cm), equilibrated with 10 mM Tris-HCI, pH 7.6; the hemoglobin was eluted with 50 mM TrisHCl at the same pH. The globin mixture from the purified hemoglobin was dissolved in 0.1% trifluoroacetic acid, containing 10% acetonitrile. The globin chains were separated by FPLC on a pro-RPC HR 5/10 reverse-phase column (Pharmacia); the solvents were 0.1% trifluoroactic acid (A) and acetonitrile (B); the flow rate was 0.5 ml/min. Up to 4 mg globin mixture was loaded for each preparative run. The fractions containing each purified globin were pooled and lyophilized. SDS-PAGE was carried out according to Laemmli (16), in a 9-15% polyacrylamide linear gradient, containing 6 M urea in the upper gel

’ Abbreviations used: FPLC, fast protein liquid chromatography; IHP, inositol hexakisphosphate; bistris, Z-[bis(2-hydroxyethyI)amino]-2(hydroxymethyl)1,3-propanediol; PAGE, polyacrylamide gel electrophoresis; MS, mass spectrometry; SDS, sodium dodecyl sulfate; Hb, hemoglobin.

ET AL. and 8 M urea in the lower gel. Running conditions and staining were as described (15). Total hydrolysis of globins and peptides was carried out in 6 N HCl, containing 0.02% phenol and 0.04% 2-mercaptoethanol. Hydrolysis tubes were sealed under vacuum and kept at 110°C for 20 h. Tryptophan was determined after hydrolysis with 3 M mercaptoethanesulfonic acid (17). Cysteine was determined as cysteic acid after oxidation with performic acid (18). Amino acid analysis was performed with an automatic singlecolumn Carlo Erba analyzer, Model 3A29. C-terminal sequences were determined by digesting the 01and fl chains with carboxypeptidases B and A, respectively. Samples (0.3-0.5 nmol) were dissolved in 0.2 M N-ethylmorpholine acetate buffer, pH 8.5, containing 1% SDS; the enzymes were at a 1:lOO molar ratio. Incubation was at 37°C. Aliquots were taken at different times and the enzymes were precipitated by adding equal volumes of 1 M acetic acid. After centrifugation, the supernatants were subjected to amino acid analysis. Alkylation of cysteyl residues was achieved by reaction with 4-vinylpyridine (19). The globins were dissolved at a concentration of 10 mg/ ml in 0.5 M Tris-HCl, pH 7.8, containing 2 mM EDTA and 6 M guanidine hydrochloride. A B-fold excess of dithiothreitol was added; the solution was kept at 37°C for 2 h; incubation with a 20.fold excess of 4+inylpyridine was then carried out for 30 min at room temperature in the dark. The reaction was stopped by addition of dithiothreitol; excess reagents were removed by reverse-phase FPLC, as described above. Tryptic digestion was carried out on pyridylethylated chains (100 nmol/ml), dissolved in 1% ammonium bicarbonate. Trypsin, dissolved in 1 mM HCL, was added at a ratio of 1:lOO (by weight); after a 3-h incubation at 37”C, another aliquot of enzyme was added, at a final ratio of 1:50 (by weight). After 6 h, the reaction was stopped by lyoph-

1.5

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1.0

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0.5

0 U

10

20

TIME, FIG.

1.

FPLC of globin chains on a reverse-phase

30

min.

column. Other details are given under Experimental

Procedures.

THE

SINGLE TABLE

HEMOGLOBIN

OF THE

I

Amino Acid Composition of the Globin Chains of Gymnodraco czcuticeps Hemoglobin

Asp Asn Thr Ser Glu Gin Pro

(D) (N) (T) (S) (E)

01chain

0 chain

11.0 ;;;

16.6 gp’

5.2 (5) 13.4 (13)

9.1 (9) 9.6 (9)

10.9 1;;

(Q) (P)

6.6 8.0 11.6 1.0 11.0 2.5 10.0 13.8 3.4 7.4 5.4 12.4 4.9 2.3 IL42

GUY (G)

Ala (A) CYS CC) Val (V) Met (M) Ile (I) Leu (L) Trr 0’) Phe (F) His (H) LYS (K) Arg (R) Trp W) No. of residues

(6) (8) (12) (1) (11) (3) (12) (14) (3) (7) (6) (13) (5) (2)

3.5 10.6 18.0 1.9 7.9 1.6 7.6 15.8 6.6 7.7 5.7 11.9 1.9 2.5 146

(3) (10) (17) (2) (7) (2) (10) (17) (6) (7) (6) (12) (2) (2)

Note. Relative molar amounts of amino acids are given. The numbers of residues from sequence are indicated in parentheses. Cys was determined as cysteic acid (18). ilization. The hydrolysates were resuspended in 0.1% trifluoroacetic acid and clarified by centrifugation. Separation of tryptic peptides ‘was obtained by HPLC, on a F-Bondapak C,, reverse-phase column (Waters, 0.39 X 30 cm), using a Beckman Model 324 gradient liquid chrom,atograph, equipped with a variablewavelength monitor. The solvents were 0.1% trifluoroacetic acid (A) and 0.08% trifluoroacetic acid in acetonitrile (B); the flow rate was 1 ml/min. Up to 20 nmol of peptide mixture was loaded for each preparative run. Tryptic peptides were numbered sequentially from the N-terminus. In situ acidic cleavage of Asp-P:ro bonds (20) was performed in intact globins by wetting the samples, immobilized on polybrene-coated filters, with 70% formic acid. The filters were enclosed in a heat-sealed plastic envelope and incubated at 38’C for 24 h; the yield was about 40%. In the o( chain, this treatment also provided partial removal of the Nterminus-blocking group. Removal of the blocking group in the N-terminal peptide of the ru chain, unavailable to Edman degradation, was performed as described (21). Amino acid sequencing of intact or acid-treated globin chains, and of the purified tryptic peptides, was performed using the automated proteinpeptide sequencer Model 477A from Applied Biosystems, equipped with a 120A analyzer for the on-line detection of phenylthioidantoin amino acids, and 0.1-1.0 nmol of sample. The N-terminus-blocking group of the o( chain was identified by fast
RESULTS Preparation Chains

of Hemoglobin

and Separation

of Globin

Cellulose acetate electrophoresis of the hemolysate showed a single hemoglobin component. The hemolysate

ANTARCTIC

FISH

Gymnodraco

acuticeps

297

was run through a DEAE-cellulose (DE 52) column, and the hemoglobin obtained in pure form by a single-step elution. Crystallization was achieved by dialysis of concentrated solutions against ammonium sulfate at 40% saturation, adjusted at pH 8.0 by addition of Tris. Crystals were formed within 24 h and measured about 1 mm in length. These procedures were carried out at the Antarctic station. Pure hemoglobin was converted to the carbon monoxide derivative, frozen at -8O”C, and shipped back to Naples for further studies. The globin mixture was analyzed by FPLC on a reversephase column. The separated (Y and /l chains accounted for 49 and 41%, respectively, of the eluted proteins (Fig. 1). The total yield of the chromatography was 77%. The molecular weight of the (Yand @chains, estimated by SDS-PAGE, was 15,200 and 16,200, respectively. The amino acid compositions are reported in Table I. Amino

Acid Sequence

The N-terminus of the (Y chain was not accessible to Edman degradation, suggesting the presence of a blocking group; sequencing of the N-terminus of the @chain proceeded for 28 steps. The (Y and p chain C-terminal sequences, determined by carboxypeptidase digestion, were -Arg-Tyr-Arg and -Gin-Tyr-His, respectively. Treatment of the globin chains with 70% formic acid resulted in the cleavage of an internal Asp-Pro bond (Aspg6-Pro96 in the LYchain and Asp,,-ProlDO in the p). After blocking the non-Pro N-terminus of the chains by reaction with o-phthalaldehyde (24), a stretch of internal sequence (28 residues of the (Y and 24 residues of the p chain, from Prog6 and Proloo, respectively) was elucidated. After acidic cleavage, the (Y chain was also sequenced without prior reaction with o-phthalaldehyde; this procedure yielded the first 19 residues of the N-terminal sequence, due to the partial removal of the blocking group. The tryptic peptides of the two chains were separated by HPLC on a reverse-phase column. Figure 2 shows the tryptic maps of (Y (A) and /3 (B) chains. In a! chain Tl, fast atom bombardment MS revealed an acetyl group at the N-terminus, similar to other fish hemoglobins. The sequences of several peptides were aligned by overlapping with N-terminal sequences, internal sequences obtained after Asp-Pro cleavage, and Cterminal sequences. The remaining peptides were aligned on the basis of sequence identities or homologies with hemoglobins of other Antarctic and non-Antarctic fishes (see Ref. (9)). The complete amino acid sequences of the two chains are reported in Fig. 3. The (Y and p chains, respectively, contain 142 and 146 residues; the molecular weight is 15,816 and 16,014, in good agreement with the values determined by SDS-PAGE. The degree of sequence identity among the cy and P chains of G. acuticeps hemoglobin and those of other Antarctic and non-Antarctic fish hemoglobins is reported in

298

TAMBURRINI

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T14

0

FIG. 2.

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,a T2

<’

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min.

HPLC of tryptic peptides of S-pyridylethylated

(Y(A) and B (B) chains. Details are given under Experimental

Procedures.

Table II. In Fig. 4, the hydropathy difference profiles (25), of the major component Hb 1 of the Antarctic fish species using a window size of five amino acid residues, show N. coriiceps neglecta of the family Nototheniidae (respecseveral identical regions when comparing the 01 and /3 tively, A and E) and C. mawsoni of the family Bathydraconidae (B and D). In contrast, the comparison with the chains of G. acuticeps hemoglobin with the (Yand p chains

A Ac-~LSDKDKAA"RALWSTISKSSDAI~~DALSRMIV\rY5 Tl

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KKMDNIEAAYADLSSLHSEKLHVDPDNFKLLSDCITIVLAAKLGSAFTAETQATFQKFLGAVMSALGKQYH , TlO , Tll T12 , T13 ,T14 T9a I I I I I

T9b

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FIG. 3. Amino acid sequence of cy (A) and /3 (B) chains of G. acuticeps hemoglobin. The one-letter notation sequences, and sequences obtained after acidic cleavage and carboxypeptidase digestion are indicated.

I -1 -7-l

is used. Tryptic

peptides, N-terminal

THE:

SINGLE

HEMOGLOBIN

OF THE

ANTARCTIC

TABLE

FISH

Gymnodraco

299

acuticeps

II

Sequence Identity (%) in (Yand /3 Chains of Fish Hemoglobins S. irideus” Species a chains N. coriiceps neglecta Hb 1 C. mawsoni Hb 1, Hb 2 G. acuticeps N. coriiceps neglecta Hb 2 S. irideus’ Hb I S. irideus” Hb IV C. carpio’

T. thynnus’

73 77 75 68 65 60 66

C. carpio”

Hb IV

59 60 58 63 66 63

57 62 62 63 60

HbI

55 53 53 62

N. coriiceps neglecta Hb2

63 69 67

G. acuticeps

82 93

C. mawsoni Hbl, Hb2

83

S. irideus” Species p chains N. coriiceps neglecta Hbl, Hb2 C. mawsoni Hb 1 G. acuticeps C. mawsoni Hb 2 S. irideus” Hb I S. irideus” Hb IV C. carpio”

T. thynnus’

66 67 63 60 51 61 60

Note. In N. coriiceps neglecta, the minor component a Non-Antarctic species.

C. carpio”

Hb IV

Hb I

57 56 56 55 64 73

63 62 59 60 59

53 53 55 54

C. mawsoni Hb2

65 67 65

G. acuticeps

80 85

C. mawsoni Hb 1

88

Hb 2 has the /3 chain in common with Hb 1; in C. mawsoni, the chain in common is the a.

chains which differentiate the minor component Hb 2 of these two Antarctic species from Hb 1 (the (Ychain in N. coriiceps neglecta (C) and the /3 chain in C. mawsoni (F)) indicates the absence of identical regions. Oxygen Binding The purified hemoglobin had no alkaline Bohr effect in the pH range 6.0-8.0, the Bohr coefficient + = A log P&A pH being close to zero (Fig. 5). The hemoglobin had a low oxygen affinity (PII = 30.9 mm Hg at pH 7.0, lO”C), which was only slightly lowered by the endogenous effecters chloride ions and organic phosphates (PIi = 37.1). The Hill coefficient varied from 2.0 to 2.5 in the range of pH investigated. The effect of temperature on the oxygen affinity was investigated in the range lo--20°C, in which the van? Hoff plot was linear. Values of AH for oxygenation at pH 7.0, after subtracting the heat of oxygen solubilization (approximately -3.0 kcal/mol. of oxygen), were -0.56 and -2.02 kcal/mol, in the presence and absence of chloride ions and IHP, respectively. At pH 8.0, in the absence of effecters, AH was -2.91 kcal/mol. Oxygen-saturation curves were obtained with the stripped hemolysate and the purified hemoglobin. As expected, no Root effect was observed, even in the presence of IHP or ATP (see inset of Fig. 5).

Carbon Monoxide Rebinding Under all experimental conditions explored, the time courses of carbon monoxide rebinding to G. acuticeps hemoglobin after flash photolysis (Fig. 6) are described by one or two well-separated exponentials. At high levels of photolysis (from 50 to 100%) a large fraction of the unliganded hemoglobin (90% or more) recombined slowly with the ligand, and a second-order rate constant of 8.0 + 2.0 X 10e4 M-l s-l was calculated; this rate was independent of pH and organic phosphates. At neutral or acidic pH, lower levels of photolysis (e.g., 15%) populated a quickly reacting species, characterized by a second-order rate constant of 1.0 to 1.5 X lo6 M-l s-l; the relative weight and the rate constant of this kinetic component were almost independent of allosteric effectors. At pH 8.5 the 15% photolysis failed to elicit the faster component to any significant extent, and, although the overall reaction rate was twice as fast as that observed after complete photolysis, it was described by a single exponential for more than 90% of the total optical density change. DISCUSSION Antarctic fishes generally have either a single hemoglobin, or a major hemoglobin component representing 90-95% of the protein and a minor component of the

300

TAMBURRINI

0

50

100

ET AL.

0

50

RESIDUE FIG. 4.

Hydropathy

difference

I

profiles with the hemoglobins

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A

I I

I I

I

l

2.0 I I

I I

2.0 -

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No.

of the Antarctic

6

species N. coriiceps neglecta (A, C, El and C. mawsoni (B, D, F).

order of 5 to lo%, in contrast with fishes of other latitudes, which generally have multiple hemoglobins (26). G. acuticeps has only one hemoglobin, which has been isolated in pure and crystalline form and subsequently thoroughly characterized, both in terms of primary structure and of oxygen- and carbon monoxide-binding properties. N-Acetylated Ser was detected at the N-terminus of the CYchain, similar to other Antarctic (8,9,11) and non-

A

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L

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7.0

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100

,-” a -E

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$0

,

01

7.0

50

8.0

PH

’ 6.0

I

I 7.0

I

1 8.0

PH FIG. 5. Oxygen equilibrium isotherms (Bohr effect) (A) and the Hill coefficient (B) as a function of pH; 0.1 M Tris or bistris buffer. Absence (0) and presence (A) of 100 mM NaCl and 3 mM IHP. PI,* was measured in millimeters Hg. The inset shows oxygen saturation of pure hemoglobin as a function of pH (Root effect); 0.1 M Tris or bistris buffer. Absence (0) and presence (A) of 3 mM IHP. Other details are given under Experimental Procedures.

0 8.0

9.0

PH

FIG. 6. Half-time of carbon monoxide recombination to G. acuticeps hemoglobin as a function of pH. Absence (squares) and presence (circles) of 3 mM organic phosphate. Closed symbols, partial photolysis; open symbols, full photolysis.

THE

SINGLE

HEMOGLOBIN

OF

THE

Antarctic fishes. The N-terminus of the p chain and the C-terminus of both chains (Val, Arg, and His, respectively) are also conserved. A very high degree of conservation has been found when examining (a) the five residues known to be invariant in both globin chains of all vertebrates; (b) the 26 residues conserved in horse and human hemoglobin and in sperm whale myoglobin; (c) the 19 heme-contact residues; (d) the “sliding contact” regions cri& or LY&; and (e) the “packing contact” regions ai& or cvZpz (27). High conservation during evolution, in the domains of structural and functional importance, is hardly unexpected. Substitutions have occurred lmore frequently, on the other hand, in domains where the structural and functional requirements are less stringent. The sequence identity among the major or single hemoglobins of Antarctic fish species is very high, even among species of different families. This is reflected in the hydropathy difference Iprofiles (Fig. 4) and is probably a sign of the strong evolutionary pressure to which every species has been exposed at the extreme southern latitudes. In addition, these profiles support the hypothesis that the minor components, where present designated Hb 2 in the literature (6, 7), are merely an evolutionary remnant, without physiologica. significance, as suggested by the much lower degree of sequenceidentity with the major hemoglobins. The oxygen binding of -the purified hemoglobin of G. acuticeps is not pH regulated. In accordance with previous results on the hemolysate (28), no alkaline Bohr effect (29) was found, even in the presence of chloride ions and organic phosphates as effecters. Consequently, the stripped hemolysate and the purified hemoglobin of G. acuticeps did not display tlhe Root effect (30, 31), which is an exaggerated Bohr eflect and is due to overstabilization of the low-affinity T-state hemoglobin; it is expressed by a dramatic decrease in oxygenation and loss of cooperativity at low pH values, where saturation of Root-effect hemoglobins with oxygen is no longer possible. The kinetics of carbon monoxide rebinding to G. acuticeps hemoglobin showed two distinctive features, i.e., the lack of effect of pH and organic phosphates and the anomalous effect of partial photolysis at alkaline pH; these will be discussed separately. The overall time course of carbon monoxide binding to this hemoglobin was insensitive to the pH of the medium and to allosteric effecters; thus kinetic evidences on this point strictly agree with equilibrium data. Moreover, the cooperative interactions that operate during ligand binding were reflected, at neutral or acidic pH, by the effect of partial photolysis which populated partially liganded hemoglobin intermediates, characterized by high affinity and a high ligand-recombination rate constant. On the other hand, the observation that at alkaline pH the partial photolysis populated the quickly reacting species only to less than 10% of the unliganded hemes is in keeping with the observed decrease of the Hill coefficient at this pH

ANTARCTIC

FISH

Gymnodraco

acuticeps

301

and suggests that the high-affinity R state of this hemoglobin is not stabilized and may in fact be destabilized by proton release. These findings are in agreement with the absence of the Bohr effect. Moreover, the fact that the high-affinity R state is paradoxically more populated in partialphotolysis experiments carried out at acid pH is the exact contrary of what is found for Root-effect hemoglobins (32). When considered in the framework of the stereochemical model proposed by Perutz and Brunori (33), the hemoglobin of G. acuticeps seemsrather unorthodox. In fact, the /3 chain does have the two residues considered to account for the Root and Bohr effects, not displayed by this hemoglobin, i.e., Ser F9 (93) and His HC3 (146) at the C-terminus. It seems likely that specific oxygen-binding features (e.g., absence of the Root and Bohr effects) depend upon complex structural interactions among molecular domains, so that the substitution of the amino acid residues responsible for the above-mentioned effects may become irrelevant. This hemoglobin is characterized by a reduced sensitivity to changes in temperature and, consequently, by a low heat of oxygenation. In terms of energy saving, this feature is likely to be an important factor of adaptation to the extreme conditions of the Antarctic environment. To our knowledge, the cold-adapted Antarctic teleost G. acuticeps is the first fish speciesin which oxygen transport, mediated by a single hemoglobin, has been found not to be modulated by pH and allosteric effecters. Although unusual, these features are in accordance with the general behavior and lifestyle of G. acuticeps, confined in a stable environment such as the Antarctic ocean with relatively constant physicochemical features. Being a slow predator on small marine organisms, it does not need a large oxygen turnover; the absence of a Bohr effect is balanced by the low oxygen affinity of the hemoglobin, which facilitates oxygen release to the tissues in conditions of acidosis, and by the small amount of energy required during the oxygenation-deoxygenation cycle. ACKNOWLEDGMENTS This study is in the framework of the Italian National Program for Antarctic Research. We thank Professor M. Brunori and Dr. A. Bellelli for helpful comments and suggestions. The outstanding contribution of Mr. V. Carratore in sequencing the protein is gratefully acknowledged. Fast atom bombardment MS was performed at the MS Center, Faculty of Medicine, University of Florence, Italy.

REFERENCES 1. Cheng, C. C., and DeVries, A. L. (1991) in Life under Extreme Conditions (di Prisco, G., Ed.), pp. 1-14, Springer-Verlag, Berlin/Heidelberg. 2. Everson, I., and Ralph, R. (1968) Br. Antarct. Suru. Bull. 15, 5962. 3. Hureau, J.-C., Petit, D., Fine, J. M., and Marneux, M. (1977) in Adaptations within Antarctic Ecosystems (Llano, G. A., Ed.), pp. 459-477, Smithsonian Institution, Washington, DC.

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TAMBURRINI

4. Wells, R. M. G., Ashby, M. D., Duncan, S. G., and Macdonald, J. A. (1980) J. Fish Biol. 17, 517-527. 5. Ruud, J. T. (1954) Nature 173,848-850. 6. di Prisco, G., and D’Avino, R. (1989) Antarct. Sci. 1, 119-124. 7. di Prisco, G., D’Avino, R., Camardella, L., Caruso, C., Romano, R., and Rutigliano, B. (1990) Polar Biol. 10, 269-274. 8. D’Avino, R., Caruso, C., Romano, R., Camardella, L., Rutigliano, B., and di Prisco, G. (1989) Eur. J. Biochem. 179, 707-713. 9. D’Avino, R., Caruso, C., Camardella, L., Schinini, M. E., Rutigliano, B., Romano, R., Carratore, V., Barra, D., and di Prisco, G. (1991) in Life under Extreme Conditions (di Prisco, G., Ed.), pp. 15-33, Springer-Verlag, Berlin/Heidelberg. R., Caruso, C., Schinini, M. E., Rutigliano, B., Romano, 10. D’Avino, R., Camardella, L., Bossa, F., Barra, D., and di Prisco, G. (1990) Comp. B&hem. Physiol. B 96,367-373. B., Romano, M., and di Prisco, G. (1991) 11. Caruso, C., Rutigliano, Biochim. Biophys. Acta 1078, 273-282. 12. di Prisco, G. (1986) Antarct. J. U.S. 21, 215-216. 13. di Prisco, G., Giardina, B., D’Avino, R., Condo, S. G., Bellelli, A., and Brunori, M. (1988) Camp. Biochem. Physiol. B 90, 585-591. 14. di Prisco, G. (1988) Comp. Biochem. Physiol. B 90,631-637. 15. D’Avino, R., and di Prisco, G. (1989) Eur. J. Biochem. 179, 699705. 16. Laemmli, U. K. (1970) Nature 227, 680-685. 17. Penke, B., Ferenczi, R., and Kovacs, K. (1974) Anal. Biochem. 60, 45-50. 18. Hirs, C. H. W. (1967) in Methods in Enzymology (Hirs, C. H. W., Ed.), Vol. 11, pp. 197-199, Academic Press, New York. 19. Friedman, M., Krull, L. H., and Cavins, J. F. (1970) J. Biol. Chem. 245, 3868-3871.

ET

AL.

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