Unique features of the hemoglobin system of the AntarcticPanagiotis Marinakis, Maurizio Tamburrini, Vito Carratore and Guido di Prisco Institute of Protein Biochemistry, CNR, Naples, Ita
Trang 1Unique features of the hemoglobin system of the Antarctic
Panagiotis Marinakis, Maurizio Tamburrini, Vito Carratore and Guido di Prisco
Institute of Protein Biochemistry, CNR, Naples, Italy
The hemolysate of the Antarctic teleost Gobionotothen
gibberifrons(family Nototheniidae) contains two
hemoglo-bins (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 a- and two b-chains) The complete amino-acid
sequence of the two hemoglobins (Hb 1, a1b12; Hb 2, a2b22)
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
Keywords: fish; Antarctic; hemoglobin; amino-acid sequence; oxygen binding
Organisms living in extreme environments, such as the
Arctic and Antarctic sea waters, are exposed to strong
constraints, among which temperature is often a driving
factor [1–4] Hemoglobin (Hb), a direct link between the
exterior and body requirements, has thus experienced a
major evolutionary pressure in these organisms to adapt
its functional features at molecular/functional level The
search for correlation between fish hematology and the
extreme conditions of the Antarctic environment leads to
a study on the biochemistry of oxygen transport, centred
on Hb molecular structure and oxygen-binding
properties, taking the ecological constraints under
con-sideration In view of the role of temperature in
modifying the oxygenation-deoxygenation cycle in
respir-ing tissues, thermodynamic analysis deserves special
attention
The largely dominant Antarctic suborder Notothenioidei
is by far the most thoroughly characterized group of fish in
the world Thirty-five species (all bottom dwellers) of the 38
so far investigated were shown to have a single major Hb (Hb 1) and often a minor one (Hb 2, 5%of the total Hb content, generally having the b-chain in common with
Hb 1) both displaying, with some exceptions, strong Bohr and Root effects [2,5] Each of the remaining three species (Trematomus newnesi and Pagothenia borchgrevinki, two active cryopelagic species; Pleuragramma antarcticum, a pelagic, sluggish but migratory fish) all belonging to the family Nototheniidae, have a unique oxygen-transport system, and each system appears adjusted to the fish specific life style, substantially different from that of the sluggish benthic species
Compared with other Notothenioidei, the Antarctic teleost, Gobionotothen gibberifrons (family Nototheniidae),
is endowed with novel hematological features A detailed study of the oxygen-transport system is herewith reported
A preliminary communication on the Hb system of this species has appeared previously [6] G gibberifrons lives at a depth range between 5 and 750 m in the waters of northern Antarctic Peninsula and of the islands located north-east
G gibberifronshas Hb 1 and Hb 2 The complete amino-acid sequence of the two components has been established, and the regulation of oxygen binding by pH, allosteric effectors (chloride and organophosphates) and temperature has been investigated
Materials and methods
Toyopearl Super Q-650S was from TosoHaas (Laboratory Service Analytical); trypsin (EC 3.4.21.4) treated with
L-1-tosylamide-2-phenylethylchloromethylketone from Cooper Biomedical; dithiothreitol from Fluka; Tris, bis-Tris, Hepes, Mes, 4-vinylpyridine and IHP from Sigma; sequanal-grade reagents from Applied Biosystems;
Correspondence to G di Prisco, Institute of Protein Biochemistry,
CNR, Via Marconi 12, I-80125 Naples, Italy.
Fax: + 39 0815936689; Tel.: + 39 0817257242;
E-mail: diprisco@dafne.ibpe.na.cnr.it
Abbreviations: Hb, haemoglobin; OPA, o-phthalaldehyde; IHP,
inositol hexakisphosphate; P 50 , partial pressure of oxygen required to
achieve Hb half saturation.
Note: The protein sequence data reported in this paper will appear in
the SWISS-PROT and TrEMBL knowledgebase under the accession
numbers P83611 (a 1 chain), P83612 (b 1 chain), P83613 (a 2 chain) and
P83614 (b2chain).
(Received 7 July 2003, revised 1 August 2003,
accepted 7 August 2003)
Trang 2HPLC-grade acetonitrile from Laboratory-Scan Analytical.
All other reagents were of the highest purity commercially
available
Specimens of G gibberifrons were caught at Dallmann
Bay and Low Island (6325¢S, 6215¢W), onboard the
research vessels R/V Hero and R/S Polar Duke Blood
samples were drawn from the caudal vein by means of
heparinized syringes Hemolysates were prepared as
des-cribed [7] Stripping of endogenous ligands was carried out
by running aliquots through a small column of Dowex AG
501 X8 (D), a mixed bed ion-exchange resin
Separation of Hbs was achieved by FPLC
anion-exchange chromatography on a Toyopearl Super Q-650S
column (1.5· 10 cm) Elution was carried out with a
gradient from 0 to 50%of buffer B (500 mM Tris/HCl,
pH 7.6) in buffer A (10 mMTris/HCl, pH 7.6) in 75 min
The flow rate was 0.5 mLÆmin)1; the absorbance was
measured at 546 nm The Hb-containing pooled fractions
were dialysed against 10 mMHepes pH 7.7 All steps were
carried out at 0–5C No oxidation was
spectrophoto-metrically detectable Hb solutions were stored in small
aliquots at)80 C until use
Globin separation was accomplished by reverse-phase
HPLC of purified Hbs on a lBondapak C18 column
(Waters, 3.9· 300 mm) Elution was carried out with a
gradient from 0 to 100%of eluent B (60%acetonitrile) in
eluent A (45%acetonitrile, containing 0.3%trifluoroacetic
acid) in 32.5 min The flow rate was 1 mLÆmin)1; the
absorbance was followed at 280 nm
Alkylation of the sulfhydryl groups with 4-vinylpyridine,
deacetylation of the a-chain N-terminus and tryptic
diges-tions were carried out as described [8,9]
Tryptic peptides were purified by reverse-phase HPLC on
a lBondapak-C18 column (Waters, 3.9· 300 mm)
Clea-vage of Asp-Pro bonds was performed on polybrene-coated
glass-fibre filters in 70%(v/v) formic acid, for 24 h at 42C
[10] Asp-Pro-cleaved globins were treated with OPA before
sequencing [11] in order to block the non-Pro N-terminus
and reduce the background
Sequencing was performed with an Applied Biosystems
Procise 492 automatic sequencer, equipped with on-line
detection of phenylthiohydantoin amino acids
The molecular mass of the S-pyridylethylated a- and
b-chains and of peptides of less than 10 kDa was
measured by MALDI-TOF mass spectrometry on a
PerSeptive Biosystems Voyager-DE Biospectrometry
Workstation Analyses were performed on premixed
solutions prepared by diluting samples (final
concentra-tion, 5 pmolÆlL)1) in four volumes of matrix, namely (a)
10 mgÆmL)1sinapinic acid in 30%acetonitrile containing
0.3%trifluoroacetic acid (v/v/v; for globin analysis), and
(b) 10 mgÆmL)1a-cyano-4-hydroxycinnamic acid in 60%
acetonitrile containing 0.3%trifluoroacetic acid (v/v/v;
for peptide analysis)
Oxygen-saturation curves were determined as described
[8] Oxygen equilibria were measured at 5C and 10 C, in
100 mM Hepes buffer (pH range 6.0–8.0) prepared at the
temperature of the oxygen-binding measurements The final
Hb concentration was 0.5–1.0 mM on a heme basis An
average standard deviation of ± 3%for values of P50was
calculated Experiments were performed in duplicate To
measure stepwise oxygen saturation, a modified gas
diffu-sion chamber (Eschweiler) was used, coupled to cascaded Wo¨sthoff pumps for mixing pure nitrogen with air [12,13] Absorbance variations between deoxygenated and oxygen-ated Hb were measured at 436 nm with an Eppendorf spectrophotometer model 1101 M pH values were meas-ured at the end of each experiment with a Radiometer BMS Mk2 thermostatted electrode Sensitivity to chloride was assessed by adding NaCl to a final concentration of
100 mM The effects of IHP were measured at a final ligand concentration of 3 mM, namely a large excess over the concentration of tetrameric Hb Oxygen affinity (denoted
by P50) and cooperativity (nH) were calculated from the linearized Hill plot of log S/(1-S) vs log PO2 at half saturation; S denotes fractional oxygen saturation The amplitude of the Bohr effect is given by the Bohr coefficient, /¼ Dlog P50/DpH
The overall oxygenation enthalpy change DH (kcalÆmol)1; 1 kcal¼ 4.184 kJ), corrected for the heat of oxygen solubilization ()3 kcalÆmol)1), was calculated by the integrated van’t Hoff equation, DH¼) 4.574[(T1T2)/ (T1–T2)]Dlogp50/1000
Results
Hb and globin purification Cellulose acetate electrophoresis showed that the hemo-lysate of G gibberifrons contains two Hbs (Hb 1 and Hb 2), accounting for 80–85%and 15–20%, respectively, of the total Hb content The two Hbs were purified by ion-exchange chromatography on a Super Q ToyoPearl column (Fig 1) Hb 2 often appeared to be contaminated by Hb 1;
a second chromatography on the same column yielded pure
Hb 2 (not shown)
Reverse-phase HPLC of the hemolysate showed two major and two minor peaks (Fig 2) HPLC of Hb 1 showed two peaks, whose elution times corresponded to those of the major peaks of the hemolysate; Hb 2 showed two peaks having elution times corresponding to those of the minor peaks The molecular mass values (Da), obtained
by MALDI-TOF mass spectrometry, were 15 597 and
Fig 1 Ion-exchange chromatography of G gibberifrons hemolysate on
a Toyopearl column Details are given in Materials and methods.
Trang 316 097 (a1and b1chains, respectively, of Hb 1), and 15 800
and 16 420 (a2and b2of Hb 2)
Amino-acid sequencing
The primary structure of the globins was established by
sequencing intact proteins, internal regions obtained after
specific hydrolysis of Asp-Pro bonds, and tryptic peptides
purified by reverse-phase HPLC
a-Chains
Direct sequencing of intact a-chains was unsuccessful,
suggesting that (as in all Antarctic fish Hbs examined so far)
the N-terminal residue is blocked, therefore not available
to direct Edman degradation The molecular masses of the
N-terminal tryptic peptides of a1 and a2, measured by
MALDI-TOF mass spectrometry, were 43 Da higher than
those found after sequencing the unblocked peptides, thus
confirming that the a-chain N-terminus is acetylated
Following cleavage of the Asp-Pro bonds, sequencing
proceeded from Pro96 to Asp127 in a1, and from Pro96
to Lys140 in a2
In a1, cleavage at Lys5 and at Arg93 by trypsin was not
complete, therefore peptides T1–T2 and T12–T13 were also
found The peptide bond after Lys116 was not cleaved at all
Also the peptide bonds after Lys61 and Lys62 were not
completely cleaved, generating peptides T10a and T10b
which coeluted Four additional peptide pairs (T1 and T1– T2; T3 and T16; T6 and T13; T7 and T12–T13) coeluted from the column; however, sequences were unambiguosly established on the basis of their different amounts
In a2, trypsin failed to cleave the peptide bond after Lys7 Two peptide pairs (T1 and T10; T5 and T9) coeluted; again, sequences were established on the basis of their different amounts Sequence 101–140 was established only after Asp-Pro cleavage
Figure 3A,B shows the complete amino-acid sequences
of the a chains Tryptic peptides were aligned on the basis of sequence homologies with known globin sequences, and with the sequences obtained following Asp-Pro cleavage Each chain is made of 142 residues The molecular masses, calculated from the sequence, are 15 605 and 15,790, for a1 and a2, respectively
b-Chains Direct sequencing proceeded for 20 and 31 residues for the b-chain of Hb 1 (b1) and Hb 2 (b2), respectively After cleavage of the Asp–Pro bond, the internal sequences from Pro100 to Leu134 in b1, and from Pro100 to Lys132 in
b2, were established
In b1, T2 and T11 were not found, and their sequence was directly established from the N-terminus of the intact globin (T2) and from the internal sequence obtained after cleavage
of the Asp-Pro bond (T11) T10 and T12 coeluted in the same chromatographic peak, and their sequence was established on the basis of their different amount
In b2, T2 and T6 coeluted in the same peak Being the sequence of T2 already known from the N-terminus, the sequence of T6 was established by difference analysis Figure 3C,D reports the complete sequences of the
b chains Tryptic peptides were aligned as described for the a chains, and with the sequences obtained from the N-terminus Each chain is made of 146 residues The molecular masses, calculated from the sequence, are
16 081 and 16 400 for b1and b2, respectively
Oxygen binding Functional studies were carried out on Hb 1 and Hb 2, determining the oxygen-binding curves as a function of pH
in the temperature range 5–10C, in the absence and presence of allosteric effectors (Fig 4 and Table 1) In the
pH range examined, the oxygen affinity of Hb 2 was lower than that of Hb 1 All samples displayed the alkaline Bohr effect, slightly enhanced by chloride and, to a higher extent,
by organophosphate The latter had a very strong effect at alkaline pH values, especially in Hb 2, which, although apparently reducing the amplitude of the Bohr coefficient (/) in the pH range examined, is indicative of a stronger overall Bohr effect In all samples oxygen binding was cooperative above pH 6.5 in the absence of effectors Chloride and, to a higher extent, phosphate, enhanced the decrease in oxygen-binding cooperativity brought about by increasing proton concentration In fact, IHP virtually abolished cooperativity from pH 7.5 downwards
All samples displayed the Root effect, which was maximal in the pH range 6.5–7.5 (Fig 5) Oxygen-satura-tion curves were determined at atmospheric pressure In the
Fig 2 Reverse-phase HPLC of G gibberifrons hemolysate, Hb 1 and
Hb 2 (A, B and C, respectively) Details are given in Materials and
methods.
Trang 4Fig 3 Amino-acid sequences of a 1 , a 2 , b 1 and b 2 globin chains (A, B, C and D, respectively) The tryptic peptides (T) and the sequence portions elucidated by automated Edman degradation from the N-terminus and after cleavage of an Asp-Pro bond are indicated below the sequences.
Trang 5absence of IHP, complete saturation was achieved at pH 7.5
with Hb 1 and Hb 2 At pH 6.0, the saturation of Hb 1 and
Hb 2 was 73 and 56%, respectively In the presence of IHP,
maximal saturation was achieved at pH 8.0 in Hb 2, and at
pH 7.5 in Hb 1 At pH 6.0, the saturation of Hb 1 and
Hb 2 was 58 and 47%, respectively In all samples, IHP
shifted the inflexion of the curve (corresponding to maximal
Root effect) towards more alkaline pH
Temperature variations had different effects on the
oxygen binding of Hb 1 and Hb 2, both in the absence
and presence of allosteric effectors (Fig 6) Compared to
Hb 1, Hb 2 showed more exothermic DH values at acidic
pH, whereas lower values were measured at pH 8.0 In the
presence of chloride, the DH values of Hb 2 were higher (in
absolute value) than those of Hb 1 in the entire pH range
Oxygen-binding studies were also carried out on intact
erythrocytes and stripped hemolysate (not shown), which
showed intermediate functional properties between those of
Hb 1 and Hb 2 Erythrocytes contain endogenous
organo-phosphates; consequently, the curves are similar to those
obtained with the stripped hemolysate in the presence of
effectors
Discussion
In the Antarctic suborder Notothenioidei, most species of the family Nototheniidae have one major and one minor Hb (Hb 1 and Hb 2, 95%and 5%of the total, respectively) [2, 4,14] The two Hbs have the b-chain in common, with the exception of those of Cygnodraco mawsoni [15] which share the a-chain Therefore, Nototheniidae (and all Notothe-nioidei) are generally characterized by reduced Hb multi-plicity compared to many teleosts of temperate waters [2] In
T newnesi, P antarcticum and P borchgrevinki higher multiplicity was observed [16–18], but these species are pelagic and migratory, differing in life style from the other notothenioids, which are in general sluggish, benthic fish
G gibberifrons is also a sluggish, benthic nototheniid Not much more is known about its life style However, in comparison with all other benthic nototheniids, it has distinct and novel hematological features The blood has the highest level (approx 15–20%) of the minor component
Hb 2 ever found in Antarctic Notothenioidei; unlike other nototheniids, in which Hb 2 tends to disappear in adults (e.g T bernacchii and D mawsoni only have Hb 2 in juveniles), the level of Hb 2 does not decrease in adult fish Moreover, unlike all other species, the two Hbs of
G gibberifrons do not have any globin in common An
Hb system where two components are made by four chains
is a unique case among Notothenioidei Four chains instead
of three might well be a feature of speciation in the pathway
of evolution of the suborder
Fig 4 Oxygen-equilibrium isotherms (Bohr effect) and subunit
coop-erativity, as a function of pH, of Hb 1 (A, B, respectively) and Hb 2 (C,
D, respectively) Experiments were carried out at 5 C in 100 m M
Hepes or Mes buffers, in the absence of effectors (s), in the presence of
100 m M NaCl (m), and of 100 m M NaCl, 3 m M IHP (j).
Table 1 Bohr coefficients (/), calculated from the oxygen-binding
curves determined in the absence of effectors and in the presence of
100 m M NaCl or 100 m M NaCl and 3 m M IHP.
No effectors NaCl (100 m M ) NaCl/IHP (100/3 m M )
Hb 1
Hb 2
Fig 5 Oxygen-saturation curves at atmospheric pressure (Root effect)
of Hb 1 (A) and Hb 2 (B) Experiments were carried out at 2 C, in
100 m M Tris/HCl or bisTris/HCl buffers, in the absence (s) and presence (d) of 3 m M IHP.
Trang 6Although from a morphological point of view G
gibber-ifronsevidently belongs to the family Nototheniidae [19–21],
Tokita et al [22] have reported dendrograms based on
genetic distances obtained from two-dimensional gel
elec-trophoresis of total protein constituents of cardiac muscle
In these dendrograms the nototheniid G gibberifrons
appears more closely related to species belonging to
the most phyletically advanced notothenioid families
Bathydraconidae and Channichthyidae, than to the clade
composed by three other nototheniid species As far as
red-blooded species are concerned, this interesting conclusion
is not fully supported by our results on Hb sequences
(Table 2) In fact, the sequence identities do not indicate
clear grouping of G gibberifrons either with species of the
same family (Trematomus bernacchii and T newnesi) or of Bathydraconidae (Gymnodraco acuticeps and C mawsoni) However, the lack of a clear relationship with the Hb sequences of other nototheniid species suggests that in this case the evolution of the oxygen-transport system has occurred in response to special needs of this species, as shown by the intermediate position taken by G gibberifrons Hbs in phylogenetic trees [23]
In line with other Antarctic Hbs, the sequence identity between the a chains of the two Hbs of G gibberifrons is 68%; it is 70% between the b-chains In summary, the a-chain and the extra b-chain of Hb 2 have much higher sequence identity with minor than with major Hbs of other Antarctic species Thus the latter extra chain also groups with minor Hbs
In Antarctic fish Hbs, Aspb94, which in human HbA establishes a strong ionic bond with Hisb146, important for the Bohr-effect mechanism [24], is generally conservatively replaced by Glu Moreover, Vala1 is always replaced by Ac-Ser, which cancels the contribution of the N-terminus to the Bohr effect These substitutions are also found in the two Hbs of G gibberifrons, characterized by the Bohr and Root effects However, these Hbs show significantly distinct Bohr coefficients and amplitude of Root effect; in particular,
Hb 2 has lower oxygen affinity than Hb 1 in the pH range examined, and phosphate modulation of the affinity is stronger in Hb 2 than Hb 1 These differences might be due
to other substitutions in the primary structure For instance,
in Hb 2 it is worth noting that position b82, which is part of the phosphate binding site [25,26], is occupied by Lys, whereas in Hb 1 the latter residue is replaced by Ala This substitution may well account for the lower effect of organophosphates in the latter Hb
Temperature dependence, which is governed by the associated overall enthalpy change, is an important feature
of the reaction of Hbs with oxygen Heat absorption and release can be considered physiologically relevant modula-ting factors, similar to hetero and homotropic ligands The two Hbs of G gibberifrons also differ in thermodynamic behaviour Hb 1 is less sensitive to temperature variations than Hb 2 which, in turn, shows strong variations of enthalpy change especially at pH below 7.5, depending on chloride and/or phosphate In particular, Hb 2 shows a progressive increase of the exothermic enthalphy change as
a function of proton concentration This feature has never been reported in fish Hbs Chloride virtually abolishes this exothermic change by providing a strong endothermic contribution to oxygen binding Although a molecular inter-pretation is hard to find, this differential thermodynamic
Fig 6 Oxygenation enthalpy of Hb 1 (A) and Hb 2 (B) DH values
were calculated in the temperature range 5–10 C from the
oxygen-binding data reported in Fig 4 and Table 1 Experimental conditions
were: 100 m M Hepes or Mes buffers, in the absence of effectors (s), in
the presence of 100 m M NaCl (m), and of 100 m M NaCl, 3 m M IHP
(j).
Table 2 Sequence identity (%) between a- and b-chains of G gibberifrons and of some other Antarctic fish Hbs T bernacchii Hb C, P borchgrevinki
Hb 0 and C mawsoni Hb 2 are minor components P borchgrevinki Hb 0 and Hb 1, in addition to C mawsoni Hb 1 and Hb 2, share the a-chain.
T bern Hb 1 T bern Hb C P borch Hb 1 P borch Hb 0 G acut Hb C maws Hb 1 C maws Hb 2 a-chains
b-chains
Trang 7regulation of the oxygenation/deoxygenation cycle (that
may play a significant role in keeping the internal
tempera-ture constant) may be an important adaptive tool
This paper reports some novel features of the oxygen
transport of a notothenioid fish species The results suggest
that G gibberifrons Hb 2 cannot merely be considered an
evolutionary remnant, as in other Antarctic Notothenioidei
[27] The functional differences suggest that Hb 2, rather
than being a vestigial or larval remnant, may indeed have a
physiological role; the two Hbs of this cold-adapted teleost
might be used alternatively to face special needs in relation
with life style and different environmental conditions (e.g
temperature fluctuations during migration) requiring fine
regulation of oxygen binding Finally, although in an
organism biosynthesis of higher amounts of an additional
Hb can be easily accomplished and may be considered a
short-time response to environmental changes, preservation
of the role of the gene duplication which has produced an
additional chain is a physiologically complex long-term
response, and may well be considered an evolutionarily
important adaptation
Acknowledgements
This study is in the framework of the Italian National Programme for
Antarctic Research.
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