Unique features of the hemoglobin system of the Antarctic
notothenioid fish
Gobionotothen gibberifrons
Panagiotis Marinakis, Maurizio Tamburrini, Vito Carratore and Guido di Prisco
Institute of Protein Biochemistry, CNR, Naples, Italy
ThehemolysateoftheAntarcticteleostGobionotothen
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-andtwob-chains). The complete amino-acid
sequence of the two hemoglobins (Hb 1, a
1
2
b
1
2
;Hb2,a
2
2
b
2
2
)
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,

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. gibberifrons has 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

All other reagents were of the highest purity commercially
available.
Specimens of G. gibberifrons were caught at Dallmann
Bay and Low Island (63°25¢S, 62°15¢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 m
M
Tris/HCl,
pH 7.6) in buffer A (10 m
M
Tris/HCl, pH 7.6) in 75 min.
The flow rate was 0.5 mLÆmin
)1
;theabsorbancewas
measured at 546 nm. The Hb-containing pooled fractions
were dialysed against 10 m
M
Hepes pH 7.7. All steps were
carried out at 0–5 °C. 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

solutions prepared by diluting samples (final concentra-
tion, 5 pmolÆlL
)1
) in four volumes of matrix, namely (a)
10 mgÆmL
)1
sinapinic acid in 30% acetonitrile containing
0.3% trifluoroacetic acid (v/v/v; for globin analysis), and
(b) 10 mgÆmL
)1
a-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 5 °Cand10°C, in
100 m
M
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 m
M
on a heme basis. An
average standard deviation of ± 3% for values of P
50
was
calculated. Experiments were performed in duplicate. To
measure stepwise oxygen saturation, a modified gas diffu-
sion chamber (Eschweiler) was used, coupled to cascaded
Wo
¨

;1kcal¼ 4.184 kJ), corrected for the heat of
oxygen solubilization ()3kcalÆmol
)1
), was calculated by the
integrated van’t Hoff equation, DH ¼ ) 4.574[(T
1
T
2
)/
(T
1
–T
2
)]Dlogp
50
/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

, 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 a
1
, and from Pro96
to Lys140 in a
2
.
In a
1
, 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 a
2
, 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

established on the basis of their different amount.
In b
2
, 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 b
1
and b
2
, 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 5–10 °C, 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.

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

NaCl and 3 m
M
IHP.
No effectors NaCl (100 m
M
) NaCl/IHP (100/3 m
M
)
Hb 1
5 °C )0.64 )0.69 )0.62
10 °C )0.57 )0.68 )0.61
Hb 2
5 °C )0.75 )0.77 )0.43
10 °C )0.89 )0.77 )0.61
Fig. 5. Oxygen-saturation curves at atmospheric pressure (Root effect)
ofHb1(A)andHb2(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)of3m
M
IHP.
Ó FEBS 2003 The hemoglobins of G. gibberifrons (Eur. J. Biochem. 270) 3985
Although from a morphological point of view G. gibber-
ifrons evidently 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

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

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.
References
1. di Prisco, G. & Tamburrini, M. (1992) The hemoglobins of marine
and freshwater fish: the search for correlations with physiological
adaptation. Comp. Biochem. Physiol. 102B, 661–671.
2. di Prisco, G. (1998) Molecular adaptations in Antarctic fish
hemoglobins. In Fishes of Antarctica. A Biological Overview (di
Prisco, G., Pisano, E. & Clarke, A., eds), pp. 339–353. Springer,
New York.
3. di Prisco, G., Tamburrini, M. & D’Avino, R. (1998) Oxygen-
transport systems in extreme environments: multiplicity and
structure/function relationship in hemoglobins of Antarctic fish.
In Cold Ocean Physiology (Po
¨
rtner, H.O. & Playle, R., eds), pp.
143–165. Cambridge University Press, Cambridge, UK.
4. Tamburrini, M. & di Prisco, G. (2000) Oxygen-transport system

tion of O
2
-binding curves of hemoproteins by means of a diffusion
chamber. Anal. Biochem. 32, 362–376.
13. Lykkeboe, G., Johansen, K. & Maloy, G.M.O. (1975) Functional
properties of hemoglobin in the teleost Tilapia grahami. J. Comp.
Physiol. 104, 1–11.
14. di Prisco, G. (1997) Physiological and biochemical adaptations
in fish to a cold marine environment. In Proceedings of the SCAR
6th Biol. Symposium, Venice (Antarctic Communities: Species,
Structure and Survival) (Battaglia, B., Valencia, J. & Walton,
D.W.H., eds), pp. 251–260. Cambridge University Press,
Cambridge, UK.
15. Caruso,C.,Rutigliano,B.,Romano,M.&diPrisco,G.(1991)
The hemoglobins of the cold-adapted Antarctic teleost Cygno-
draco mawsoni. Biochim. Biophys. Acta 1078, 273–282.
16. D’Avino, R., Caruso, C., Tamburrini, M., Romano, M., Ruti-
gliano, B., Polverino de Laureto, P., Camardella, L., Carratore,
V. & di Prisco, G. (1994) Molecular characterization of the
functionally distinct hemoglobins of the Antarctic fish Tremato-
mus newnesi. J. Biol. Chem. 269, 9675–9681.
17. Tamburrini, M., D’Avino, R., Fago, A., Carratore, V., Kunz-
mann, A. & di Prisco, G. (1996) The unique hemoglobin system of
Pleuragramma antarcticum, an Antarctic migratory teleost.
Structure and function of the three components. J. Biol. Chem.
271, 23780–23785.
18. Riccio, A., Tamburrini, M., Carratore, V. & di Prisco, G. (2000)
Functionally distinct haemoglobins of the cryopelagic Antarctic
teleost Pagothenia borchgrevinki. J. Fish Biol. 57A, 20–32.
19. Iwami, T. (1985) Osteology and relationships of the family