Báo cáo khoa học: Correlation between functional and structural changes of reduced and oxidized trout hemoglobins I and IV at different pHs doc

Correlation between functional and structural changes of reduced
and oxidized trout hemoglobins I and IV at different pHs
A circular dichroism study
Rosita Gabbianelli
1
, Giovanna Zolese
2
, Enrico Bertoli
2
and Giancarlo Falcioni
1
1
Dipartimento di Biologia M.C.A., Universita
`
di Camerino, Camerino, Italy;
2
Istituto di Biochimica, Facolta
`
di Medicina,
Universita
`
Politecnica delle Marche, Ancona, Italy
Circular dichroism (CD) spectra of two major hemoglobin
components (Hb), HbI and HbIV, from Oncorhyncus
mykiss (formerly Salmo irideus) trout were evaluated in
the range 250–600 nm. HbI is characterized by a complete
insensitivity to pH changes, while HbIV presents the Root
effect. Both reduced [iron(II) or oxy] and oxidized (met)
forms of the two proteins were studied at different pHs, 7.8
and 6.0, to obtain information about the pH effects on
the structural features of these hemoglobins. Data obtained

show that oxy and met-HbI are almost insensitive to pH
decrease, remaining in the R conformational state also at
low pH. On the contrary, the pH decrease induces similar
structural changes, characteristics of ligand dissociation
and R fi T transition, both in the reduced and in the
oxidized HbIV. The structural changes, monitored by CD,
are compared with the peroxidative activity of iron(II)-Hb
and met-Hb forms and with the superoxide anion scav-
enger capacity of the proteins.
Keywords: trout hemoglobin derivatives; hemoglobin per-
oxidase activity; superoxide anion; circular dichroism; pH
effect.
The hemoglobin system of the Oncorhyncus mykiss (for-
merly Salmo irideus) trout is made up of four electro-
phoretically distinct components, two of which [trout
hemoglobin (Hb)I (% 20%) and trout HbIV (60%)] repre-
sent quantitatively a large fraction of the whole pigment. In
the last years, the properties of these two major components
(HbI and HbIV) have been investigated in considerable
detail under various experimental conditions [1,2]. Their
structural and functional characterization has indicated
some striking differences between the two proteins that have
been related to their different physiological role [2]. HbI is
characterized by the presence of cooperative phenomena
and complete absence of pH and organic phosphate effects,
while in HbIV, oxygen affinity and cooperativity depend on
pH and organic phosphates (Root effect). In air (pO
2
¼
155 mmHg) and at pH 7.8, iron(II) HbIV is entirely in the

oxygenated form, while at pH 6.0 it is almost completely in
the deoxy form, as a consequence of the Root effect [2]. On
the contrary, iron(II) HbI is fully oxygenated at both pHs.
Moreover, both hemoglobins are stable towards dissoci-
ation even in the ligated form; the value of the tetramer–
dimer dissociation constant is between 10 and 50 times less
than that of human hemoglobin measured under similar
conditions [2].
The main function of HbI is to assure the basic level of
O
2
to active tissues, providing a normal oxygen supply
in emergency, while a basic role of HbIV is to release
O
2
against high hydrostatic pressure in the swim bladder.
Our previous study by circular dichroism (CD) demon-
strated large structural differences in HbI and HbIV ([3] and
references cited therein), which are likely related to their
different physiological roles as gas carriers.
Trout HbIV and HbI are also characterized by different
peroxidative activities [4,5], which are dependent from the
pH of the medium and/or the iron oxidation state. In fact, it
is known that the hemoglobin molecules present peroxida-
tive properties [5–7], which may be important for the life
span of red blood cells (RBCs), continuously exposed to
extracellular and intracellular sources of reactive oxygen
species (ROS), which are a potential cause of oxidative
injury and could have a role in erythrocytes senescence [7].
The toxicity of H

2
O
2
, a reactive oxygen species involved
in cellular injury under various pathophysiological condi-
tions, is known to be enhanced in the presence of
hemoglobin [8]. H
2
O
2
binds to and reacts with Hb,
generating the highly reactive ferrylhemoglobin intermedi-
ate, which in turn oxidizes the substrate [6]. The final
products of the reaction are superoxide radical and met-Hb.
However, our previous studies on human hemoglobin
demonstrated that the presence of Hb reduces the level of
superoxide in the medium [7]. Met-Hb was shown to be
more efficient in reducing the level of this radical with
respect to oxyHb, and this difference was more marked at
low pH values [7]. It is known that, during reversible oxygen
binding, Hb undergoes a slow autoxidation to met-Hb,
producing superoxide, which is released into the heme
pocket [9] The superoxide released in the heme pocket reacts
with globin, producing a secondary radical [9].
Correspondence to R. Gabbianelli, Dipartimento di Biologia M.C.A.,
Universita
`
di Camerino, Via Camerini 2–62032 Camerino (Mc), Italy.
Fax: + 39 073 7636216, Tel.: + 39 073 7403208,
E-mail:

Abbreviations: HbI, hemoglobin I; HbIV, hemoglobin IV; ROS,
reactive oxygen species; RBCs, red blood cells.
(Received 23 December 2003, revised 15 March 2004,
accepted 25 March 2004)
Eur. J. Biochem. 271, 1971–1979 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04109.x
As heme’s interaction with globin appears to be import-
ant both for autoxidation [9] and H
2
O
2
binding [6], a
systematic study of the heme–globin interaction, at different
pH and Hb oxidative state [iron(II)- or met-Hb], could be
important to increase the knowledge of the structural basis
of these interactions.
Our previous CD study [3] demonstrated that the heme–
globin interaction in oxy-HbI and oxy-HbIV are quite
different. The aim of the present paper will be to charac-
terize the heme–globin interaction in iron(II)- and met-
HbIV and HbI, at different pH values, by CD. These studies
will be compared with the peroxidative and superoxide
anion scavenger activities of these proteins. The CD
spectroscopy will be particularly useful because this tech-
nique permits the evaluation of the optical activity of heme
proteins [10,11], which result from different kinds of heme
interaction with the protein matrix.
Three different wavelength regions (near-UV, far-UV
and visible) can offer different degrees of information. These
CD regions are largely used to study the tertiary and
quaternary structure of heme proteins [10,11].

A direct comparison for differences in structure between
trout HbI and HbIV in solution will increase the knowledge
on the physiological roles of these Hbs and on the molecular
adaptation mechanisms of these aquatic organisms living
under particular environmental conditions.
Materials and methods
All reagents were of analytical grade. Preparation of trout
hemoglobin components were performed as previously
described [12]. Iron(III) hemoproteins were obtained by the
addition of K
3
[FeCN)
6
] (molar ratio 2 : 1) to the oxygen-
ated derivative; excess oxidizing agent and ferrocyanide
were removed by gel filtration through a Sephadex G-25
column eluted with 50 m
M
Tris/HCl pH 7.8 or 50 m
M
Bis/
Tris, pH 6.0. The hemoglobin concentration was deter-
mined by the pyridine–hemochromogen method [13].
Circular dichroism
CD spectra were recorded on a Jasco spectropolarimeter
under nitrogen flux at 6 °C. In the near UV and Soret region
the Hb concentration was 0.1 mgÆmL
)1
, while in the range
490–670 nm the concentration was 0.5 mgÆmL

)1
. Measure-
ments were carried out in 1 cm path length quartz cuvettes.
The molar ellipticity is always expressed, on a molar heme
basis, as degreeÆcm
2
Ædmol
)1
. Readings were carried out
against a reference cuvette containing the same components
without protein. Data were acquired at 6 °C, in order to
increase Hb stability [14] and to mimic environmental living
conditions of the trout. All spectra are the averages of four
experiments, where three different recording were accumu-
lated for each sample.
Peroxidase activity assay
The assay for peroxidase activity was performed as reported
by Everse et al. [6] using guaiacol as substrate. Fifty
millimolar sodium phosphate/citrate buffer (0.9 mL) at
pH 5.4 and containing Hb and 10 m
M
guaiacol was used.
The reaction was started by the addition of 157 m
M
H
2
O
2
solution (0.1 mL) and monitored by absorbance changes at
470 nm. The absorbance change was due to Hb-catalyzed

oxidation of the substrate by hydrogen peroxide.
Chemiluminescence measurements
Chemiluminescence measurements were performed by
lucigenin as chemiluminogenic probe, and superoxide
radicals were produced by xanthine/xanthine oxidase sys-
tem as previously described [15]. Briefly, the chemilumines-
cence (CL) was measured in automatic LB 953 (Berthold
Co., Wildbad, Germany) in a reaction mixture containing
0.9 UÆmL
)1
xanthine oxidase, 40 lgÆmL
)1
of hemoprotein
and 150 lmolÆL
)1
lucigenin in 1 mL of the chosen buffer.
The reaction was started by the injection of xanthine at the
final concentration of 50 l
M
andfollowedfor60sas
previously described [16]. Values obtained are expressed as
counts per second (c.p.s.).
Results
Circular dichroism
L-Band (260 nm region) and 270–300 nm region. The
270–300 nm region of CD spectra was used to study changes
in the Hb quaternary structure at the a
1
b
2

interface [3,17].
Within the near UV region (1250–300 nm) CD bands are due
to aromatic amino acids, S-S bridges and heme groups [11],
and are poorly characterized. Near this region, the
L
-band
(centered around 260 nm) is considered to be sensitive to the
interactions between the heme and the surrounding globin,
being influenced by the attached ligand and thereby by the
spin state of the iron atom [11]. According to Perutz et al.
[18], the region around 285 nm is considered as indicative of
the R fi T transition: in the T-form, the ellipticity is more
negative than in the R-form [10,11,19]. The ellipticity change
in this band is independent from the ligand state of heme, but
it is indicative only of R fi T transition [10].
CD spectra acquired in the near-UV and Soret regions
(250–320 nm and 320–470 nm, respectively) for iron(II)-
HbI and iron(II)-HbIV at both pHs and in air are shown in
Figs 1 and 2. In the range 250–320 nm (Fig. 1), CD spectra
are baseline corrected to zero ellipticity at 320 nm, accord-
ing to Henry et al. [20]. In the region 320–470 nm (Fig. 2),
spectra are corrected to zero ellipticity at 470 nm. In line
with our previous data [3] iron(II)-HbI (Figs 1B and 2B)
and iron(II)-HbIV (Figs 1A and 2A) spectra in the range
250–470 nm show similar positive bands, resembling the
dichroic characteristics of other oxy-hemoglobins [21]. In
our experimental conditions, the ellipticity of the L band
(centered around 260 nm) is directly related to pH
(Fig. 1A,C) for both iron(II)-HbIV and met-HbIV. Only
small changes between iron(II)- and met-HbIV, at both pH

values, are evident (Fig. 1A,C). L-Band ellipticity is directly
related to pH decrease also in iron(II)-HbI and met-HbI
(Fig. 1B,D), although this effect is more evident for met-
HbI. Following iron oxidation (Fig. 1D) almost no changes
in this band were observed at pH 7.8 [comparing this value
to the L-band of iron(II)-HbI at the same pH].
In both iron(II)- and met-HbIV, the change from pH 7.8
to 6.0 induces a shift towards a more negative ellipticity
(correlatedtoRfi T transition) in the region of 285 nm
1972 R. Gabbianelli et al. (Eur. J. Biochem. 271) Ó FEBS 2004
Fig. 1. Near-UV CD spectra of iron(II)-HbIV (A), iron(II)-HbI (B),
met-HbIV (C) and met-HbI (D) in 50 m
M
Tris/HCl, pH 7.8, (solid line)
and 50 n
M
Bis/Tris, pH 6.0 (broken line). Temperature ¼ 6 °C.
Fig. 2. Soret CD spectra of iron(II)-HbIV (A), iron(II)-HbI (B), met-
HbIV (C) and met-HbI (D) in 50 m
M
Tris/HCl, pH 7.8 (solid line) and
50 n
M
Bis/Tris, pH 6.0 (broken line). Temperature ¼ 6 °C.
Ó FEBS 2004 Structural changes in trout hemoglobins (Eur. J. Biochem. 271) 1973
(Fig. 1A,C). For iron(II)-HbIV, this behavior is in agree-
ment with the presence of the Root effect, where the
pH decrease induces deoxygenation and transition to the
T-form. However, met-HbIV, compared with iron(II)-
HbIV at the same pH, always shows a slightly more

negative ellipticity (Fig. 1A,C). In trout iron(II)-HbI, the
pH decrease induces hardly any changes in the negative
ellipticity value at 285 nm (Fig. 1B); while in met-HbI, at
pH 7.8 (Fig. 1D) a slight increase in the negative ellipticity
compared with the same protein at pH 6.0 is evident. On the
other hand, met-HbI at pH 6.0 shows a similar ellipticity to
iron(II)-HbI at both pHs.
Visible region. The Soret region for both iron(II) and met-
HbIV at pH 6.0 and 7.8 are shown in Fig. 2A,C. Changes
in the CD Soret region (near 400 nm) were related to the
interaction of the heme prosthetic group with the surround-
ing aromatic residues and to modifications in the spatial
orientation of these amino acids with respect to heme [22].
These modifications affect porphyrin transitions and p–p*
transitions in the surrounding aromatic residues. However,
the protein-induced heme distortions from planarity and the
contributions of polarizable groups (near the heme) have
been recently postulated to participate to the ellipticity in the
Soret region [23].
According to some authors ([11,23] and references cited
therein) a blue shift in the Soret band is a consequence of
R fi Ttransitionanditreflects theinteractionbetweena
1
and
b
1
subunits. It may be due to tertiary structural changes in
regions including aromatic residues and it may be involved in
the interactions between these subunits [24]. However, this
bandissensitivealsotoliganddetachment(deligation)[17,19].

In the Soret region at pH 7.8, HbIV in the oxygenated-
form is characterized by a band at 418 nm, in agreement
with our previous data [3]. A similar band is present in other
oxy-hemoglobins, such as human HbA [10,25]. A significant
red shift (to about 433 nm) and a decreased intensity in this
peak was measured in both iron(II)- (Fig. 2A) and met-
HbIV (Fig. 2C), as a consequence of pH decrease, although
the superimposition of two different bands in met-HbIV at
pH 6.0 is evident (Fig. 2C). Compared with the iron(II)
form at the same pH, met-HbIV shows a small blue shift in
the Soret band (from 418 to 416 nm at pH 7.8, while the
position of the peak measured at pH 6.0 is more difficult to
calculate, due to its form) and an increased positive
ellipticity (Fig. 2A,C). In iron(II)-HbI (Fig. 2B), the Soret
band is also localized at 418 nm, in line with previous results
[3]. In this protein, the pH lowering induces a slight decrease
in the Soret band ellipticity, without wavelength shift
(Fig. 2B). Met-HbI (Fig. 2D), compared with the iron(II)-
form, shows a small blue shift and an increased ellipticity in
this band, at both pH values.
The two major peaks in the region 500–600 nm (Q
0
and
Q
m
) give indications on the constraints at the heme site and
reflect the symmetric properties of the heme-iron bound
material [11]. In particular, they are correlated to the
asymmetry of the proximal bond. In fact, a decreased
symmetry leads to an enhanced intensity in the Q

0
band
[25,26]. The splitting of these bands has been regarded as a
lowering of the heme symmetry in HbA-CO [17,27], due to
nonperpendicular iron–ligand bond above the xy plane of
the heme group.
Fig. 3. Circular dichroism spectra in the visible region of iron(II)-HbIV
(A), iron(II)-HbI (B), met-HbIV (C) and met-HbI (D) in 50 m
M
Tris/
HCl pH 7.8 (solid line) and 50 n
M
Bis/Tris, pH 6.0 (broken
line). Temperature ¼ 6 °C.
1974 R. Gabbianelli et al. (Eur. J. Biochem. 271) Ó FEBS 2004
Both iron(II)-HbI (Fig. 3B) and iron(II)-HbIV (Fig. 3A)
at pH 7.8 (oxygenated), show very sharp Q
0
bands with
similar intensities, while Q
m
shows a lower ellipticity in HbI.
The spectra obtained are similar to those observed in many
vertebrate hemoglobins and myoglobins [10,17,21,25]. The
Q bands of iron(II)-HbIV (Fig. 3A) and -HbI (Fig. 3B) at
pH 7.8 show some variations in relative intensity, suggesting
small differences in the constraints at the heme pocket. In
HbI, the spectrum is only slightly modified by pH lowering
(Fig. 3B), while HbIV at pH 6.0 (Fig. 3A) shows a band
centered around 545 nm and a shoulder around 572 nm

(Fig. 3A). This spectrum is quite similar to that of deoxy
human HbA [19], suggesting similar changes at the heme
site in HbA and trout HbIV.
Following oxidation, met-HbIV (Fig. 3C) shows spectra
similar to iron(II)-HbIV at both pHs tested. These data
indicate only small modifications at the heme site. On the
contrary, when met-HbI is compared with iron(II)-HbI, it
shows (Fig. 3D) a slightly decreased intensity in the Q
0
band
(around 572 nm) with respect to Q
m
band (around 545 nm),
suggesting a modified symmetry in the proximal bond. This
behavior is similar at both pHs.
Peroxidase activity
The peroxidase activity was followed by monitoring the
increase in absorbance at 470 nm. Guaiacol is a methoxy-
phenol that oxidizes to a radical, followed by dimerization
[6]. Figure 4 shows the peroxidase activity of iron(II)- and
met-forms of HbI and HbIV. It is evident that the enzymatic
activity decreases according to the order iron(II)-HbIV >
met-HbIV > met-HbI > iron(II)-HbI.
Chemiluminescence measurements
Chemiluminescence (CL) measurements were performed by
using lucigenin as chemiluminogenic probe for superoxide
radical produced by xanthine/xanthine oxidase system. The
reaction of lucigenin with superoxide radical gives rise to
chemiluminescence whose level indicates the presence of
superoxide in the medium under study. Table 1 shows

results obtained on met- and iron(II)-Hbs at two different
pH values, 7.8 and 6.0. The first parameter reported in
Table 1 is the duration of the reaction, which was very
different in the two buffers used. At pH 7.8, the reaction
time was always about 15 s, while at pH 6 this time was
40 s. The maximum peak value was 1.640 ± 0.001 ( · 10
7
)
c.p.s. for Tris and 2.453 ± 0.0020 ( · 10
5
) c.p.s. for Bis/Tris
buffer, respectively. The presence of 40 lgÆmL
)1
of Hb
[iron(II)- or met-derivatives of both hemoglobins] does not
significantly reduce the CL level when the experiments were
performed in 50 m
M
Tris/HCl pH 7.8. The CL reduction
wasnolargerthan5%ineachsample.Whenthe
experiments were performed in 50 m
M
Bis/Tris pH 6, the
CL reduction was significantly different for HbI and HbIV:
iron(II)-HbI and met-HbI induce a 23.2 and 50.1%
reduction of the peak intensity, respectively, when com-
pared with the peak of the buffer. Iron(II)-HbIV and met-
HbIV induce a 35.8 and 62.1% reduction of the peak
intensity, respectively.
Discussion

In red blood cells, Hb can readily generate or interact with
free radicals [28]. In fact, during spontaneous Hb autoxi-
dation to met-Hb, the superoxide anion is produced. Most
superoxide is reduced by superoxide dismutase to H
2
O
2
.
Catalase and glutathione peroxidase eliminate H
2
O
2
.
Hemoglobin also presents peroxidative properties (hydro-
gen peroxide removal activity), which was recognized more
than 30 years ago and could be important for the cellular
lifespan [5,6].
The mechanism of hydrogen peroxide removal by
iron(II)-Hb (oxyHb and deoxyHb) and met-Hb results in
the formation of ferrylhemoglobin (ferrylHb) and oxo-
ferrylhemoglobin (oxoferrylHb), respectively. Both are
strong oxidizing agents, which can be source of cellular and
tissue damage [8]. A recent work [8] suggested that ferryl-Hb
takes an electron from a second molecule of H
2
O
2
and is
reduced to met-Hb, while the H
2

O
2
is oxidized to super-
oxide anion:
HbðIIÞþH
2
O
2
! HbFeðIVÞ¼
O þ H
2
O
2
! HbFeðIIIÞþO
À
2
! heme degradation
Ferryl-Hb [formed by iron(II)-Hb] can be also capable of
withdraw an electron from a suitable substrate, resulting in
the formation of metHb and a substrate radical [6]. The
superoxide anion produced at the heme pocket is thought
to easily react with porphyrin molecule, resulting in heme
degradation and iron release [8]. However, H
2
O
2
-induced
heme degradation products were demonstrated with
iron(II)-Hb, but not with met-Hb [8,29]. The inability to
produce heme degradation products by met-Hb with H

2
O
2
,
was explained by the reaction of oxoferrylHb with H
2
O
2
with the production of met-Hb and oxygen instead of
superoxide anion [8]. Our data performed, at pH 5.4, in the
presence of H
2
O
2
and guaiacol (as reductant) indicate a
larger peroxidase activity of HbIV, with respect to HbI, in
Fig. 4. Peroxidase activity of different hemoglobins using guaiacol as
substrate, monitored by the increase in optical density at 470 nm. For
experimental details see Materials and methods. (j), iron(II)-HbI; (h),
met-HbI; (r), iron(II)-HbIV; (e), met-HbIV.
Ó FEBS 2004 Structural changes in trout hemoglobins (Eur. J. Biochem. 271) 1975
line with a previous work [5]. However, these results show a
decrease of the peroxidase activity in the order: iron(II)-
HbIV > met-HbIV > met-HbI > iron(II)-HbI. The larger
peroxidative activity of iron(II)-HbIV with respect to met-
HbIV is in line with previous data obtained with human
HbA derivatives [7]. The unexpected HbI results could be
explained suggesting a restricted accessibility to guaiacol for
the heme pocket of HbI. In fact, it is known that, although
H

2
O
2
binds directly to the heme iron, the guaiacol can have
different kind of interactions with Hb, because it could be
too voluminous to penetrate in the heme pocket [6]. It is
known that one important difference between the two trout
Hbs is present on the a-subunit, where the distal Val residue
(E11) (present in both a and b pockets of human Hb) is
substituted in HbIV by a Thr, and in HbI by an Ile. Because
the Val(E11) residue is known to affect the ligand accessi-
bility to heme pocket, its substitution with the bulky
hydrophobic side chain of Ile could impose a certain steric
hindrance to the a-pocket. On the other hand, the polar Thr
could likely facilitate the access of guaiacol (o-methoxyphe-
nol) to a-pocket by binding it with a hydrogen bond. The
b-pocket of HbI is mainly affected by the interaction of
Tyr(F1)85b with Ala70b and Ala74b [30]. As these residues
are maintained in HbIV, similar b-pocket features are
expected in both proteins. However, other amino acid
substitutions could affect pockets structure, e.g. the change
of Cys(F9)93b of human Hb with Ser in HbIV and Ala in
HbI; or the change of His(HC3)146b with Phe in HbI are
known to affect the physiological behavior of the proteins
[31]. However, these residues may affect also the b-pocket
structure [30,31]. According to Perutz and Brunori [31], the
polar Ser in HbIV prefers an external position to the pocket.
On the contrary, an internal position could by hypothesized
for the hydrophobic Ala of HbI. Moreover, the residue b66,
present in the b-pocket [32] is a Val for HbI and a Thr for

HbIV, so that also in this case the polarity of the pocket is
increased for HbIV. Although it is impossible to simply
foresee the effect of these changes on the pocket structure,
they could suggest a general lower accessibility of a quite
bulky and polar molecule a guaiacol to HbI heme pockets.
A structural modification of the heme pocket could be
hypothesized also to explain the larger peroxidase activity
shown by met-HbI, compared with iron(II)-HbI (Fig. 4), in
the presence of substrate (guaiacol).
The lower reactivity of HbI towards reactive oxygen
species is confirmed also by the reaction with the superoxide
anion obtained by the xanthine/xanthine oxidase reaction,
both at pH 7.8 and pH 6, although a larger activity of both
Hbs is evident at the acidic pH, in line with previous data
obtained with human HbA [7,33]. Also in this case the
larger reactivity shown by HbIV, when compared with HbI
in the same iron oxidative state, could suggest a lower
accessibility of the HbI heme pocket to superoxide anion.
Moreover, a different pocket accessibility to the relatively
small and charged superoxide could be related also to the
pH-dependent reactivity of the proteins.
Differences between HbI and HbIV in the reactions with
H
2
O
2
and superoxide anion are likely to be related to
possible different features of the heme–globin interaction,
which can be modified in iron(II)- and met-Hbs and as
consequence of pH changes. As the comparison between the

structural characteristics of HbI and HbIV, at two different
pH values, had been never studied, we have performed these
studies by CD, whose spectra acquired in near UV and
visible regions are particularly sensitive to heme surround-
ing. In line with a previous work [3], the near-UV and visible
regions of HbI and HbIV (oxygenated forms, pH 7.8) are
characterized by quite different spectra, more evident at the
a
1
b
2
interface, as suggested by the CD spectra in the region
270–290 nm (Fig. 1).
Data presented in this work demonstrate that CD spectra
(Figs 1–3), acquired under different conditions of pH and
oxidation, show different patterns of modifications for the
two trout hemoglobin components (HbI and HbIV), which
are likely to be related to their different functional properties
[2]. HbIV spectral modifications induced by a pH decrease,
are quite similar to those reported in the literature for
human HbA under deoxygenation [17,18] (Figs 1–3).
HbIV deoxygenation is evident by the L-band which
responds strongly to spin state [20,21] because it shows a
large positive ellipticity for low-spin Hbs and a very reduced
contribution for high-spin Hbs. Deoxygenation is evident
Table 1. Lucigenin-amplified chemiluminescence of the xanthine/xanthine oxidase reaction in the presence of 40 lgÆmL
)1
of oxy and met derivatives of
both hemoglobins. The system contained 0.9 U of xanthine oxidase per mL and 150 l
M

lucigenin; the reaction was started by injecting xanthine at
the final concentration of 50 mmol in 50 m
M
Tris pH 7.8 or 50 m
M
Bis/Tris pH 6.0. Values obtained are expressed in count per second (c.p.s.).
Sample
Duration
(s)
T
half rise
(s)
T
max
(s)
T. half fall
(s)
Peak value
(c.p.s.)
CL reduction
(%)
50 m
M
Tris pH 7.8 15 < 2.40 6.60 (1.640 ± 0.001) · 10
7
Fe(II)-HbIV 15 < 2.40 7.20 (1.599 ± 0.006) · 10
7
2.5
Met-HbIV 15 < 2.40 7.20 (1.555 ± 0.005) · 10
7

5.1
Fe(II)-HbI 15 < 2.40 7.20 (1.610 ± 0.005) · 10
7
1.8
Met-HbI 15 < 2.40 7.18 (1.599 ± 0.005) · 10
7
2.5
50 m
M
Bis/Tris pH 6.0 40 < 3.00 25.80 (2.453 ± 0.002) · 10
5
Fe(II)-HbIV 40 < 1.20 22.80 (1.575 ± 0.010) · 10
5
* 35.8
Met-HbIV 40 < 1.80 24.60 (0.929 ± 0.010) · 10
5
* 62.1
Fe(II)-HbI 40 < 3.00 24.00 (1.884 ± 0.010) · 10
5
* 23.2
Met-HbI 40 < 5.40 26.40 (1.207 ± 0.010) · 10
5
* 50.1
*P<0.05.
1976 R. Gabbianelli et al. (Eur. J. Biochem. 271) Ó FEBS 2004
also by the large red shift of the Soret band (433 nm at
pH 6.0 and 418 nm at pH 7.8), in line with the reported
spectral characteristics of human deoxy-HbA [17,18]. This
red shift is most likely not linked to changes in the
quaternary structure, as a similar deoxygenation-induced

shift was also observed in the Soret peak of monomeric
Lucina pectinata Hb [25]. Moreover, although the data
reported here seem not to be in line with previous data by
Perutz et al. [18], which show that the Soret band in the
T-form is slightly blue shifted and higher in intensity than
the R-form, the measured effect in the Soret band is likely
due to the superimposition of deoxygenation and the
proton-dependent R fi T transition of HbIV. This possi-
bility could be consistent with the results indicating that in
deoxygenated human Hb, the difference CD spectra shows
a maximum in the Soret peak at 437 nm for the R-form,
and at 433 nm for the T-form [24].
The near-UV and visible CD spectral features are very
similar in HbIV and in human HbA. However, the CD
Soret band of human deoxy-HbA shows an increased
ellipticity when compared with the oxygenated derivative
[17,34]. The spectrum of deoxy-HbIV (pH 6.0) follows an
opposite behavior (Fig. 2A). A previous work performed
on human pathologic Hbs related the decrease in the Soret
band ellipticity to a decreased cooperativity [35]. This
possible interpretation of the unexpected decrease in the
Soret band ellipticity in deoxy-HbIV is in agreement with
data indicating that an acid pH causes a reduction in
cooperativity (together with a marked reduction in ligand
affinity) in a fish hemoglobin, exhibiting the Root effect
[1,2,34].
Comparison between iron(II)-HbIV and met-HbIV at
pH 7.8 in the regions of the L-band (about 260 nm), 280–
290 nm and the Soret band indicate quite similar structural
features between these two ligated Hbs showing a similar

R structure. The small, but evident differences between the
spectra of iron(II)-HbIV and met-HbIV pH 7.8 around
285 nm (Fig. 1A,C) may be related to larger values of the
equilibrium constant L ¼ [T]/[R] for met-HbIV (in terms of
a two-state concerted model).
The CD spectra obtained at pH 6.0 for met-HbIV
indicate a shift to the T-form, high spin iron, a mixed
population of six and five coordinated molecules (as
suggested by the Soret band) and a decreased cooperativity
(as suggested by the decreased ellipticity of the Soret band).
These results on HbIV are in line with a previous work on
human HbA by Perutz et al. [14], which suggested that the
high-spin ligands as H
2
O (which occupies the sixth coordi-
nation state in met-Hbs) favor the transition to the T state
more than the low-spin ligands.
HbI at pH 7.8 and pH 6.0 is reported to be completely
oxygenated and cooperative [2]. As expected, the CD
spectra recorded at pH 6.0 show the characteristic bands of
a ligated Hb, at each wavelength range tested. The small
spectral changes induced by low pH on iron(II)-HbI
(decreased intensity, either negative or positive, of each
band), in the region 250–470 nm could be due to a possible
protein destabilization, which may affect the quaternary
structure of the protein and, as a consequence, the a
1
b
2
and

a
1
b
1
contacts.
Oxidation does not induce important structural modifi-
cations in the HbI spectra, although a slightly more negative
ellipticity in the 285 nm region, at pH 7.8, can indicate a
larger value of the equilibrium constant L ¼ [T]/[R].
However, the modified ratio Q
0
/Qm (visible region) indicates
a slightly different symmetry at the axial bond with the
proximal histidine, as a consequence of iron oxidation.
Comparison with HbIV spectra suggests the possibility that
the modified ratio Q
0
/Qm could be due to a partial change to
the unligated form following oxidation, at both pH values.
At pH 6.0, the low ellipticity value of the L-band (centered
around 260 nm) for met-HbI suggests a high spin iron.
This is in agreement with the presence, at low pH, of the
high-spin ligand, water, bound to iron(III) subunits [36].
According to Perutz et al. [14] in the absence of organic
phosphates, the R structure is dominant in all Hbs in which
the irons are six-coordinated, but the spin state can modify
the allosteric equilibrium between the two structures,
shifting the equilibrium constant L ¼ [T]/[R] to a higher
value. However, this is not the case of met-HbI at pH 6.0, as
indicated by the CD spectra obtained which show no

changes indicative of R fi T transition in the region of
285 nm and no blue shift in the Soret band.
Conclusions
Data presented in this work demonstrate different structural
features for the heme–globin interaction in HbIV and HbI,
which could be related to their different activities towards
ROS. However, CD data did not give any indication about
the different reactivity shown by both Hbs towards
superoxide anion at pH 7.8 and 6. For this reason it is
suggested that the larger reaction with the superoxide at pH
6 could be linked to the pH-induced decrease of negative
charge density on dissociable groups on the proteins.
Differences in HbI and HbIV behavior are likely related
to the known differences [36,37] in number and position of
amino acids with charged lateral groups.
The larger activity of met-HbI, with respect to iron(II)-
HbI, in the presence of the guaiacol (Fig. 4) could be due to
the inhibition of R fi T transition in met-HbI. We suggest
that the persistence of R form could hinder the structural
changes, induced by the low pH, which could affect the
interaction of the guaiacol large molecule at the heme pocket.
A modification of the heme pocket structure is suggested also
by the modified ratio Q
0
/Qm (visible region), indicating a
changed symmetry at the axial bond with the proximal
histidine. This modified symmetry seems to be related to a
partial dissociation of ligand by met-HbI, which could
permit a binding of more H
2

O
2
molecules with heme iron
and, as a consequence, a higher rate of peroxidative reaction
by the R-form of met-HbI pH 5.4 with respect to the R-form
of iron(II)-HbI at pH 5.4. This hypothesis is also suggested
by the comparison with data obtained for HbIV: at low pH
the iron(II) form is completely deoxygenated (as confirmed
by CD data, in particular by Soret band), and met-HbIV is
only partially delegated (see Soret band). For HbIV the
peroxidative reaction follows the order iron(II)-HbIV >
met-HbIV, suggesting that in the case of this protein, the
deligation also affects the rate of the reaction.
We stress that our CD data give new information about
ligation features and/or R fi T transition of HbIV and
HbI: HbIV appears to dissociate ligands, as a consequence
of pH also decreases in the oxidized form, even if the shift to
Ó FEBS 2004 Structural changes in trout hemoglobins (Eur. J. Biochem. 271) 1977
the non ligated form is incomplete. Moreover, met-HbIV
spectra(atpH6.0)showanRfi T transition, which is
related to the change from a low- to high-spin iron. This
behavior is in line with data indicating that tetramers with
high-spin ligand water of iron(III) subunits, show the most
T-state behavior [38].
CD spectra show that iron(II)- and iron(III)-HbI struc-
tural features are almost insensitive to pH decrease, as the
protein remains in the ligated form. This result is expected in
iron(II)-HbI, which remains in the R-form and low-spin
iron. However, a particularly high stability of the R-form is
evident for met-HbI at pH 6.0, also in the presence of

high-spin iron, in contrast with the expected transition to the
Tform.
In spite of the efficiency of the antioxidant defense
system, the trout RBCs can be exposed to a considerable
flux of reactive oxygen species, implying a red cell oxidative
stress. Our data could suggest that trout HbI, which shows a
reduced sensitivity to reactive oxygen species (such as
superoxide anion and H
2
O
2
), with respect to HbIV, could
have a role in the maintenance of normal oxygen supply
during ROS production.
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