Báo cáo khoa học: Nitric oxide formation from the reaction of nitrite with carp and rabbit hemoglobin at intermediate oxygen saturations pdf

Nitric oxide formation from the reaction of nitrite
with carp and rabbit hemoglobin at intermediate
oxygen saturations
Frank B. Jensen
Institute of Biology, University of Southern Denmark, Odense M, Denmark
Nitrite (NO
À
2
) is naturally present at low concentra-
tions in vertebrates, where it originates as an oxidative
metabolite of nitric oxide (NO) produced by nitric
oxide synthases [1] with some contribution from the
diet [2]. In fish, nitrite can also be taken up from the
ambient water via active transport across the gills [3].
Recent research has suggested that nitrite constitutes
a reservoir of NO activity that can be activated under
hypoxic conditions [4,5]. NO can be regenerated from
nitrite by acidic disproportionation [6] and by enzy-
matic reduction via xanthine oxidoreductase [7], mito-
chondria [8], or deoxygenated hemoglobin [4,9,10] and
myoglobin [11]. The deoxyhemoglobin-mediated for-
mation of NO from nitrite has attracted particular
interest because this reaction may provide the red cells
with the ability to both sense O
2
conditions (through
the degree of hemoglobin deoxygenation) and produce
a vasodilator (NO) that when released from the red
cells can increase blood flow according to need [4,9].
This idea is supported by in vivo and in vitro studies
Keywords

deoxyhemoglobin; nitric oxide; nitrite;
nitrosylhemoglobin; oxyhemoglobin
Correspondence
F. B. Jensen, Institute of Biology, University
of Southern Denmark, Campusvej 55,
DK-5230 Odense M, Denmark
Fax: +45 6593 0457
Tel: +45 6550 2756
E-mail:
(Received 11 March 2008, revised 29 April
2008, accepted 30 April 2008)
doi:10.1111/j.1742-4658.2008.06486.x
The nitrite reductase activity of deoxyhemoglobin has received much recent
interest because the nitric oxide produced in this reaction may participate
in blood flow regulation during hypoxia. The present study used spectral
deconvolution to characterize the reaction of nitrite with carp and rabbit
hemoglobin at different constant oxygen tensions that generate the full
range of physiological relevant oxygen saturations. Carp is a hypoxia-toler-
ant species with very high hemoglobin oxygen affinity, and the high R-state
character and low redox potential of the hemoglobin is hypothesized to
promote NO generation from nitrite. The reaction of nitrite with deoxyhe-
moglobin leads to a 1 : 1 formation of nitrosylhemoglobin and methemo-
globin in both species. At intermediate oxygen saturations, the reaction
with deoxyhemoglobin is clearly favored over that with oxyhemoglobin,
and the oxyhemoglobin reaction and its autocatalysis are inhibited by
nitrosylhemoglobin from the deoxyhemoglobin reaction. The production of
NO and nitrosylhemoglobin is faster and higher in carp hemoglobin with
high O
2
affinity than in rabbit hemoglobin with lower O

2
affinity, and it
correlates inversely with oxygen saturation. In carp, NO formation remains
substantial even at high oxygen saturations. When oxygen affinity is
decreased by T-state stabilization of carp hemoglobin with ATP, the reac-
tion rates decrease and NO production is lowered, but the deoxyhemo-
globin reaction continues to dominate. The data show that the reaction of
nitrite with hemoglobin is dynamically influenced by oxygen affinity and
the allosteric equilibrium between the T and R states, and that a high O
2
affinity increases the nitrite reductase capability of hemoglobin.
Abbreviations
deoxyHb, deoxygenated hemoglobin; Hb, hemoglobin; HbNO, nitrosylhemoglobin; metHb, methemoglobin; NO, nitric oxide; oxyHb,
oxygenated hemoglobin; P
50
,O
2
tension at 50% SO
2
; PO
2
, oxygen tension; SO
2,
O
2
saturation.
FEBS Journal 275 (2008) 3375–3387 ª 2008 The Author Journal compilation ª 2008 FEBS 3375
documenting that nitrite causes vasodilation and
increases blood flow, consistent with its conversion
into NO by hemoglobin and ⁄ or red cells [4,12–14].

The reactions of nitrite with oxygenated hemoglobin
(oxyHb) and deoxygenated hemoglobin (deoxyHb) are
very different, and it is only the reaction with deoxyHb
that produces NO. The reaction of nitrite with fully
oxygenated hemoglobin (Hb) proceeds via an initial
slow ‘lag’ phase followed by an autocatalytic increase
in reaction rate. The mechanism is complex and
involves a series of steps where reactive intermediates
such as H
2
O
2
,NO
2
and ferrylhemoglobin are produced
[15–17]. The stoichiometry for the overall reaction
reveals that oxyHb is oxidized to ferric Hb [methemo-
globin (metHb)] and nitrite is oxidized to nitrate [15]:
4HbðFe
2þ
ÞO
2
þ 4NO
À
2
þ 4H
þ
! 4HbðFe
3þ
Þþ4NO

À
3
þ O
2
þ 2H
2
O
ð1Þ
The reaction of nitrite with fully deoxygenated Hb
leads to the oxidation of deoxyHb to metHb, whereas
nitrite becomes reduced to NO. The NO subsequently
binds to an adjacent ferrous heme to form nitrosyl-
hemoglobin (HbNO) [4,9,18]:
HbðFe
2þ
ÞþNO
À
2
þ H
þ
! HbðFeÞ
3þ
þ NO þ OH
À
ð2Þ
HbðFe
2þ
ÞþNO ! HbðFe
2þ
ÞNO ð3Þ

The deoxyHb reaction has a sigmoid, autocatalytic-
like reaction kinetics, where the reaction rate increases
during the reaction, which has been ascribed to an
allosteric transition from the T structure to the R
structure induced by metHb and HbNO formation and
a lower redox potential (i.e. a better ability to reduce
nitrite) for deoxygenated hemes in the R structure than
in the T structure [19].
In the arterial-venous circulation, Hb cycles between
full and intermediate oxygen saturations, and Hb will
never become fully deoxygenated. It is therefore
important to understand how the reaction of nitrite
with Hb proceeds at intermediate oxygen saturations.
However, unlike the many studies with fully oxygen-
ated or fully deoxygenated Hb, the reaction at interme-
diate oxygen saturations has only recently been
explored in human Hb [20]. Furthermore, because
nitrite reduction to NO is important mainly during
hypoxia, the reaction may have particular relevance in
species that are naturally exposed to hypoxia.
Hypoxia-tolerant fish, such as carp, have become
evolutionarily adapted to cope with severe hypoxia,
partly by having hemoglobin with very high O
2
affinity
[21]. This can be hypothesized to give the Hb a high
R-state character and a low redox potential, which
should promote deoxyHb-mediated nitrite reduction to
NO. The present study tested the idea that NO forma-
tion from nitrite is enhanced in hemoglobin with a

high O
2
affinity compared to hemoglobin with a low
O
2
affinity. The reaction of nitrite with carp Hb was
characterized at natural red cell pH and ionic strength
at several different constant O
2
tensions (Po
2
), which
produced O
2
saturations (So
2
) that ranged from the
fully deoxygenated Hb through a series of intermediary
So
2
values to the fully oxygenated Hb. Parallel results
were obtained using rabbit Hb under the same experi-
mental conditions, which enabled a direct comparison
to be made between carp Hb and a mammalian Hb
with lower O
2
affinity. The experiments also scruti-
nized the influence of decreasing O
2
affinity in carp Hb

via T-state stabilization by ATP and the effects of
changes in O
2
tension ⁄ saturation during the reaction.
The data revealed that the reactivity is dynamically
influenced by oxygen affinity and the allosteric equilib-
rium between the T and R states, and that the deoxy-
Hb reaction dominates over the oxyHb reaction at
intermediate O
2
saturations.
Results
Oxygen-binding properties
Carp Hb in 0.05 molÆL
)1
Tris buffer (pH 7.3) and
0.1 molÆL
)1
KCl had a very high oxygen affinity and a
low cooperativity, as reflected by an O
2
tension at
50% So
2
(P
50
) of 1.2 mmHg and an n value of 1.03.
Under the same conditions, the P
50
value in rabbit Hb

was 5.1 mmHg and n was 1.8 (results not shown).
Addition of ATP at an [ATP] ⁄ [Hb] ratio of 5
([ATP] ⁄ [Hb
4
] = 20) increased the P
50
of carp Hb to
6 mmHg and the n value to 2.7, showing that ATP
both lowered O
2
affinity and increased cooperativity.
Reaction of nitrite with carp Hb at different O
2
saturations
Nitrite was added at an [NO
À
2
] ⁄ [Hb] ratio of 2.7 and
the concentrations of deoxyHb, oxyHb, metHb and
HbNO in the course of the reaction were evaluated by
spectral deconvolution. The least squares curve-fitting
procedure [22] gave accurate fits to the spectral data,
and the overall R
2
of experimental fits was
0.99950 ± 0.00002 (mean ± SEM, n = 260 fits) for
carp Hb and 0.9990 ± 0.00009 (mean ± SEM,
Nitrite–hemoglobin reactions at different O
2
saturations F. B. Jensen

3376 FEBS Journal 275 (2008) 3375–3387 ª 2008 The Author Journal compilation ª 2008 FEBS
n = 115) for rabbit Hb. Examples of absorbance spec-
tra of carp Hb at specified time-points following the
addition of nitrite are given in Fig. 1 to illustrate the
spectral changes that occurred during the reaction of
nitrite with deoxyHb (Fig. 1A) and with Hb with an
initial So
2
of 46% (Fig. 1B).
When nitrite reacted with carp deoxyHb in a nitro-
gen atmosphere, the concentration of deoxyHb
decreased to zero in approximately 30 min. The reac-
tion products HbNO and metHb concomitantly
increased in a 1 : 1 stoichiometry, and HbNO reached
a maximum of half the total Hb concentration
(Fig. 2A), which is in agreement with reaction
Eqns (2,3) above. After deoxyHb had declined to zero,
the concentration of HbNO started to decrease slowly,
while the concentration of metHb increased, pointing
to dissociation of some of the NO bound to ferrous
heme and continued oxidation of ferrous heme to
ferric heme (Fig. 2A). There was a small amount of
oxyHb present (So
2
= 2%), apparently because the
traces of O
2
present in the N
2
gas [O

2
£ 5 parts per
million (p.p.m.) = 0.0037 mmHg] were sufficient to
produce detectable traces of oxyHb as a result of the
very high oxygen affinity of carp Hb.
At intermediate So
2
values, nitrite had the possi-
bility of reacting with deoxyHb and oxyHb simul-
taneously. Furthermore, NO formed in the deoxyHb
reaction could react with either deoxyHb to form
HbNO or with oxyHb to form metHb and NO
À
3
. The
data revealed a clear preference for nitrite reacting
with deoxyHb. The concentration of deoxyHb
decreased faster than the concentration of oxyHb, and
deoxyHb reached zero within 40–50 min, well before
oxyHb approached zero. This was evident when the
reaction occurred at initial So
2
values of 35%
(Fig. 2A), 46% (Fig. 2C), 65% (Fig. 2D) and 78%
(Fig. 2E), showing that the reaction of nitrite with
deoxyHb was favored over that with oxyHb in the full
range of physiologically relevant intermediate So
2
values. The reaction at intermediate So
2

led to the
production of a higher concentration of metHb than
of HbNO (Fig. 2B–E), but the formation of NO and
HbNO remained significant, even at 78% So
2
(Fig. 2E). The concentration of HbNO peaked when
deoxyHb reached zero (Fig. 2B–E), whereafter HbNO
slowly decreased.
The reaction of nitrite with fully oxygenated Hb
(100% So
2
) led to the complete conversion of oxyHb
to metHb (Fig. 2F), which agreed with the exp-
ected stoichiometries for the oxyHb reaction (Eqn 1
above). The reaction progressed more rapidly at
100% So
2
than at intermediate values of So
2
. The
considerably faster decline in oxyHb at 100% So
2
(Fig. 2F) than at intermediate So
2
(Fig. 2B–E)
showed that the oxyHb reaction was inhibited at
intermediate So
2
values. The reaction at 100% So
2

was only slightly quicker than the reaction with
deoxyHb (Fig. 2A,F). During the autocatalytic phase
of the reaction of nitrite with fully oxygenated Hb,
intermediates such as ferrylHb are transiently pro-
duced in small amounts. Reference spectra of these
minor intermediates were not included in the present
analysis, and spectral deconvolution instead proposed
the transient appearance of small amounts of
deoxyHb and HbNO (fitting artifacts) during the
autocatalytic phase (Fig. 2F).
In order to study how an increase in oxygenation
in the middle of the reaction influenced the subse-
quent reaction course, nitrite was allowed to react
with carp Hb at low So
2
values (10%) for 12 min,
whereafter Po
2
was abruptly increased (Fig. 2G).
Absorbance Absorbance
SO
2
= 2%
S
O
2
= 46%
PO
2
= 1.17 mmHg

450 500 550 600 650 700
0.0
0.5
1.0
1.5
2.0
2.5
0 min
2 min
5 min
8 min
11 min
14 min
17 min
23 min
32 min
41 min
50 min
59 min
68 min
77 min
deoxyHb
mixture of HbNO
and metHb
450 500 550 600 650 700
0.0
0.5
1.0
1.5
2.0

2.5
Wavelength (nm)
0 min
2 min
5 min
8 min
11 min
14 min
17 min
23 min
32 min
41 min
50 min
59 min
74 min
90 min
110 min
140 min
180 min
A
B
Fig. 1. Spectral changes during the reaction of nitrite with carp
hemoglobin at different oxygen saturations. (A) Reaction of nitrite
with deoxyHb (oxygen saturation = 2%). (B) Reaction of nitrite with
hemoglobin with an initial oxygen saturation of 46%. Absorbance
spectra were obtained at specified time-points following nitrite
addition for up to 180 min. The hemoglobin concentration was
155 l
M on heme basis, and the nitrite ⁄ heme concentration ratio
was 2.7. The temperature was 25 °C. Measurements were made

in 0.05
M Tris buffer, with 0.1 M KCl, at a pH of 7.3.
F. B. Jensen Nitrite–hemoglobin reactions at different O
2
saturations
FEBS Journal 275 (2008) 3375–3387 ª 2008 The Author Journal compilation ª 2008 FEBS 3377
0 20 40 60 80 100 120 140 160 180
0
20
40
60
80
100
120
140
160
oxyHb
metHb
HbNO
deoxyHb
0 20 40 60 80 100 120 140 160 180
0
20
40
60
80
100
120
140
160

0 20 40 60 80 100 120 140 160 180
0
20
40
60
80
100
120
140
160
0 20 40 60 80 100 120 140 160 180
0
20
40
60
80
100
120
140
160
0 20 40 60 80 100 120
0
20
40
60
80
100
120
140
160

Time
(
min
)

0 20 40
0
20
40
60
80
100
120
140
160
Time (min)
C
oncentration (μM)
C
oncentration (μM)
C
oncentration (μM)
[NO
–
2
]/[Hb] = 2.7
Carp Hb
0 20 40 60 80 100 120 140 160
0
20

40
60
80
100
120
140
160
Oxygenation during reaction at low So
2
Time (min)
SO
2
= 2% SO
2
= 35%
S
O
2
= 46% SO
2
= 65%
S
O
2
= 78%
S
O
2
= 100%
A

B

C D
E F G
Fig. 2. Time-dependent changes in the concentrations of oxygenated hemoglobin, methemoglobin, nitrosylhemoglobin and deoxygenated
hemoglobin during the reaction of nitrite with carp hemoglobin at different oxygen saturations. Initial oxygen saturations (S
O
2
) were: (A) 2%,
(B) 35%, (C) 46%, (D) 65%, (E) 78% and (F) 100%. Panel G shows the effects of an acute oxygenation (P
O
2
increase) during the reaction at
low S
O
2
. The hemoglobin concentration was 155 lM, and the nitrite ⁄ heme concentration ratio was 2.7. The temperature was 25 °C. Mea-
surements were made in 0.05
M Tris buffer, with 0.1 M KCl, at a pH of 7.3.
Nitrite–hemoglobin reactions at different O
2
saturations F. B. Jensen
3378 FEBS Journal 275 (2008) 3375–3387 ª 2008 The Author Journal compilation ª 2008 FEBS
The elevated Po
2
produced a sharp increase in oxy-
Hb and decreased deoxyHb to zero. This was associ-
ated with a significant slowing down of the
subsequent reaction (now occurring with oxyHb),

revealing that the oxyHb reaction was retarded in
spite of full oxygenation of the remaining functional
Hb (Fig. 2G).
Reaction of nitrite with rabbit Hb at different
O
2
saturations
The reaction of nitrite with rabbit Hb (Fig. 3) was
considerably slower than with carp Hb (Fig. 2) (note
the different time axis scale in the two figures). This
applied to all So
2
values tested except for 100%
So
2
, where the reaction rates in the two species were
comparable. At 2% So
2
, the profile for the decrease
in rabbit deoxyHb was definitely sigmoid (Fig. 3A).
DeoxyHb was reduced to zero in 380 min, and HbNO
and metHb rose in parallel in a practically 1 : 1 stoi-
chiometric relationship (Fig. 3A). At intermediate So
2
values, the reaction of deoxyHb was clearly preferred
over that with oxyHb, even though the difference was
less marked than for carp (compare So
2
= 46% for
rabbit in Fig. 3C with that for carp in Fig. 2C).

When rabbit Hb reacted with nitrite at an So
2
of
67%, the reaction entered an autocatalytic phase
when deoxyHb approached zero, and the remaining
oxyHb was quickly converted into metHb (Fig. 3D).
This autocatalysis for the oxyHb reaction was absent
at lower So
2
values (28% and 46%; Fig. 3B,C),
where oxyHb only decreased slowly and remained
Concentration (µM) Concentration (µM)
Time (min)
Time (min)
oxyHb
metHb
HbNO
deoxyHb
A
0 50
100 150 200 250 300 350 400
0 50
100 150 200 250 300 350 400
0 50
100 150 200 250 300 350 400
0
50
100
0 50
100

150
200
0
20
40
60
80
100
120
140
160
0
20
40
60
80
100
120
140
160
B

C
0
20
40
60
80
100
120

140
160
Rabbit Hb
0
20
40
60
80
100
120
140
160
E
0
20
40
60
80
100
120
140
160
D
Time (min)
SO
2
= 2%
S
O
2

= 46%
S
O
2
= 28%
S
O
2
= 67%
S
O
2
= 100%
Fig. 3. Time-dependent changes in the concentrations of oxygenated hemoglobin, methemoglobin, nitrosylhemoglobin and deoxygenated
hemoglobin during the reaction of nitrite with rabbit hemoglobin at different oxygen saturations. Initial oxygen saturations (S
O
2
) were:
(A) 2%, (B) 28%, (C) 46%, (D) 67% and (E) 100%. The hemoglobin concentration was 155 l
M, and the nitrite ⁄ heme concentration ratio was
2.7. The temperature was 25 °C. Measurements were made in 0.05
M Tris buffer, with 0.1 M KCl, at a pH of 7.3.
F. B. Jensen Nitrite–hemoglobin reactions at different O
2
saturations
FEBS Journal 275 (2008) 3375–3387 ª 2008 The Author Journal compilation ª 2008 FEBS 3379
present after deoxyHb had reached zero. At 100%
So
2
the reaction of rabbit Hb with nitrite was fast

and autocatalytic, producing a marked difference in
the reaction rate between the fully oxygenated
(Fig. 3E) and deoxygenated (Fig. 3A) Hb.
The production of NO and HbNO in rabbit Hb
decreased with increasing So
2
. Peak HbNO concentra-
tions were reached by the time that deoxyHb reached
zero, whereafter HbNO decreased (Fig. 3). At interme-
diate So
2
values, the HbNO levels were lower than
observed for carp Hb. At 67% So
2
, HbNO was pro-
duced in only small amounts and disappeared com-
pletely when the reaction entered the autocatalytic
phase (Fig. 3D).
Reaction in presence of ATP
The addition of ATP to carp Hb at an [ATP] ⁄ [Hb]
ratio of 5 ([ATP] ⁄ [Hb
4
] = 20) stabilized the T struc-
ture and lowered O
2
affinity, which caused oxyHb to
be completely absent in the N
2
atmosphere (Fig. 4A).
The presence of ATP slowed down the reaction of

nitrite with fully deoxygenated Hb, whereby the
decline in [deoxyHb] to zero lasted some 90 min
(Fig. 4A). The initial reaction seemed to result in the
formation of HbNO in excess of metHb, but subse-
quently the concentrations of reaction products
increased in parallel, and at the end of the experiment,
both HbNO and metHb were present at approximately
Concentration (µM) Concentration (µM)
0 20 40 60 80
100 120 140 160 180
0 20 40 60 80
100 120 140 160 180
0 20 40 60 80
100 120 140 160 180
0 20 40 60 80
100 120 140 160 180
0
20
40
60
80
100
120
140
160
A

[ATP]/[Hb] = 5
0
20

40
60
80
100
120
140
160
B
0
20
40
60
80
100
120
140
160
C
Time
(
min
)

0
20
40
60
80
100
120

140
160
D
Time
(
min
)

[NO
–
2
]/[Hb] = 2.7
oxyHb
metHb
HbNO
deoxyHb
[ATP]/[Hb] = 5
Carp Hb
SO
2
= 0%
S
O
2
= 100%
S
O
2
= 32% [ATP]/[Hb] = 5
SO

2
= 70% [ATP]/[Hb] = 5
Fig. 4. Effect of ATP on the reaction of nitrite with carp hemoglobin at different oxygen saturations. Concentration profiles of oxygenated
hemoglobin, methemoglobin, nitrosylhemoglobin and deoxygenated hemoglobin are shown for reactions that occurred at initial oxygen satu-
rations of (A) 0%, (B) 32%, (C) 70% and (D) 100%. The [ATP] ⁄ [Hb] ratio was 5 on a heme basis (equal to a ratio of 20 on tetramer basis).
The hemoglobin concentration was 155 l
M, and the nitrite ⁄ heme concentration ratio was 2.7. The temperature was 25 °C. Measurements
were made in 0.05
M Tris buffer, with 0.1 M KCl, at a pH of 7.3.
Nitrite–hemoglobin reactions at different O
2
saturations F. B. Jensen
3380 FEBS Journal 275 (2008) 3375–3387 ª 2008 The Author Journal compilation ª 2008 FEBS
half the initial deoxyHb concentration (Fig. 3A), as
expected from Eqns (2,3).
The presence of ATP also decelerated the reaction
kinetics at intermediate So
2
values (Fig. 4B,C). How-
ever, as observed in the absence of ATP, the reaction of
nitrite with deoxyHb was favored over that with oxyHb
(Fig. 4B,C). The protracted reaction meant that the
maximum HbNO concentration was delayed (Fig. 4).
The reaction of nitrite with fully oxygenated carp Hb
(So
2
= 100%) was only slightly slower in the presence
of ATP (Fig. 4D) than in the absence of ATP (Fig. 2F).
Reaction rates
Differentiation of the deoxyHb and oxyHb concentra-

tion profiles for carp (Figs 2 and 4) gave the reaction
rates for the deoxyHb and oxyHb reactions with nitrite
at different So
2
values (Fig. 5). In the absence of ATP,
the rate for the reaction of nitrite with deoxyHb
initially increased to reach a peak at 5 min, whereafter
the rate decreased to eventually reach zero, when all
deoxyHb was used up (Fig. 5A). This behavior has
been suggested to reflect the faster reaction of nitrite
with deoxy hemes in the R structure than in the
T structure [19,20]. Thus, the reaction rate was not
maximal at the start of the reaction, where the concen-
tration of deoxy hemes in the T structure was
maximal, but rather later in the reaction when the for-
mation of HbNO and metHb (both tending to assume
the R conformation) had caused an allosteric T to
R transition. Both the initial rate and the maximal rate
for the reaction of nitrite with deoxyHb decreased
when the deoxyHb concentration decreased with
increasing values of So
2
(Fig. 5A).
deoxyHb reaction rate
[ATP]/[Hb] = 5
Time (min)
0
2
4
6

8
10
12
14
deoxyHb reaction rate
A
2%
35%
46%
64%
78%
0 20 40 60 80 100 120 140 160 180 0 20 40 60 80 100 120 140 160 180
0 20 40 60 80 100 120 140 160 180 0 20 40 60 80 100 120 140 160 180
0
2
4
6
8
10
12
14
B
0%
32%
70%
–dC/dt (µM min
–1
)–dC/dt (µM min
–1
)

oxyHb reaction rate
[ATP]/[Hb] = 5
100%
32%
70%
0
2
4
6
8
10
12
14
D
Time (min)
0
2
4
6
8
10
12
14
C
oxyHb reaction rate
100%
35%
46%
64%
78%

Carp Hb
SO
2
SO
2
SO
2
SO
2
Fig. 5. Instantaneous reaction rates for the reaction of nitrite with deoxygenated and oxygenated carp hemoglobin at different oxygen satu-
rations in the absence (A, C) and presence (B, D) of ATP. Reaction rates were obtained by differentiation of concentration profiles for
deoxyHb and oxyHb during the reaction, as exemplified in Fig. 2 (absence of ATP) and Fig. 4 (presence of ATP).
F. B. Jensen Nitrite–hemoglobin reactions at different O
2
saturations
FEBS Journal 275 (2008) 3375–3387 ª 2008 The Author Journal compilation ª 2008 FEBS 3381
The addition of ATP to stabilize the T state and to
impede the T to R transition caused the disappearance
of the well-defined peak for the deoxyHb reaction rate
and decreased the absolute reaction rates (Fig. 5B). At
0 and 32% So
2
, the initial reaction rate was now the
highest recorded rate (Fig. 5B).
Assuming that the initial rate for the reaction of
nitrite with fully deoxygenated Hb depends on a sec-
ond-order reaction between nitrite and Hb, the initial
second-order rate constant can be calculated by divid-
ing the initial reaction rate with [deoxyHb] and
[NO

À
2
]. This gave values of 2.5 and 1.0 m
)1
Æs
)1
for
carp Hb in the absence and presence of ATP, respec-
tively, and 0.06 m
)1
Æs
)1
for rabbit Hb, which illustrates
the high reactivity of carp Hb and the decreased rate
of reaction with T-state stabilization and lowered O
2
affinity.
The reaction of nitrite with fully oxygenated carp
Hb at 100% So
2
was clearly autocatalytic. The reac-
tion rate initially showed a sharp increase, reached a
marked peak and then displayed a decrease, as the
reaction approached completion (Fig. 5C). This pat-
tern was also observed in the presence of ATP
(Fig. 5D), and the absolute rates were only marginally
lower, and the peak was only slightly delayed, com-
pared with the absence of ATP. Interestingly, the dis-
tinct autocatalysis observed for the oxyHb reaction at
100% So

2
was completely absent at all tested interme-
diate So
2
values, both in the absence and presence of
ATP (Fig. 5C,D).
Dependency of HbNO production on So
2
The maximal [HbNO] showed a significant correla-
tion with the initial So
2
under all experimental con-
ditions (Fig. 6). HbNO formation was greatest at
zero So
2
, and as So
2
gradually increased, the yield
of HbNO gradually decreased. The relationships
between [HbNO]
max
and So
2
were curvilinear and
converged at the extreme So
2
values (0% and
100%), but differed at intermediate So
2
values

(Fig. 6). This revealed that the production of HbNO
depended on So
2
, the species-specific O
2
affinity
(carp against rabbit) and the relative stabilization of
the T state versus the R state of Hb (presence and
absence of ATP). According to the stoichiometrics
for the deoxyHb reaction (Eqns 2,3), the HbNO
concentration could maximally increase to half of
the deoxyHb concentration that was present at the
start of the experiment. Therefore, because the initial
deoxyHb concentration decreased with increasing So
2
(i.e. at 50% So
2
it would only be half the value at
0% So
2
), the possible maximum for HbNO also
decreased with increasing So
2
(represented by the
upper dotted straight line in Fig. 6). The observed
maximal HbNO values were lower than this possible
maximum at intermediate So
2
(Fig. 6). This was
expected because at intermediate So

2
the NO pro-
duced could react both with deoxyHb to form
HbNO (Eqn 3) and with oxyHb to form metHb and
nitrate, whereby the entire production of NO needed
not end up as HbNO. Furthermore, some NO could
dissociate from HbNO and ⁄ or escape the system.
The difference between the observed and the possible
maximum was relatively limited in carp Hb com-
pared with rabbit Hb, but it increased in carp by T-
state stabilization with ATP (Fig. 6).
Discussion
The results of the present study show that the reaction
of nitrite with deoxyHb is favored over that with oxy-
Hb at intermediate So
2
values and that the formation
of NO and HbNO from the reaction with deoxyHb is
substantial in carp Hb, even at relatively high values
of So
2
. The data support the idea that the high O
2
affinity of carp Hb is associated with an elevated
nitrite reductase capability compared to mammalian
Hb with a lower O
2
affinity.
Initial oxygen saturation (%)
[HbNO]

max
(µ
M)
0 20 40 60 80 100
0
10
20
30
40
50
60
70
80
Carp Hb
Carp Hb + ATP
Rabbit Hb
Fig. 6. The maximal HbNO concentration during the reaction of
nitrite with hemoglobin depends on initial oxygen saturation and on
oxygen affinity. The maximal HbNO concentration is plotted as
a function of the initial oxygen saturation for reactions of carp Hb
(
, high initial O
2
affinity: P
50
= 1.2 mmHg) and rabbit Hb (s, lower
initial O
2
affinity: P
50

= 5.1 mmHg), and for carp Hb in the presence
of ATP (
), where oxygen affinity is lowered (P
50
= 6 mmHg) by
T-state stabilization of the Hb. The upper dotted line represents the
possible maximum HbNO value if all NO formed during the reaction
of nitrite with Hb at intermediate oxygen saturations binds to
vacant deoxy hemes and no NO reacts with oxyHb or escapes the
system.
Nitrite–hemoglobin reactions at different O
2
saturations F. B. Jensen
3382 FEBS Journal 275 (2008) 3375–3387 ª 2008 The Author Journal compilation ª 2008 FEBS
Reactions at extreme oxygen saturations
The reaction of nitrite with fully oxygenated Hb
proceeded via an initial lag phase followed by an auto-
catalytic increase in reaction rate (Figs 2F, 4D and
5C,D) as previously observed for mammalian Hb and
for fish Hb [15,23,24]. The length of the lag phase
depends inversely on the concentration of nitrite rela-
tive to Hb, and under the present experimental condi-
tions ([NO
À
2
] ⁄ [Hb] = 2.7) it was relatively short. The
autocatalytic increase in reaction rate is caused by the
formation of reactive oxidizing free radicals, such as
NO
2

, in intermediary steps of the oxyHb reaction
[16,17].
It has recently been pointed out that the reaction of
nitrite with fully deoxygenated human Hb has a sig-
moid curve pattern that reveals an autocatalytic-like
kinetics, with an initial increase in reaction rate fol-
lowed by a decrease in rate as the deoxyHb reactant
slowly becomes depleted [19,25]. This was also observed
in rabbit deoxyHb (Fig. 3A) and in carp Hb (Fig. 5A),
and can be related to the T to R transition in the pro-
tein and to a higher reactivity of deoxy hemes in the R
state than in the T state as a result of the lower redox
potential of unreacted R-state hemes [19,20,25].
The reaction of nitrite with fully oxygenated Hb is
typically much faster than the reaction with fully deox-
ygenated Hb when nitrite is present in excess to Hb
[18,20,23]. This difference was indeed established for
rabbit Hb (Fig. 3A,E), but interestingly was not
observed in carp Hb, where the reactions were com-
pleted in a comparable time when ATP was absent
(Fig. 2A,F). The comparatively fast deoxyHb reaction
in carp agrees with the idea that the very high oxygen
affinity of carp Hb gives the Hb more R-state charac-
ter and lowers the heme redox potential, which
increases the deoxyHb reactivity. This interpretation is
supported by the induction of a considerably slower
deoxyHb reaction when the oxygen affinity was
decreased by T-state stabilization with ATP, which
established the normally observed faster reaction of
nitrite with fully oxygenated Hb compared with fully

deoxygenated Hb (Fig. 4A,D). The slowing down of
the deoxyHb reaction by ATP is similar to the effect
of inositol hexaphosphate [19,25] or 2,3-diphosphogly-
cerate [26] in human Hb, and it correlates with the
increase in redox potential induced by these phos-
phates [27,28].
Equations (2,3) predict that the reaction of nitrite
with fully deoxygenated Hb converts deoxyHb into
equal amounts of HbNO and metHb at half the con-
centration of the initial deoxyHb concentration. This
is, however, not always found. Some studies report the
expected 1 : 1 formation of HbNO and metHb [25],
whereas others report a production of metHb that sig-
nificantly exceeds the production of HbNO [18,26,29].
Deviation from the 1 : 1 reaction product formation
can result from O
2
contamination [25] or the forma-
tion of reaction intermediates other than metHb and
HbNO [26]. In carp and rabbit there was practically
equal formation of metHb and HbNO, and the sum of
metHb and HbNO concentrations by the time that
deoxyHb reached zero was very close to the initial
deoxyHb concentration (Figs 2A, 3A and 4A). Thus,
there was no indication of large concentrations of
intermediates, as recently suggested in human Hb [26],
and the data comply well with the mechanism
proposed by Eqns (2,3).
Reactions at intermediate oxygen saturations
At intermediate values of So

2
, nitrite may react with
both oxyHb and deoxyHb, but the deoxyHb reaction
is clearly favored, and deoxyHb is used up well before
oxyHb in carp (Figs 2 and 4). This striking feature
could not be predicted from the available knowledge
on the reactions with fully oxygenated and deoxygen-
ated Hb, which strengthens the importance of studying
the reaction at intermediate values of So
2
. A retarded
decay in oxyHb compared with deoxyHb also applies
to rabbit Hb (Fig. 3) and to human Hb [20], but at
any given intermediate So
2
value the difference is more
pronounced in carp Hb than in the mammalian Hbs.
The clear preference for the deoxyHb reaction in carp
Hb is associated with substantial NO production.
Interestingly, the levels of HbNO observed for carp at
intermediate So
2
values are much higher than those
seen in rabbit Hb (Fig. 6) and reported for human Hb
[20], whereas the fractional HbNO levels in rabbit and
human Hb are comparable in spite of experimental dif-
ferences between the two studies (much higher nitrite
concentrations were used in the human study). Thus,
there is a genuine difference between carp Hb and the
two mammalian Hbs. The higher O

2
affinity in carp
Hb than in the mammalian Hbs provides carp Hb with
a lower redox potential that makes it a better nitrite
reductase, which translates into higher HbNO levels.
This influence of O
2
affinity is further supported by
the formation, in carp, of a higher amount of HbNO
when the O
2
affinity is high (absence of ATP) than
when it is lowered by ATP (Fig. 6). There are, how-
ever, other mechanistic details that contribute to the
difference between species. This particularly concerns
the potential influence of reaction products from the
deoxyHb reaction with the oxyHb reaction and vice
versa.
F. B. Jensen Nitrite–hemoglobin reactions at different O
2
saturations
FEBS Journal 275 (2008) 3375–3387 ª 2008 The Author Journal compilation ª 2008 FEBS 3383
It has been shown that HbNO formed in the deoxy-
Hb reaction delays and reduces autocatalysis of the
oxyHb reaction [20]. In human Hb, an autocatalytic
phase of the oxyHb reaction is absent below 43% So
2
but present at 48% So
2
and above [20]. A similar situ-

ation was found in rabbit Hb, where autocatalysis was
absent at 46% So
2
but present at 67% So
2
(Fig. 3). In
carp Hb, autocatalysis was absent at all intermediate
So
2
values tested, including 78% So
2
(Fig. 2). Given
that HbNO inhibits autocatalysis of the oxyHb reac-
tion, the higher HbNO levels in carp can explain this
complete absence of autocatalysis for the oxyHb reac-
tion at all intermediate So
2
values (Fig. 5C,D). Inhibi-
tion of the oxyHb reaction by HbNO is, furthermore,
in accordance with the slow oxyHb reaction and
absence of autocatalysis when full oxygenation is
induced after the deoxyHb reaction has run for a while
to elevate HbNO (Fig. 2G). The inhibition of autoca-
talysis by HbNO may feedback positively on HbNO
levels because the reactive intermediates formed during
the autocatalytic phase of the oxyHb reaction have
been suggested to oxidize HbNO to metHb with the
release of NO [20]. In human Hb, this oxidative denit-
rosylation leads to the disappearance of HbNO when
oxyHb enters the autocatalytic phase of Hb oxidation

(i.e. when the reaction occurs at So
2
values of 48%
and above); and when deoxyHb is suddenly oxygen-
ated in the presence of nitrite, all the HbNO produced
also vanishes [20]. In carp, HbNO does not disappear
at any of the explored intermediate So
2
values or upon
acute oxygenation during the reaction (Figs 2 and 4).
These results agree with the idea that the absent oxy-
Hb autocatalysis in carp Hb limits HbNO depletion.
The gradual decrease in HbNO concentration fol-
lowing the sudden oxygenation of carp Hb (Fig. 2G)
can be ascribed to the reaction of O
2
with HbNO. This
reaction involves a rate-limiting dissociation of NO
from HbNO followed by the binding of O
2
to ferrous
heme and subsequent NO-mediated oxidation of oxy-
Hb to form metHb and nitrate [30]. Only in the pres-
ent case will the Hb oxidation be both NO-mediated
and nitrite-mediated, as a result of the presence of
nitrite. It may also be considered that part of the
HbNO decrease could result from an oxygenation-
induced allosteric transfer of NO from the heme to
Cys-b93 forming S-nitroso-Hb, as proposed in mam-
malian Hbs [31]. This particular cysteine, which is

highly conserved in Hbs from mammals and birds, is,
however, absent in carp and other fish Hbs [32].
The decrease in HbNO observed at low Po
2
after
deoxyHb became depleted (Figs 2 and 4) can also be
related to the dissociation of small amounts of NO
from HbNO. At this time of the reaction there are no
unligated ferrous hemes (deoxyHb = 0), and the off-
loaded NO can only react with oxyHb or escape the
system, whereby the amount of HbNO slowly
decreases.
Physiological perspectives
A main conclusion of the present work is that the
high-O
2
-affinity Hb of hypoxia-tolerant carp produces
a greater amount of NO from nitrite than does mam-
malian Hb with lower O
2
affinity. This characteristic
suggests that the reaction between Hb and nitrite may
be particularly relevant in ectothermic species that
periodically experience hypoxia in their environment.
The preferential reaction of nitrite with deoxyHb,
rather than with oxyHb, at intermediate So
2
has a par-
allel at the red cell membrane level. In carp, nitrite is
preferentially transported into the red cells at low So

2
,
whereas it enters oxygenated red cells only minimally
at physiological pH [3,24]. Therefore, carp possess
mechanisms at both cellular and molecular levels that
guide nitrite towards the reaction with deoxyHb to
produce NO. These characteristics would appear ideal
for a role of nitrite-derived red cell NO in blood flow
regulation during hypoxia. It is uncertain, however, to
what extent NO activity will be able to escape the red
cells and induce vasodilation. NO binds to deoxygen-
ated ferrous heme with very high affinity, and the rate
of dissociation is low, whereby Hb exerts a NO scav-
enging role rather than a NO liberating role. NO is
tightly bound to carp Hb and neither Po
2
changes nor
conformation changes seem able to liberate NO from
HbNO within the physiological circulation time. In
spite of this dilemma, there is accumulating evidence
that some NO can escape autocapture by Hb and pro-
duce vasodilation [4,12–14]. The mechanism of this is
as yet unknown, but export of NO activity from the
red cells could be eased via a localized reaction
between deoxyHb and nitrite at the membrane, the
intermediacy of S-nitroso compounds, or the forma-
tion of N
2
O
3

that diffuses out to form NO outside the
red cells [33,34]. Future research will need to clarify
these possibilities.
For fish the reaction of nitrite with Hb has an addi-
tional physiological perspective. Aquatic environments
can experience elevated nitrite concentrations, and this
can cause very high plasma nitrite concentrations
because freshwater fish take up nitrite via active trans-
port across the gills [3]. The data from the present
study suggest that high plasma nitrate concentrations
should induce not only methemoglobinemia but also
the formation of substantial amounts of NO and
HbNO at the intermediate So
2
values found in venous
Nitrite–hemoglobin reactions at different O
2
saturations F. B. Jensen
3384 FEBS Journal 275 (2008) 3375–3387 ª 2008 The Author Journal compilation ª 2008 FEBS
blood. This is indeed what was recently reported dur-
ing the in vivo exposure of zebrafish to nitrite [22].
Materials and methods
Preparation of hemoglobin
Carp (Cyprinus carpio) were anaesthetized in MS 222 (ethyl
3-aminobenzoate methanesulfonate; Sigma, Steinheim,
Germany) and blood was sampled from the caudal vessels
into heparinized syringes. Freshly drawn blood from rabbit
(Oryctolagus cuniculus) was obtained from the Biomedical
Laboratory, University of Southern Denmark. The blood
was centrifuged and plasma and buffy coat were removed.

The red blood cells were washed twice in cold 0.9% NaCl
and subsequently hemolyzed in cold distilled water. The Hb
solutions were purified by passage through a Sephadex G25
superfine (Amersham, Uppsala, Sweden) gel filtration col-
umn that was equilibrated and eluted with a stripping buf-
fer containing 0.05 m Tris buffer, pH 7.3, and 0.1 m KCl to
simulate natural erythrocyte pH and ionic strength [24].
The Hb solutions were divided among a series of tubes and
stored at )80 °C. For experiments, individual Hb tubes
were thawed, and the Hb was diluted with stripping buffer
to obtain the experimental Hb concentration of approxi-
mately 155 lmolÆhemeÆL
)1
.
Experimental set-up and experiments
Experiments were conducted at 25 °C in a specially con-
structed glass tonometer with an inbuilt 1-cm light path
3-mL cuvette. The tonometer received a continuous flow of
humidified gas from cascaded Wo
¨
sthoff (Bochum, Germany)
Digamix gas mixing pumps. The required O
2
tension
(Po
2
) was obtained by mixing air and N
2
(O
2

£
5 p.p.m. = 0.0037 mmHg) in the appropriate ratio. Three
millilitres of Hb was transferred to the tonometer and
equilibrated for 1 h in the shaking tonometer to ensure full
equilibration of the Hb to the gas atmosphere of the system.
The tonometer was then transferred to a Cecil CE2041 spec-
trophotometer (Cambridge, UK) for recording Hb absor-
bance in the inbuilt tonometer cuvette. A spectral scan was
made from 480 to 700 nm in 0.2-nm steps. Then, 9.1 lLofa
140 mm NaNO
2
solution was added to obtain a [NO
À
2
] ⁄ [Hb]
ratio of 2.7. The tonometer was quickly shaken to ensure
instant mixing and then repositioned in the spectrophoto-
meter. Subsequent spectral scans were run at specified time-
points during the reaction. Gas flow to the tonometer was
maintained throughout the entire experiment, which ensured
that the Po
2
was constant during the experiment. The spec-
trophotometer cuvette was kept at a temperature of 25 °C.
The pH of Hb solutions was measured using the capillary pH
electrode of a Radiometer (Copenhagen, Denmark) BMS3
electrode set-up connected to a PHM84 research pH meter.
Series 1 experiments examined the reaction of nitrite with
carp Hb and rabbit Hb in stripping buffer at constant Po
2

values. Different Po
2
values were used in separate experi-
ments, in order to obtain data with Hb O
2
saturation (So
2
)
values that covered the full range from 0% to 100% So
2
.
Series 2 investigated the influence of stabilizing the T state
of carp Hb with ATP, which is a natural allosteric effector
in fish erythrocytes [35]. An 80 mmolÆL
)1
ATP stock solu-
tion was prepared by dissolving the adenosine 5¢-triphos-
phate disodium salt (Sigma-Aldrich, Steinheim, Germany)
in distilled water and titrating the solution to pH 7.3 with
NaOH. ATP was added from this stock solution to obtain
an [ATP] ⁄ [Hb] ratio of 5 ([ATP] ⁄ [Hb
4
] = 20) in the Hb
solution. The reaction of nitrite with carp Hb in the pres-
ence of ATP was tested at different constant Po
2
values.
Series 3 assessed the influence of increasing the Po
2
in

carp Hb after the reaction with nitrite had been started, to
evaluate the effects of a rapid change in So
2
on the course
of the reaction. A Po
2
increase was obtained by switching a
low-Po
2
gas supply to 100% air with subsequent shaking of
the tonometer.
Data analysis
The Hb solution at any time during the reaction of nitrite
with Hb was assumed to be a mixture of deoxyHb, oxyHb,
metHb and HbNO. The concentrations of these four Hb
derivatives were evaluated by spectral deconvolution of indi-
vidual spectra, using a least squares curve-fitting procedure
and reference spectra of carp and rabbit deoxyHb, oxyHb,
metHb and HbNO, as described previously [22]. The reac-
tion kinetics was evaluated from plots of the concentrations
of the four Hb derivatives as function of time. The reaction
rates for the nitrite reaction with deoxyHb and oxyHb were
obtained by differentiation of the concentration versus
time relationships, using commercial software (origin 7;
OriginLab Corporation, Northampton, MA, USA). The
So
2
(%) of functional Hb was calculated from [oxy-
Hb] ⁄ ([oxyHb]+[deoxyHb]). The oxygen-binding properties
of carp Hb and rabbit Hb were determined by using So

2
values measured at the beginning of experiments to plot
log(So
2
⁄ 100)So
2
) versus log Po
2
(Hill plots), from which
the O
2
tension at 50% So
2
(P
50
) and cooperativity of O
2
binding (Hill’s n) were calculated.
Acknowledgements
The work was supported by the Danish Natural
Science Research Council.
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