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

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].

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

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

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

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

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

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

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

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

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).

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

= 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

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

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

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

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%

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

(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

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

, 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

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

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

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

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.

£
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

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

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