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

The reaction of nitrite with carp Hb was characterized at natural red cell pH and ionic strength at several different constant O2 tensions Po2, which produced O2 saturations So2 that ran

with carp and rabbit hemoglobin at intermediate

oxygen saturations

Frank B Jensen

Institute of Biology, University of Southern Denmark, Odense M, Denmark

Nitrite (NO2) 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 O2 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: fbj@biology.sdu.dk

(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 nitrosylhedeoxyhe-moglobin 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 O2 affinity than in rabbit hemoglobin with lower O2 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 producreac-tion 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 O2 affinity increases the nitrite reductase capability of hemoglobin

Abbreviations

deoxyHb, deoxygenated hemoglobin; Hb, hemoglobin; HbNO, nitrosylhemoglobin; metHb, methemoglobin; NO, nitric oxide; oxyHb, oxygenated hemoglobin; P50, O2tension at 50% S O2; P O2, oxygen tension; S O2,O2saturation.

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 H2O2, NO2and 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]:

4HbðFe2þÞO2þ 4NO2 þ 4Hþ

! 4HbðFe3þÞ þ 4NO3 þ O2þ 2H2O 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]:

HbðFe2þÞ þ NO2 þ Hþ! HbðFeÞ3þþ NO þ OH

ð2Þ

HbðFe2þÞ þ NO ! HbðFe2þÞNO 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 deoxygenoxygen-ated 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 O2affinity [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 O2 affinity compared to hemoglobin with a low

O2 affinity The reaction of nitrite with carp Hb was characterized at natural red cell pH and ionic strength

at several different constant O2 tensions (Po2), which produced O2 saturations (So2) that ranged from the fully deoxygenated Hb through a series of intermediary

So2 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 O2 affinity The experiments also scruti-nized the influence of decreasing O2affinity in carp Hb via T-state stabilization by ATP and the effects of changes in O2 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 oxydeoxy-Hb reaction at intermediate O2saturations

Results Oxygen-binding properties Carp Hb in 0.05 molÆL)1 Tris buffer (pH 7.3) and 0.1 molÆL)1KCl had a very high oxygen affinity and a low cooperativity, as reflected by an O2 tension at 50% So2 (P50) of 1.2 mmHg and an n value of 1.03 Under the same conditions, the P50value 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]⁄ [Hb4] = 20) increased the P50 of carp Hb to

6 mmHg and the n value to 2.7, showing that ATP both lowered O2affinity and increased cooperativity

Reaction of nitrite with carp Hb at different O2 saturations

Nitrite was added at an [NO2]⁄ [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 R2 of experimental fits was 0.99950 ± 0.00002 (mean ± SEM, n = 260 fits) for carp Hb and 0.9990 ± 0.00009 (mean ± SEM,

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 So2of 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 (So2= 2%), apparently because the traces of O2 present in the N2 gas [O2£ 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 So2 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 NO3 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 So2 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 So2

values The reaction at intermediate So2 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% So2 (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% So2) 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% So2 than at intermediate values of So2 The considerably faster decline in oxyHb at 100% So2

(Fig 2F) than at intermediate So2 (Fig 2B–E) showed that the oxyHb reaction was inhibited at intermediate So2 values The reaction at 100% So2 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 So2 values (10%) for 12 min, whereafter Po2 was abruptly increased (Fig 2G)

SO2 = 2%

SO

2 = 46%

PO2 = 1.17 mmHg

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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.

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Oxygenation during reaction at low So2

Time (min)

SO2 = 2% SO2 = 35%

SO

2 = 65%

SO

2 = 78%

SO2 = 100%

C D

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 O2) were: (A) 2%, (B) 35%, (C) 46%, (D) 65%, (E) 78% and (F) 100% Panel G shows the effects of an acute oxygenation (P O2increase) during the reaction at low S O 2 The hemoglobin concentration was 155 l M , 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.

The elevated Po2 produced a sharp increase in

oxy-Hb and decreased deoxyoxy-Hb 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

O2saturations

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 So2 values tested except for 100%

So2, where the reaction rates in the two species were

comparable At 2% So2, 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 So2

values, the reaction of deoxyHb was clearly preferred over that with oxyHb, even though the difference was less marked than for carp (compare So2= 46% for rabbit in Fig 3C with that for carp in Fig 2C) When rabbit Hb reacted with nitrite at an So2 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 So2 values (28% and 46%; Fig 3B,C), where oxyHb only decreased slowly and remained

oxyHb metHb HbNO deoxyHb

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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.

present after deoxyHb had reached zero At 100%

So2 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 So2 Peak HbNO

concentra-tions were reached by the time that deoxyHb reached

zero, whereafter HbNO decreased (Fig 3) At

interme-diate So2 values, the HbNO levels were lower than

observed for carp Hb At 67% So2, 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]⁄ [Hb4] = 20) stabilized the T struc-ture and lowered O2 affinity, which caused oxyHb to

be completely absent in the N2 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

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oxyHb metHb HbNO deoxyHb

[ATP]/[Hb] = 5

Carp Hb

SO2 = 0%

SO2 = 100%

SO2 = 32% [ATP]/[Hb] = 5

SO2 = 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.

half the initial deoxyHb concentration (Fig 3A), as

expected from Eqns (2,3)

The presence of ATP also decelerated the reaction

kinetics at intermediate So2 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

(So2= 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 reacconcentra-tion

rates for the deoxyHb and oxyHb reactions with nitrite

at different So2values (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 So2(Fig 5A)

deoxyHb reaction rate [ATP]/[Hb] = 5

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SO2

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).

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% So2, 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

[NO2] This gave values of 2.5 and 1.0 m)1Æs)1 for

carp Hb in the absence and presence of ATP,

respec-tively, and 0.06 m)1Æs)1 for rabbit Hb, which illustrates

the high reactivity of carp Hb and the decreased rate

of reaction with T-state stabilization and lowered O2

affinity

The reaction of nitrite with fully oxygenated carp

Hb at 100% So2 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% So2 was completely absent at all tested

interme-diate So2 values, both in the absence and presence of

ATP (Fig 5C,D)

Dependency of HbNO production on So2

The maximal [HbNO] showed a significant

correla-tion with the initial So2 under all experimental

con-ditions (Fig 6) HbNO formation was greatest at

zero So2, and as So2 gradually increased, the yield

of HbNO gradually decreased The relationships

between [HbNO]max and So2 were curvilinear and

converged at the extreme So2 values (0% and

100%), but differed at intermediate So2 values

(Fig 6) This revealed that the production of HbNO

depended on So2, the species-specific O2 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 So2

(i.e at 50% So2 it would only be half the value at

0% So2), the possible maximum for HbNO also

decreased with increasing So2 (represented by the upper dotted straight line in Fig 6) The observed maximal HbNO values were lower than this possible maximum at intermediate So2 (Fig 6) This was expected because at intermediate So2 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 So2 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 So2 The data support the idea that the high O2 affinity of carp Hb is associated with an elevated nitrite reductase capability compared to mammalian

Hb with a lower O2affinity

Initial oxygen saturation (%)

0 20 40 60 80 100

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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 O2affinity: P50= 5.1 mmHg), and for carp Hb in the presence

of ATP ( ), where oxygen affinity is lowered (P50= 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.

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 ([NO2]⁄ [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

NO2, 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 concon-centration 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 O2 contamination [25] or the forma-tion of reacforma-tion 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 So2, 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 So2 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 So2value 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 So2 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 O2 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 O2 affinity is further supported by the formation, in carp, of a higher amount of HbNO when the O2 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

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% So2

but present at 48% So2 and above [20] A similar

situ-ation was found in rabbit Hb, where autocatalysis was

absent at 46% So2but present at 67% So2(Fig 3) In

carp Hb, autocatalysis was absent at all intermediate

So2 values tested, including 78% So2 (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 So2 values (Fig 5C,D)

Inhibi-tion of the oxyHb reacInhibi-tion 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 So2 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 So2values 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 oxy-Hb limits oxy-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 O2with HbNO This

reaction involves a rate-limiting dissociation of NO

from HbNO followed by the binding of O2 to ferrous

heme and subsequent NO-mediated oxidation of

oxy-Hb to form metoxy-Hb 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 Po2 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-O2-affinity Hb of hypoxia-tolerant carp produces

a greater amount of NO from nitrite than does mam-malian Hb with lower O2 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 So2has a par-allel at the red cell membrane level In carp, nitrite is preferentially transported into the red cells at low So2, 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 Po2changes 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 N2O3that 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 So2 values found in venous