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 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-tolerant 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 deoxyhemoglobin leads to a : formation of nitrosylhemoglobin and methemoglobin 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 reaction rates decrease and NO production is lowered, but the deoxyhemoglobin 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 Nitrite (NOÀ ) is naturally present at low concentra2 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], mitochondria [8], or deoxygenated hemoglobin [4,9,10] and myoglobin [11] The deoxyhemoglobin-mediated formation 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 Abbreviations deoxyHb, deoxygenated hemoglobin; Hb, hemoglobin; HbNO, nitrosylhemoglobin; metHb, methemoglobin; NO, nitric oxide; oxyHb, oxygenated hemoglobin; P50, O2 tension at 50% SO2; PO2, oxygen tension; SO2, O2 saturation FEBS Journal 275 (2008) 3375–3387 ª 2008 The Author Journal compilation ª 2008 FEBS 3375 Nitrite–hemoglobin reactions at different O2 saturations F B Jensen 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, NO2 and ferrylhemoglobin are produced [15–17] The stoichiometry for the overall reaction reveals that oxyHb is oxidized to ferric Hb [methemoglobin (metHb)] and nitrite is oxidized to nitrate [15]: 4HbFe2ỵ ịO2 ỵ 4NO ỵ 4Hỵ ! 4HbFe3ỵ ị ỵ 4NO ỵ O2 ỵ 2H2 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 nitrosylhemoglobin (HbNO) [4,9,18]: evolutionarily adapted to cope with severe hypoxia, partly by having hemoglobin with very high O2 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 formation 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 experimental conditions, which enabled a direct comparison to be made between carp Hb and a mammalian Hb with lower O2 affinity The experiments also scrutinized the influence of decreasing O2 affinity 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 equilibrium between the T and R states, and that the deoxyHb reaction dominates over the oxyHb reaction at intermediate O2 saturations HbFe2ỵ ị ỵ NO þ Hþ ! HbðFeÞ3þ þ NO þ OHÀ ð2Þ HbFe2ỵ ị ỵ NO ! HbFe2ỵ ịNO Oxygen-binding properties 3ị The deoxyHb reaction has a sigmoid, autocatalyticlike 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 oxygenated or fully deoxygenated Hb, the reaction at intermediate 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 3376 Results Carp Hb in 0.05 molỈL)1 Tris buffer (pH 7.3) and 0.1 molỈL)1 KCl 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 P50 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 ([ATP] ⁄ [Hb4] = 20) increased the P50 of carp Hb to mmHg and the n value to 2.7, showing that ATP both lowered O2 affinity and increased cooperativity Reaction of nitrite with carp Hb at different O2 saturations Nitrite was added at an [NOÀ ] ⁄ [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, FEBS Journal 275 (2008) 3375–3387 ª 2008 The Author Journal compilation ª 2008 FEBS Nitrite–hemoglobin reactions at different O2 saturations F B Jensen A 2.5 deoxyHb Absorbance 2.0 min min 11 14 17 23 32 41 50 59 68 77 SO2 = 2% 1.5 1.0 mixture of HbNO and metHb 0.5 0.0 450 B 500 550 600 650 700 min min 11 14 17 23 32 41 50 59 74 90 110 140 180 2.5 PO2 = 1.17 mmHg Absorbance 2.0 SO2 = 46% 1.5 1.0 0.5 0.0 450 500 550 600 Wavelength (nm) 650 700 Fig 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 The hemoglobin concentration was 155 lM 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 n = 115) for rabbit Hb Examples of absorbance spectra of carp Hb at specified time-points following the addition of nitrite are given in Fig to illustrate the spectral changes that occurred during the reaction of nitrite with deoxyHb (Fig 1A) and with Hb with an initial So2 of 46% (Fig 1B) When nitrite reacted with carp deoxyHb in a nitrogen atmosphere, the concentration of deoxyHb decreased to zero in approximately 30 The reaction products HbNO and metHb concomitantly increased in a : 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 £ 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 possibility of reacting with deoxyHb and oxyHb simultaneously Furthermore, NO formed in the deoxyHb reaction could react with either deoxyHb to form HbNO or with oxyHb to form metHb and NOÀ 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 expected stoichiometries for the oxyHb reaction (Eqn 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 produced 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 subsequent 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) FEBS Journal 275 (2008) 3375–3387 ª 2008 The Author Journal compilation ª 2008 FEBS 3377 Nitrite–hemoglobin reactions at different O2 saturations A B 160 SO2 = 2% 140 Concentration (μM) F B Jensen Carp Hb SO2 = 35% 140 120 120 oxyHb metHb HbNO deoxyHb 100 80 100 80 60 60 [NO– ]/[Hb] = 2.7 40 40 20 20 0 C 160 20 40 60 80 100 120 140 160 180 80 100 120 140 160 180 60 80 100 120 140 160 180 SO2 = 65% 140 120 120 100 100 80 80 60 60 40 40 20 20 0 20 40 60 80 100 120 140 F 160 SO2 = 78% 140 160 180 20 G 160 40 160 140 140 SO2 = 100% Oxygenation during reaction at low So2 120 120 120 100 100 100 80 80 80 60 60 60 40 40 40 20 20 20 Concentration (μM) 60 160 140 Concentration (μM) 40 D 160 SO2 = 46% E 20 0 20 40 60 80 Time (min) 100 120 20 40 Time (min) 20 40 60 80 100 120 140 160 Time (min) Fig 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 (SO2) were: (A) 2%, (B) 35%, (C) 46%, (D) 65%, (E) 78% and (F) 100% Panel G shows the effects of an acute oxygenation (PO2 increase) during the reaction at low SO2 The hemoglobin concentration was 155 lM, 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 3378 FEBS Journal 275 (2008) 3375–3387 ª 2008 The Author Journal compilation ª 2008 FEBS Nitrite–hemoglobin reactions at different O2 saturations F B Jensen 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 : stoichiometric 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 The elevated Po2 produced a sharp increase in oxyHb and decreased deoxyHb to zero This was associated 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 O2 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 So2 values tested except for 100% So2, where the reaction rates in the two species were A B 160 Rabbit Hb SO2 = 2% 140 160 120 100 100 80 80 60 60 40 40 20 Concentration (µM) 120 20 0 C 50 100 150 200 250 300 350 400 D 160 SO2 = 46% 140 Concentration (µM) SO2 = 28% 140 50 100 150 200 250 E 160 80 120 SO2 = 67% 100 oxyHb metHb HbNO deoxyHb 400 140 120 100 350 160 140 120 300 SO2 = 100% 100 80 80 60 60 40 40 40 20 20 20 0 60 50 100 150 200 250 300 350 400 Time (min) 50 100 150 Time (min) 200 50 100 Time (min) Fig 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 (SO2) were: (A) 2%, (B) 28%, (C) 46%, (D) 67% and (E) 100% The hemoglobin concentration was 155 lM, 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 FEBS Journal 275 (2008) 3375–3387 ª 2008 The Author Journal compilation ª 2008 FEBS 3379 Nitrite–hemoglobin reactions at different O2 saturations F B Jensen 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 concentrations were reached by the time that deoxyHb reached zero, whereafter HbNO decreased (Fig 3) At intermediate So2 values, the HbNO levels were lower than observed for carp Hb At 67% So2, HbNO was produced in only small amounts and disappeared completely when the reaction entered the autocatalytic phase (Fig 3D) A 160 The addition of ATP to carp Hb at an [ATP] ⁄ [Hb] ratio of ([ATP] ⁄ [Hb4] = 20) stabilized the T structure 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 (Fig 4A) The initial reaction seemed to result in the formation of HbNO in excess of metHb, but subsequently the concentrations of reaction products increased in parallel, and at the end of the experiment, both HbNO and metHb were present at approximately B Carp Hb SO2 = 0% [ATP]/[Hb] = 140 Concentration (µM) Reaction in presence of ATP – [NO 120 160 ]/[Hb] = 2.7 120 100 100 80 80 60 60 40 40 20 20 0 C SO2 = 32% [ATP]/[Hb] = 140 20 40 60 80 100 120 140 160 180 D 160 SO2 = 70% [ATP]/[Hb] = 20 40 60 80 100 120 160 180 160 180 160 140 SO2 = 100% 120 120 [ATP]/[Hb] = 100 100 80 80 60 60 40 40 20 20 140 Concentration (µM) 140 oxyHb metHb HbNO deoxyHb 0 20 40 60 80 100 Time (min) 120 140 160 180 20 40 60 80 100 120 140 Time (min) Fig 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 saturations of (A) 0%, (B) 32%, (C) 70% and (D) 100% The [ATP] ⁄ [Hb] ratio was on a heme basis (equal to a ratio of 20 on tetramer basis) The hemoglobin concentration was 155 lM, 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 3380 FEBS Journal 275 (2008) 3375–3387 ª 2008 The Author Journal compilation ª 2008 FEBS Nitrite–hemoglobin reactions at different O2 saturations F B Jensen 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) However, 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 concentration profiles for carp (Figs and 4) gave the reaction rates for the deoxyHb and oxyHb reactions with nitrite A 14 B 14 deoxyHb reaction rate 12 –dC/dt (µM min–1) at different So2 values (Fig 5) In the absence of ATP, the rate for the reaction of nitrite with deoxyHb initially increased to reach a peak at 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 concentration of deoxy hemes in the T structure was maximal, but rather later in the reaction when the formation 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) SO2 10 S O2 0% 32% 70% 4 0 20 40 60 80 100 120 140 160 180 D 14 oxyHb reaction rate 12 –dC/dt (µM min–1) [ATP]/[Hb] = 10 2% 35% 46% 64% 78% Carp Hb C deoxyHb reaction rate 12 20 SO2 100% 35% 46% 64% 78% 60 80 100 120 140 160 180 14 oxyHb reaction rate 12 10 40 [ATP]/[Hb] = 10 SO2 100% 32% 70% 4 2 0 20 40 60 80 100 120 140 160 180 Time (min) 20 40 60 80 100 120 140 160 180 Time (min) Fig Instantaneous reaction rates for the reaction of nitrite with deoxygenated and oxygenated carp hemoglobin at different oxygen saturations 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 (absence of ATP) and Fig (presence of ATP) FEBS Journal 275 (2008) 3375–3387 ª 2008 The Author Journal compilation ª 2008 FEBS 3381 Nitrite–hemoglobin reactions at different O2 saturations F B Jensen Dependency of HbNO production on So2 The maximal [HbNO] showed a significant correlation with the initial So2 under all experimental conditions (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 3382 80 Carp Hb Carp Hb + ATP Rabbit Hb 70 [HbNO] max (µM) 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 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 second-order reaction between nitrite and Hb, the initial second-order rate constant can be calculated by dividing the initial reaction rate with [deoxyHb] and [NOÀ ] This gave values of 2.5 and 1.0 m)1Ỉs)1 for carp Hb in the absence and presence of ATP, respectively, and 0.06 m)1Ỉs)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 reaction rate initially showed a sharp increase, reached a marked peak and then displayed a decrease, as the reaction approached completion (Fig 5C) This pattern 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, compared with the absence of ATP Interestingly, the distinct autocatalysis observed for the oxyHb reaction at 100% So2 was completely absent at all tested intermediate So2 values, both in the absence and presence of ATP (Fig 5C,D) 60 50 40 30 20 10 0 20 40 60 80 100 Initial oxygen saturation (%) Fig 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 O2 affinity: P50 = 1.2 mmHg) and rabbit Hb (s, lower initial O2 affinity: P50 = 5.1 mmHg), and for carp Hb in the presence of ATP ( ), where oxygen affinity is lowered (P50 = 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 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 produced 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 compared with rabbit Hb, but it increased in carp by Tstate 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 oxyHb 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 O2 affinity FEBS Journal 275 (2008) 3375–3387 ª 2008 The Author Journal compilation ª 2008 FEBS F B Jensen Reactions at extreme oxygen saturations The reaction of nitrite with fully oxygenated Hb proceeded via an initial lag phase followed by an autocatalytic 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 relative to Hb, and under the present experimental conditions ([NOÀ ] ⁄ [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 sigmoid curve pattern that reveals an autocatalytic-like kinetics, with an initial increase in reaction rate followed 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 protein 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 deoxygenated 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 completed 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 character 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-diphosphoglycerate [26] in human Hb, and it correlates with the increase in redox potential induced by these phosphates [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 concentration of the initial deoxyHb concentration This is, however, not always found Some studies report the Nitrite–hemoglobin reactions at different O2 saturations expected : formation of HbNO and metHb [25], whereas others report a production of metHb that significantly exceeds the production of HbNO [18,26,29] Deviation from the : reaction product formation can result from O2 contamination [25] or the formation 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 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 and 4) This striking feature could not be predicted from the available knowledge on the reactions with fully oxygenated and deoxygenated 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 So2 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 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 differences 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, however, 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 FEBS Journal 275 (2008) 3375–3387 ª 2008 The Author Journal compilation ª 2008 FEBS 3383 Nitrite–hemoglobin reactions at different O2 saturations F B Jensen It has been shown that HbNO formed in the deoxyHb 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 situation was found in rabbit Hb, where autocatalysis was absent at 46% So2 but 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 reaction, the higher HbNO levels in carp can explain this complete absence of autocatalysis for the oxyHb reaction at all intermediate So2 values (Fig 5C,D) Inhibition 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 autocatalysis 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 denitrosylation 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 oxygenated in the presence of nitrite, all the HbNO produced also vanishes [20] In carp, HbNO does not disappear at any of the explored intermediate So2 values or upon acute oxygenation during the reaction (Figs and 4) These results agree with the idea that the absent oxyHb autocatalysis in carp Hb limits HbNO depletion The gradual decrease in HbNO concentration following the sudden oxygenation of carp Hb (Fig 2G) can be ascribed to the reaction of O2 with 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 oxyHb to form metHb and nitrate [30] Only in the present 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 oxygenationinduced allosteric transfer of NO from the heme to Cys-b93 forming S-nitroso-Hb, as proposed in mammalian 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 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 3384 unligated ferrous hemes (deoxyHb = 0), and the offloaded 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 mammalian 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 So2 has a parallel 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 deoxygenated ferrous heme with very high affinity, and the rate of dissociation is low, whereby Hb exerts a NO scavenging role rather than a NO liberating role NO is tightly bound to carp Hb and neither Po2 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 produce 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 formation of N2O3 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 additional 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 transport 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 FEBS Journal 275 (2008) 3375–3387 ª 2008 The Author Journal compilation ª 2008 FEBS F B Jensen blood This is indeed what was recently reported during 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 column that was equilibrated and eluted with a stripping buffer 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 approximately 155 lmolỈhemL)1 Experimental set-up and experiments Experiments were conducted at 25 °C in a specially constructed glass tonometer with an inbuilt 1-cm light path 3-mL cuvette The tonometer received a continuous flow of humidied gas from cascaded Wosthoff (Bochum, Germany) ă Digamix gas mixing pumps The required O2 tension (Po2) was obtained by mixing air and N2 (O2 £ p.p.m = 0.0037 mmHg) in the appropriate ratio Three millilitres of Hb was transferred to the tonometer and equilibrated for 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 spectrophotometer (Cambridge, UK) for recording Hb absorbance in the inbuilt tonometer cuvette A spectral scan was made from 480 to 700 nm in 0.2-nm steps Then, 9.1 lL of a 140 mm NaNO2 solution was added to obtain a [NOÀ ] ⁄ [Hb] ratio of 2.7 The tonometer was quickly shaken to ensure instant mixing and then repositioned in the spectrophotometer Subsequent spectral scans were run at specified timepoints during the reaction Gas flow to the tonometer was maintained throughout the entire experiment, which ensured that the Po2 was constant during the experiment The spectrophotometer 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 Nitrite–hemoglobin reactions at different O2 saturations Series experiments examined the reaction of nitrite with carp Hb and rabbit Hb in stripping buffer at constant Po2 values Different Po2 values were used in separate experiments, in order to obtain data with Hb O2 saturation (So2) values that covered the full range from 0% to 100% So2 Series 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 mmolỈL)1 ATP stock solution was prepared by dissolving the adenosine 5¢-triphosphate 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 ([ATP] ⁄ [Hb4] = 20) in the Hb solution The reaction of nitrite with carp Hb in the presence of ATP was tested at different constant Po2 values Series assessed the influence of increasing the Po2 in carp Hb after the reaction with nitrite had been started, to evaluate the effects of a rapid change in So2 on the course of the reaction A Po2 increase was obtained by switching a low-Po2 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 individual 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 reaction 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 So2 (%) of functional Hb was calculated from [oxyHb] ⁄ ([oxyHb]+[deoxyHb]) The oxygen-binding properties of carp Hb and rabbit Hb were determined by using So2 values measured at the beginning of experiments to plot log(So2 ⁄ 100)So2) versus log Po2 (Hill plots), from which the O2 tension at 50% So2 (P50) and cooperativity of O2 binding (Hill’s n) were calculated Acknowledgements The work was supported by the Danish Natural Science Research Council 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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. .. in the concentrations of oxygenated hemoglobin, methemoglobin, nitrosylhemoglobin and deoxygenated hemoglobin during the reaction of nitrite with carp hemoglobin at different oxygen saturations. .. in the concentrations of oxygenated hemoglobin, methemoglobin, nitrosylhemoglobin and deoxygenated hemoglobin during the reaction of nitrite with rabbit hemoglobin at different oxygen saturations