Biochemical Characterization of Human
S-Nitrosohemoglobin
EFFECTS ON OXYGEN BINDING AND TRANSNITROSATION*
Rakesh P.
Patel
§,
Neil
Hogg¶,
Netanya Y.
Spencer¶,
B.
Kalyanaraman¶,
Sadis
Matalon§
, and
Victor M.
Darley-Usmar
§**
From the
Department of Pathology, Molecular and
Cellular Division, the § Center for Free Radical Biology,
and the
Department of Anesthesiology, Physiology, and
Biophysics, University of Alabama at Birmingham, Birmingham, Alabama
35294-0019 and the ¶ Biophysics Research Institute, Medical
College of Wisconsin, Milwaukee, Wisconsin 53226
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ABSTRACT |
S-Nitrosation of cysteine
93 in
hemoglobin (S-nitrosohemoglobin (SNO-Hb)) occurs in
vivo, and transnitrosation reactions of deoxygenated SNO-Hb are
proposed as a mechanism leading to release of NO and control of blood
flow. However, little is known of the oxygen binding properties of
SNO-Hb or the effects of oxygen on transnitrosation between SNO-Hb and
the dominant low molecular weight thiol in the red blood cell, GSH.
These data are important as they would provide a biochemical framework
to assess the physiological function of SNO-Hb. Our results demonstrate
that SNO-Hb has a higher affinity for oxygen than native Hb. This
implies that NO transfer from SNO-Hb in vivo would be
limited to regions of extremely low oxygen tension if this were to
occur from deoxygenated SNO-Hb. Furthermore, the kinetics of the
transnitrosation reactions between GSH and SNO-Hb are relatively slow,
making transfer of NO+ from SNO-Hb to GSH less likely as a
mechanism to elicit vessel relaxation under conditions of low oxygen
tension and over the circulatory lifetime of a given red blood cell.
These data suggest that the reported oxygen-dependent
promotion of S-nitrosation from SNO-Hb involves biochemical
mechanisms that are not intrinsic to the Hb molecule.
 |
INTRODUCTION |
The main function of Hb is transporting oxygen from the lungs to
respiring tissues. Oxygen is released when Hb undergoes a conformational change from the "R" state (i.e.
"relaxed" or high oxygen affinity form) to the "T" state
(i.e. "tense" or low oxygen affinity form). Numerous
allosteric mechanisms are present that regulate oxygen delivery by
either increasing or decreasing the oxygen affinity of Hb. These
include regulation by pH, carbon dioxide, and inorganic phosphates (1,
2). For example, by binding to the T state, which results in
stabilization of this conformer, phosphates such as adenosine
triphosphate or 2,3-bisphosphoglycerate decrease the oxygen affinity of
whole blood relative to purified Hb. These physiological regulatory
mechanisms work in concert to ensure that oxygen delivery satisfies
metabolic demand.
Efficient oxygen delivery is also a function of the amount of
blood flowing through the arteries and arterioles, which, in turn, is
regulated by the action of agents that cause either vasorelaxation or
vasoconstriction. Nitric oxide is critical in vascular homeostasis and
an important physiological modulator of blood flow due to its ability
to cause vessel relaxation (3). Nitric oxide is synthesized in
endothelial cells via the oxidation of L-arginine, a
reaction catalyzed by the type III nitric-oxide synthase and stimulated
by physiological processes such as blood flow over the endothelium (4).
Once formed, NO diffuses to the underlying smooth muscle cells, where
it binds rapidly to the heme group of the enzyme soluble guanylate
cyclase. This activates the enzyme and sets up a sequence of events
eventually resulting in relaxation of the vessel (5-9). A
controversial aspect of this hypothesis is that NO rapidly reacts with
oxy-Hb1 (k
107-108 M
1
s
1) (10), and given the high intra-erythrocytic Hb
concentration (5-10 mM heme), it is difficult to
understand how any NO would be available to initiate the signaling
pathway that results in relaxation of vascular smooth muscle (11).
Recent investigations suggest that, in part, the reaction of NO with
the heme group of Hb is restricted by partitioning into the hydrophobic
compartments of cell membranes, thus making diffusion of the free
radical into the red blood cell the limiting process (12). In addition,
several investigators have suggested that, in vivo, NO is
transported in the thiol-bound, oxidized, and more stable form of an
S-nitrosothiol. These compounds are insensitive to a direct
reaction with heme groups and have been detected in vivo as
both proteins (e.g. S-nitrosoalbumin) and small
thiol-containing amino acids or peptides (e.g.
S-nitrosoglutathione) (13-20).
Through the formation of S-nitrosothiols, Stamler and
co-workers (13, 14) have recently proposed an intriguing hypothesis by
which Hb can regulate blood flow and overcome problems associated with
scavenging of NO by heme. A novel derivative of the heme protein,
S-nitrosohemoglobin (SNO-Hb), was identified as a potential modulator of blood flow in vivo (13), and further
investigations have suggested a role for this protein in regulating
fetal blood pressure as well (21). S-Nitrosohemoglobin is a
derivative in which NO is covalently bonded to the cysteine residue at
position 93 on the
-chain (Cys-
93) (22). The nitrosyl group
bonded to this thiol may be reduced to NO, under certain conditions, to
induce relaxation of pre-capillary vessels and to inhibit platelet aggregation (13, 14, 23). It was suggested that when Hb is in the R
conformational state, the S-nitroso (SNO) group is oriented
toward the interior of the protein and stabilized by shielding from the
aqueous phase. However, when Hb undergoes transition to the T
conformational state, as has been shown to occur in pre-capillary networks, it was proposed that the SNO moiety becomes exposed to the
solvent, thereby allowing transnitrosation to glutathione or other
thiols present in the red blood cell. The subsequent formation of
S-nitrosoglutathione (GSNO) may then promote vessel relaxation (13, 14). In this way, SNO-Hb can increase blood flow and
hence oxygen delivery at sites where it becomes deoxygenated. S-Nitrosohemoglobin offers a novel solution by which NO can
modulate blood flow without the problems associated with
Hb-dependent scavenging of NO.
This hypothesis requires that NO be released following a decrease in
the oxygen concentration and thus in the degree of Hb oxygenation in
the pre-arterioles. Although it was demonstrated that ligand-induced
allosteric changes in Hb affect S-nitrosation of Cys-
93,
the published studies have not addressed the effect on the oxygen
affinity of Hb. The level of S-nitrosation in
vivo (<0.1% of oxy-Hb being S-nitrosated) is
insignificant compared with the total oxy-Hb present in the erythrocyte
(13). Therefore, the effects of S-nitrosation on oxygen
delivery to tissues are negligible. However, since the hypothesis
requires that only the deoxygenated conformer of Hb release NO (13,
14), then the oxygen affinity of SNO-Hb (despite its low concentration)
is critical to the role of Hb as an NO carrier. This is an important
issue because previous studies on the role of Cys-
93 have shown that blockage of the thiol via alkylation or the formation of mixed disulfides increases the oxygen affinity of Hb (24, 25). Furthermore, the naturally occurring Hb variant Hb Okazaki, where Cys-
93 is substituted with an arginine residue, also has an increased oxygen affinity (26). Since the formation of SNO-Hb represents a
physiologically relevant Hb derivative in which Cys-
93 is modified,
we have examined the effect of S-nitrosation of Hb on its
oxygen binding properties.
In addition, it is not clear how the oxygen-sensitive mechanisms
underlying conformation-dependent NO release function. It has been proposed that transnitrosation reactions between deoxy-SNO-Hb and GSH to form GSNO, the mediator necessary to elicit the vasorelaxant signal, occur. However, transnitrosation reactions are, in general, relatively slow, with the exact kinetics depending on the
physiochemical properties of the specific thiols involved. We have
therefore also examined the kinetics and equilibria of transnitrosation reactions between Hb and GSNO and fitted the results to a bimolecular reversible reaction.
These data show that S-nitrosation increases the oxygen
affinity of Hb, ensuring that if a Hb deoxygenation-sensitive mechanism of NO release exists, then SNO-Hb would only promote vasorelaxation at
sites of very low oxygen concentrations, i.e. at sites where the Hb existed primarily in the deoxygenated state. Furthermore, the
kinetics of the transnitrosation reaction between SNO-Hb and GSNO are
too slow to explain the vasodilatatory effects of SNO-Hb. These data
are discussed in the context of SNO-Hb as a potential modulator of
blood flow.
 |
EXPERIMENTAL PROCEDURES |
Reagents--
All reagents were purchased from Sigma. SNOC and
SNAP were synthesized as previously reported (27). Human HbA was
purified as described below.
Preparation of Hemoglobin and S-Nitrosohemoglobin--
Human
hemoglobin was prepared as described previously (10).
S-Nitrosohemoglobin was synthesized by incubating oxy-Hb
(200 µM; all stated Hb concentrations are in terms of
heme) with SNOC (2 mM) at 20 °C for 20-40 min in 2%
borate and 1 mM DTPA. In some experiments, SNAP was used
instead of SNOC, in which case the incubation time was 1-2 h. All
solutions were protected from light. Hb was separated from SNOC or SNAP
by gel filtration using Sephadex G-25 (10 × 1 ml;
pre-equilibrated with phosphate-buffered saline containing 1 mM DTPA, pH 7.4). When SNOC was used, ~10-20% of the Hb
was oxidized to methemoglobin (metHb). This was attributed to the
small, but unavoidable concentration of nitrite remaining in our SNOC
preparations. No significant metHb (measured by visible spectroscopy)
was formed when SNAP was used. Oxy-Hb concentrations were determined by
UV-visible spectroscopy using the extinction coefficient per heme group
577 nm = 14.6 mM
1
cm
1 (10). S-Nitrosation of Hb was quantified
by the Saville reaction, with all mercury-dependent nitrite
formation being protein-precipitable (13, 27). Differing levels of
S-nitrosation were obtained by altering the incubation times
between oxy-Hb and either SNOC or SNAP. Cys-
93 thiols on Hb were
measured using either dithionitrobenzoic acid or
2,2'-dithionitropyridine (28). Hb samples (5 µM heme) were incubated with dithionitrobenzoic acid or 2,2'-dithionitropyridine for 20 min at 20 °C, and absorbance at 412 or 386 nm, respectively, was measured. Reduced thiol concentrations were calculated using
412 nm = 12 mM
1
cm
1 or
386 nm = 14 mM
1 cm
1.
Oxygen Affinities of Hemoglobin and
S-Nitrosohemoglobin--
Oxygen binding curves of Hb and SNO-Hb were
determined using a tonometer. Hemoglobin samples (30-50
µM heme, 3 ml) were incubated initially with a metHb
reductase system (29) at 20 °C to remove metHb formed during
synthesis of SNO-Hb. The metHb reductase system was found to have no
effect on the oxygen affinities of Hb or on the stability of the SNO
group (data not shown). Furthermore, similar results were obtained in
instances when the metHb reductase system was not required,
i.e. when SNAP was used to synthesize SNO-Hb. Complete
reduction to oxy-Hb occurred within 20 min as determined
spectrophotometrically. Hb samples were then deoxygenated until
deoxy-Hb was formed (determined spectrophotometrically). Samples were
deoxygenated until
A560 nm/A577 nm = 0.73-0.74. This value was determined from a spectrum of pure deoxy-Hb
synthesized by addition of sodium dithionite to a solution of oxy-Hb.
Deoxygenation was accompanied by a decrease in sample volume to between
1.5 and 2 ml. Aliquots of air (0.5-1 ml) were then injected into the
tonometer, and the absorbance spectrum (500-700 nm) was recorded after
each addition. Oxygen partial pressures were determined by measuring
the atmospheric pressure at the time of the experiment using a digital
barometer (Fisher). All oxygen binding curves were measured under
identical conditions in 50 mM sodium phosphate buffer, pH
7.4, containing 250 µM DTPA at 20 °C and at a constant
partial pressure of carbon dioxide.
Effect of Allosteric Mediators on the Oxygen Affinities of Hb and
SNO-Hb--
The effect of temperature was assessed by measuring the
oxygen binding curve at either 20 or 37 °C. Once deoxygenated, Hb solutions were incubated at the required temperature in a
spectrophotometer for 15 min before addition of air. The modulation of
oxygen binding by inorganic phosphates was ascertained by adding
inositol hexaphosphate at a 100-fold excess concentration relative to
tetrameric Hb, prior to measurement of oxygen affinity. The effect of
pH was assessed by conducting the experiments in 50 mM
sodium phosphate buffer containing 250 µM DTPA at either
pH 6.5 or 7.4.
Transnitrosation Reactions between SNO-Hb and GSH--
To
monitor transnitrosation reactions between GSH and deoxy-SNO-Hb, Hb was
first deoxygenated in a tonometer, and then GSH (at a concentration 25 times that of heme) was added anaerobically using a gas-tight syringe.
Interactions with a mixture of oxygenated and deoxygenated forms were
also evaluated by partially oxygenated SNO-Hb before addition of GSH.
In either case, the effect of GSH on the oxygenation state of Hb was
monitored continuously over 5 min by measuring the absorbance spectrum.
In parallel experiments, GSNO formation was determined either
immediately or 5 min after GSH addition by adding trichloroacetic acid
(20% v/v) to Hb in the tonometer. The solution was then centrifuged to
remove precipitated protein, and GSNO was measured in the supernatant
by the Saville assay.
The reaction between GSNO and either oxy-Hb or deoxy-Hb was also
monitored by measuring the rate of loss of GSNO by HPLC. All
experiments were performed in 50 mM phosphate buffer, pH
7.4, containing 100 µM DTPA. GSNO concentrations were
determined by HPLC using a reverse-phase Kromasil C18
column (Alltech Associates, Inc.) and a mobile phase of 0.05%
trifluoroacetic acid/methanol (94:6). GSNO was detected
spectrophotometrically at 210 nm using a diode array detector and
quantified using authentic GSNO. The identity of GSNO was confirmed by
analyzing the full UV-visible spectrum of the putative GSNO peak using
the diode array detector.
Kinetic Analysis of Second-order Reversible
Reactions--
Transnitrosation reactions are second-order reversible
reactions (Equation 1).
|
(Eq. 1)
|
The kinetics of such processes can be described by two
second-order rate constants: kf for the forward
reaction and kr for the reverse reaction. The
dependence of [A] on time (t) is given by
Equation 2,
|
(Eq. 2)
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with terms defined in Equations 3-6.
|
(Eq. 3)
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(Eq. 4)
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(Eq. 5)
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(Eq. 6)
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The subscripts 0 and
indicate the concentrations of
reactants at zero time and infinite time, respectively. The data
obtained were fitted to the above equations and rate constants for the forward and reverse reactions, i.e. kf
and kr calculated.
 |
RESULTS |
Oxygen Affinity of SNO-Hb--
The oxygen affinities of Hb and
SNO-Hb were determined using a tonometer. Fig.
1 shows the absorbance spectra of Hb
(panel A) and SNO-Hb (panel B) obtained when the
corresponding deoxygenated derivatives were titrated with oxygen. In
both cases, after removal of oxygen, a typical absorbance spectrum of
deoxy-Hb was observed (
max = 555 nm), which, upon
addition of oxygen, gradually changed to give the spectrum of oxy-Hb
(
max = 544 and 577 nm). Isosbestic points at 524, 548, 569, and 585 nm were observed for both native Hb and SNO-Hb, indicating
that nitrosation of Cys-
93 does not affect the spectral changes that
occur upon oxygen binding to the heme. In addition, no spectral
evidence of formation of nitrosylhemoglobin (i.e. NO bound
to ferrous heme groups) was observed (with the methodology used,
nitrosylation of ~5% of the total heme would have been detectable).
The total concentration of solvent-exposed thiols (i.e.
NO-bound thiol + free thiol) was the anticipated 2 thiols/tetramer
(Table I).

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Fig. 1.
Changes in absorbance spectra on addition of
oxygen to deoxy-Hb (A) or deoxy-SNO-Hb
(B). Hemoglobin samples were deoxygenated by
evacuation and reoxygenated by addition of oxygen. The
arrows show the direction of absorbance changes on addition
of increasing amounts of air, indicating the transition from deoxy-Hb
to oxy-Hb. Reactions were carried out in 50 mM sodium
phosphate buffer, pH 7.4, containing 250 µM DTPA at
20 °C and at a constant partial pressure of carbon dioxide.
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Table I
Concentration of the S-nitroso group and free thiols after reaction of
Hb with SNAP or N-ethylmaleimide
The free or S-nitrosated thiols in native oxy-Hb,
oxy-SNO-Hb, or oxy-N-ethylmaleimide-Hb were determined as
described under "Experimental Procedures." The data shown are
representative of SNO-Hb synthesized with varying degrees of
S-nitrosation. In this case, SNO-Hb was synthesized by
incubation of oxy-Hb (200 µM) with SNAP (2 mM) in 2% borate buffer and 1 mM DTPA at
20 °C for 30 min. In all cases, the total thiol content was
2/tetramer. The S-nitroso group was measured by the Saville
reaction, and reduced thiols using dithionitrobenzoic acid. Reactions
were carried out in phosphate-buffered saline with 1 mM
DTPA at 20 °C. Values represent means ± S.E.
(n = 3).
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The concentration of oxy-Hb for a given pO2 was
calculated from the spectra shown in Fig. 1, and the oxygen binding
curves for Hb and SNO-Hb were thus calculated and are shown in Fig.
2A. Native Hb displayed a characteristic
sigmoidal oxygen binding curve. S-Nitrosohemoglobin (0.98 SNO groups/tetramer) also had a sigmoidal oxygen binding curve, which
was, however, left shifted compared with native Hb (Fig.
2A). These data show that, relative to native Hb, SNO-Hb has
a higher oxygen affinity and therefore, under physiological conditions,
requires an environment of substantially lower oxygen concentration
before releasing oxygen via a deoxygenation-sensitive mechanism as
described previously (13, 14). Fig. 2B summarizes a series
of experiments in which the oxygen affinities of Hb and SNO-Hb, with
differing levels of S-nitrosation, were determined. Oxygen
affinities were assessed by comparing the p50
values, i.e. the oxygen tensions at which 50% of the Hb was
bound with oxygen. Unmodified Hb had a p50 of
7 ± 0.12 mm Hg (mean ± S.E., n = 12). In
each case, S-nitrosation of Hb significantly increased the oxygen affinity (indicated by a decrease in the
p50), with the p50 of
SNO-Hb (0.6 SNO group/tetramer, i.e. 30% of
93 thiols being S-nitrosated (this number refers to a distribution of
SNO groups, as it is not possible to distinguish between specific populations of Hb containing 1 or 2 SNO groups/tetramer)) being 4.3 ± 0.27 mm Hg (mean ± S.E., n = 5).
Interestingly, S-nitrosation of up to one of the Cys-
93
residues proportionately increased the oxygen affinity; however,
further modification did not affect the oxygen affinity (Fig.
2B).

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Fig. 2.
S-Nitrosation of Hb increases the
oxygen affinity. Oxygen binding curves of Hb ( ) and SNO-Hb
(0.98 SNO group/tetramer; ) are shown in A and were
obtained from the data shown in Fig. 1. Fractional saturation
(Y) represents [oxy-Hb]/[total Hb]. B shows
the effect of S-nitrosation of Hb on the
p50 value for oxygen binding.
p50 values were determined from the respective
oxygen binding curves and plotted against the degree of
S-nitrosation of Hb. The data shown were obtained from up to
20 separate experiments and with human Hb obtained from three different
donors. *, p < 0.001 by analysis of variance. The
line shown between 0 and 1 SNO group/tetramer was fitted
using linear regression analysis (y = 7.01-4.09x, r2 = 0.99).
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Previous studies found that the SNO group of SNO-Hb was less stable
when Hb was in the deoxygenated state, resulting in nitrosyl-Hb formation, and that this was related to the ability of deoxy-SNO-Hb to
promote vasorelaxation (13). Since the method used to determine the
oxygen affinity involved initially deoxygenation followed by
oxygenation, formation of nitrosyl-Hb would affect the results obtained. However, no spectral evidence of nitrosyl-Hb or metHb was
seen during the deoxygenation-reoxygenation cycle. Furthermore, the
concentration of S-nitrosation was measured before and after completion of the oxygen saturation curve. Over the time taken to
deoxygenate and then to reoxygenate the Hb (~20-30 min), no significant change in the concentration of the Cys-
93-NO adduct could be detected. Further analysis over longer time periods indicated that the rate of spontaneous decomposition of deoxy-SNO-Hb was ~8
nM/min, representing 0.03% of the total bound NO/min in
this preparation of SNO-Hb. From these results, we conclude that
spontaneous release of NO from SNO-Hb is unlikely to be a
physiologically relevant mechanism, where a given Hb molecule will be
cycling between the oxygenated and deoxygenated forms on a time scale in the order of seconds.
Effect of Allosteric Mediators on the Oxygen Affinity of
SNO-Hb--
The increased oxygen affinity of SNO-Hb suggests that it
is less likely than native Hb to become deoxygenated and hence release NO under physiological conditions. However, in vivo,
numerous regulatory mechanisms modulate the oxygen binding affinity of Hb in the red blood cell. It is possible that S-nitrosation
of Hb alters the sensitivity to the various allosteric regulatory mechanisms present such that SNO-Hb may be able to deliver oxygen under
physiological conditions. We therefore tested the effects of pH,
temperature, and inorganic phosphates on the oxygen affinity of SNO-Hb
and compared it with that of native Hb.
The effects on oxygen affinity of each of the allosteric modulators are
shown in Table II and were determined by
measuring the difference in log p50 of SNO-Hb
and Hb elicited by the different allosteric mediators. An increase in
temperature is known to decrease the oxygen affinity and thus
facilitate oxygen delivery. Table II shows that the change in log
p50 of Hb and SNO-Hb on increasing the
temperature from 20 to 37 °C was the same. Inorganic phosphates decrease the oxygen affinity of Hb (1, 2). Inositol hexaphosphate induced similar increases in the p50 of Hb and
SNO-Hb, indicating that S-nitrosation of Cys-
93 does not
affect binding of inorganic phosphates to Hb. Finally, we assessed the
effect of pH (also called the Bohr effect). The relatively acidic
conditions in respiring tissues lower the p50 of
oxygen binding by Hb, with the net result being oxygen delivery. This
occurs in part through binding of protons to histidine residues present
in both the
- and
-chains, a property that also serves to buffer
plasma. Decreasing the pH from 7.4 to 6.5 decreased the oxygen
affinities of both Hb and SNO-Hb to similar extents, with the effect
being approximately equal for both forms of Hb (Table II). This shows
that S-nitrosation does not significantly affect the binding
and release of protons by Hb. It has been calculated that binding of
protons by His-
146 accounts for ~40% of the alkaline Bohr effect
(30). The fact that a change in pH exerts similar effects on the oxygen
affinities of SNO-Hb and Hb indicates that S-nitrosation of
Hb does not significantly alter the biological properties of this
histidine residue, which is closely situated to Cys-
93 (14). Thus,
SNO-Hb and Hb are modulated to similar extents by allosteric effector
mechanisms.
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Table II
Allosteric modulators have similar effects on the oxygen affinities of
Hb and SNO-Hb
The oxygen affinities of Hb and SNO-Hb (0.6-2 SNO groups/tetramer)
were measured in the presence and absence of allosteric mediators. The
change in oxygen affinity induced by the different allosteric
regulators was calculated by measuring the difference in log
p50. These were then compared to assess the
regulatory effects on SNO-Hb relative to Hb. Values represent means
(n = 2) ± S.E. (n = 3, as indicated).
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Cooperative Nature of Oxygen Binding to SNO-Hb--
A feature of
oxygen binding by Hb is that it is cooperative in nature. The effect of
S-nitrosation on this property was calculated by Hill plot
analysis of the oxygen binding data. Fig.
3 compares the Hill plots for Hb and
SNO-Hb. The Hill coefficient at the 50% oxygen saturation level for
each Hb was calculated. S-Nitrosation (0.6 SNO
group/tetramer) did not significantly decrease the Hill coefficient
(2.83 ± 0.11 compared with 3.17 ± 0.11 for unmodified Hb).

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Fig. 3.
Hill plots of oxygen binding curves of Hb and
SNO-Hb. The cooperative nature of oxygen binding to Hb ( ) and
SNO-Hb ( ) was examined by Hill plot analysis. Hill plots were
constructed by plotting the logarithm of Y/1 Y versus the logarithm of oxygen tension (mm Hg).
In the example shown, SNO-Hb in which 30% of the Cys- 93 residues
are nitrosated is represented. Hill coefficients were calculated by
measuring the gradient at the p50,
i.e. at log(Y/1 Y) = 0. Oxygen
binding curves were determined as described in the legends to Figs. 1
and 2.
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We can conclude from the minimal effects of S-nitrosation on
the Hill coefficient that the observed changes in oxygen affinity do
not involve altered heme interactions. Previous studies have shown that
Cys-
93 is located close to the
1
2-interface and that its covalent
modification inhibits the formation of essential salt links between
His-
146 and Asp-
94 and of various hydrogen bonds (14). The net
effect is a destabilization of the T state and hence an increase in
oxygen affinity as well as a decrease in the Hill coefficient. Since
S-nitrosation of Hb does not alter the Hill coefficient, the
increase in oxygen affinity does not appear to be modulated by
affecting the stability of salt links.
Effect of Glutathione on Release of NO from SNO-Hb--
It has
been suggested that transnitrosation reactions between SNO-Hb and low
molecular weight thiols, such as GSH, mediate the transfer of the
vasorelaxant signal from Hb to the vasculature (13, 14). The mechanism
proposed involves transnitrosation from deoxy-SNO-Hb to GSH, with
reaction with the oxy derivative being too slow and thus negligible
(13). To examine this point further, GSH was added to a solution of
SNO-Hb (2 SNO groups/tetramer) that was partially oxygenated (~60%,
i.e. Y = 0.6), and the value of Y
was measured over 5 min of incubation. Since unmodified Hb has a lower
oxygen affinity compared with SNO-Hb (Fig. 2), it is predicted that
transnitrosation from SNO-Hb to GSH, forming Hb and GSNO, will decrease
the value of Y. Addition of GSH (heme/GSH molar ratio
1:25) to SNO-Hb did not significantly change the value of
Y (Y = 0.59 ± 0.1 before addition of
GSH and 0.61 ± 0.1 after 5 min of incubation; mean ± S.E.,
n = 5). These data indicate that extensive
transnitrosation reactions are not occurring over the time scale, but
this approach would not be able to detect the release of 50-100
nM NO needed to elicit vasorelaxation.
To gain insight into the kinetics of this process, transnitrosation
between GSNO and oxy-Hb or deoxy-Hb was monitored by more sensitive
HPLC techniques (Fig. 4). Incubation of
GSNO with oxy-Hb caused a time-dependent reduction in GSNO
concentration (Fig. 4A). The kinetics of GSNO decay fit well
to a second-order reversible reaction (Equation 1 under "Experimental
Procedures" and Fig. 4A). Fig. 4A shows the
experimentally derived points (represented by symbols)
together with fits of these data to a second-order reversible process
(represented by lines). The values of both the forward
(kf) and reverse (kr) rate
constants for oxy-Hb were reasonably constant (0.08-0.2
M
1 s
1 for kf
and 0.06-0.13 M
1 s
1 for
kr) over a large range of initial GSNO
concentrations (0-0.7 mM). This reaction conforms well to
the model and precludes a simple first-order process. The average rate
constants for this reaction were 0.13 ± 0.02 M
1 s
1 (kf)
and 0.1 ± 0.01 M
1 s
1
(kr). This analysis gives an equilibrium constant
for the reaction of 1.3, close to unity. This means that under
equilibrium conditions and at the approximately equal concentrations of
GSH and Hb that occur in the red blood cell, the nitroso functional group will be evenly distributed between Hb and GSH.

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Fig. 4.
Transnitrosation between GSNO and Hb under
oxygenated and deoxygenated conditions. A, GSNO (700 ( ), 450 ( ), 300 ( ), 150 ( ), and 50 ( ) µM)
was incubated with oxy-Hb (1 mM total heme) at 37 °C in
50 mM phosphate buffer, pH 7.4, containing 100 µM DTPA. GSNO concentration was monitored by HPLC. The
data were fitted (solid lines) to a second-order reversible
model (see "Experimental Procedures"), assuming 0.5 hemoglobin
thiols/heme, to give both forward (kf) and reverse
(kr) rate constants. For the above data, the values
were 0.13, 0.08, 0.13, 0.14, and 0.20 M 1
s 1 (for kf), respectively, and 0.10, 0.06, 0.10, 0.13, and 0.10 M 1
s 1 (for kr), respectively.
B, GSNO (1220 ( ), 620 ( ), 450 ( ), and 250 ( )
µM) was incubated with deoxy-Hb ( , 2 mM;
all others, 1 mM) at 37 °C in 50 mM
phosphate buffer, pH 7.4, containing 100 µM DTPA under
strict anaerobic conditions. GSNO concentration was monitored by HPLC.
The data were fitted (solid lines) to a second-order
reversible model, assuming 0.5 hemoglobin thiols/heme, to give both
forward (kf) and reverse (kr)
rate constants. For the above data, the values were 0.05, 0.01, 0.02, and 0.11 M 1 s 1 (for
kf), respectively, and 0.01, <0.001, <0.001, and
0.05 M 1 s 1 (for
kr), respectively. It should be noted that the
concentrations given above for GSNO under anaerobic conditions are the
recovered initial concentrations that were used for the fitting
procedure. The amount added was significantly greater.
|
|
The kinetics of transnitrosation under anaerobic conditions
(i.e. between deoxy-Hb and GSNO) were also determined as
shown in Fig. 4B. The decay of GSNO conformed well to
second-order kinetics at high concentrations of GSNO and hemoglobin,
but less well at low concentrations. In addition, the rate constants
derived from the fits varied dramatically as a function of GSNO
concentration (between ~0.01 and 0.1 M
1
s
1 for kf). This suggests that a
simple second-order reversible process cannot explain the reaction
between GSNO and deoxy-Hb. One mitigating factor precluding accurate
kinetic analysis of transnitrosation between GSNO and deoxy-Hb is the
observation that GSNO binds to the heme group of deoxy-Hb.
Consequently, the amount of GSNO available for transnitrosation will be
modulated by the equilibrium of binding to the heme. Accurate kinetic
analysis can only be performed if all reactions are taken into account, and we are currently pursuing this objective. The rate constants for
the forward reactions (kf) for both oxy-Hb and
deoxy-Hb are slower, but of a similar magnitude to those previously
reported (35).
To determine the rate of GSNO formation from SNO-Hb under physiological
conditions, simulation of this process were undertaken. Using the rate
constants detailed above, the formation of GSNO, under aerobic
conditions, from the reaction between 5 µM SNO-Hb and 5 mM GSH was calculated to be ~3 nM
s
1. These concentrations were chosen to approximate the
conditions within an erythrocyte. These data indicate that GSNO
formation from SNO-Hb is a process capable of slowly generating
physiologically relevant concentrations of GSNO (20-100
nM). Although the reactions of deoxy-Hb are kinetically
more complex, our data indicate that under anaerobic conditions, the
formation of GSNO will be slower than under aerobic conditions.
 |
DISCUSSION |
The diffusability of NO and the high concentrations of Hb in the
circulation indicate that reaction between these two species will occur
in vivo. In support of this, nitrosyl-Hb, a direct product
of the reaction between deoxy-Hb and NO, has been detected in the
venous circulation (31). The corresponding reaction with oxy-Hb results
in formation of metHb and nitrate (32). Since metHb is reduced back to
ferrous Hb by the metHb reductase system in erythrocytes and nitrate is
an inert end product of NO metabolism, this reaction has been suggested
to play a role in the safe removal of NO. However, this reaction also
poses a problem regarding how NO, in the presence of Hb, can elicit
vasorelaxation and other effects synonymous with this free radical.
The detection of the novel Hb derivative SNO-Hb in vivo and
the subsequent description of mechanisms by which this protein can
mediate blood flow via NO release have brought new insights into this
paradox. This impacts upon the emerging area of NO biology addressing
the role of S-nitrosation reactions (i.e.
addition of NO+) as opposed to nitrosylation
(i.e. addition of NO). It was suggested that, in the
presence of GSH, the stability of the SNO group of SNO-Hb was dictated
by the oxygenation state of this heme protein (13). A mechanism was
formulated in which deoxy-SNO-Hb rapidly underwent transnitrosation
reactions with GSH to form GSNO (14).
In this study, we have further evaluated these mechanisms by
examining the biochemical properties of SNO-Hb, focusing on the effect
of S-nitrosation of Cys-
93 on the oxygen binding
characteristics of Hb and on transnitrosation reactions with GSH. This
is important since both the oxygen affinity of Hb and the
characteristics of transnitrosation reactions involving this protein
are intrinsically linked to the deoxygenation-sensitive mechanism of NO
release. Our results show that S-nitrosation of Hb increases
its oxygen affinity (Fig. 2). As mentioned earlier, the relatively low
concentrations of SNO-Hb in vivo suggest that although
SNO-Hb has a higher oxygen affinity, this will not affect the
efficiency of oxygen uptake and delivery by a given erythrocyte, and
thus, S-nitrosation cannot be considered another allosteric
modulator of oxygen uptake and delivery. In marked contrast, the
effects on the ability of the small population of SNO-Hb molecules to
deliver NO to tissues is highly significant and must be taken into
account since the oxygen dissociation curve of SNO-Hb is left-shifted.
Thus, in vivo, a deoxygenation-sensitive mechanism for NO
release from SNO-Hb would only be effective at sites where the oxygen
tension would drop significantly to allow SNO-Hb to release its oxygen. This would most likely occur in regions of hypoxia, and recent studies
indicate that in the rat mesenteric vasculature, the
pO2 can drop significantly (~50%) across the
endothelium, suggesting that SNO-Hb may become deoxygenated in this
tissue (33).
Since native Hb is symmetrical, both Cys-
93 residues are presumably
equivalent in terms of their chemical reactivity.
S-Nitrosation did not mediate oxidation of the thiol over
the time course of these experiments. Interestingly, however, the
effects on the oxygen affinity were mediated wholly by modification of
only one of the two available Cys-
93 residues (Fig. 2). It is not
clear how S-nitrosation of Cys-
93 increases the oxygen
affinity on a molecular level. Although blocking this residue by
alkylation has similar effects on the oxygen affinity, these data also
show that unlike S-nitrosation, maximal increases in the
oxygen affinity are only attained after both Cys-
93 residues are
blocked by N-ethylmaleimide (24).
We also evaluated transnitrosation reactions between the oxygenated and
deoxygenated forms of SNO-Hb and GSH, which have been proposed to play
an important role in Hb-dependent regulation of blood flow
(13, 14). Consistent with reported data on the kinetics of
transnitrosation reactions in general and more specifically between
GSNO and Hb (34), we found that the rate of this reaction is relatively
slow, but can yield biologically relevant concentrations of GSNO.
However, this is too slow to account for the vasodilatatory properties
of SNO-Hb over the time taken for a given red blood cell to pass
through the pre-capillary vasculature. Accurate estimation of the
transnitrosation of GSH by deoxy-SNO-Hb was difficult to achieve
because of the slow rates involved and the additional binding of the
S-nitrosothiol to another site on the deoxy-Hb molecule
(data not shown).
The concentration of SNO-Hb has been shown to be higher in arterial as
opposed to venous blood, implying release of NO in the
pre-capillary beds (13). Our data suggest that a
deoxygenation-dependent mechanism is unlikely and that the
biochemical mechanism that accounts for this observation does not
reside within the Hb molecule. An alternative explanation is that the
rates of SNO-Hb formation are oxygen-dependent and
therefore change throughout the circulation. Indeed, SNO-Hb synthesis
has been reported to be an oxygen-dependent process (35),
and this and our findings that SNO-Hb will be constantly destroyed by
GSH indicate that concentrations of this Hb derivative will be higher
in the arterial circulation.
 |
ACKNOWLEDGEMENTS |
We thank J. S. Beckman, B. A. Freeman, and M. T. Wilson for helpful discussions.
 |
FOOTNOTES |
*
This work was supported by the American Heart Association
and the American Diabetes Association (to V. M. D.-U.), National Institutes of Health Grants GM55792 (to N. H.) and HL31197 and HL51173
(to S. M.), and Office of Naval Research Grant N00014-97-1-0309 (to
S. M.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
**
To whom correspondence should be addressed: Dept. of Pathology,
University of Alabama at Birmingham, Volker Hall, Rm. GO19, 1670 University Blvd., Birmingham, AL 35294-0019. Tel.: 205-975-9686; Fax:
205-934-1775; E-mail: darley{at}path.uab.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
oxy-Hb, oxygenated
hemoglobin;
deoxy-Hb, deoxygenated hemoglobin;
SNO-Hb, S-nitrosohemoglobin;
deoxy-SNO-Hb, deoxygenated
S-nitrosohemoglobin;
metHb, methemoglobin or ferric
hemoglobin;
GSNO, S-nitrosoglutathione;
SNOC, S-nitrosocysteine;
SNAP, S-nitroso-N-acetylpenicillamine;
DTPA, diethylenetriaminepentaacetic acid;
HPLC, high pressure liquid
chromatography.
 |
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