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J Biol Chem, Vol. 274, Issue 35, 24742-24748, August 27, 1999
From the S-Nitrosated hemoglobin (SNO-Hb) is
of interest because of the allosteric control of NO delivery from
SNO-Hb made possible by the conformational differences between the R-
and T-states of Hb. To better understand SNO-Hb, the oxygen binding
properties of S-nitrosated forms of normal and sickle cell
Hb were investigated. Spectral assays and electrospray ionization mass
spectrometry were used to quantify the degree of
S-nitrosation. Hb A0 and unpolymerized Hb S
exhibit similar shifts toward their R-state conformations in response
to S-nitrosation, with increased oxygen affinity and decreased cooperativity. Responses to 2,3-diphosphoglycerate were unaltered, indicating regional changes in the deoxy structure of SNO-Hb
that accommodate NO adduction. A cycle of deoxygenation/reoxygenation does not cause loss of NO or appreciable heme oxidation. There is,
however, appreciable loss of NO and heme oxidation when oxygen-binding experiments are carried out in the presence of glutathione. These results indicate that the in vivo stability of SNO-Hb and
its associated vasoactivity depend on the abundance of thiols and other
factors that influence transnitrosation reactions. The increased oxygen
affinity and R-state character that result from
S-nitrosation of Hb S would be expected to decrease its
polymerization and thereby lessen the associated symptoms of sickle
cell disease.
Hemoglobin (Hb)1 is of central importance to human health
in its role as a respiratory protein. Another chapter in the study of
the human health significance of Hb is beginning, focused on NO uptake
and delivery by Hb and the role this plays in the control of blood
pressure and other NO-dependent reactions. Nitrosation of
sulfhydryl groups on the Hb tetramer creates S-nitrosated Hb (SNO-Hb), which has been shown to play an
important role in NO uptake and delivery (1). S-Nitrosated
forms of proteins such as Hb can be formed via interaction with
nitrosating agents formed upon interaction of NO and oxygen
(NOx) and by NO-exchange reactions
(transnitrosations) with nitrosated forms of low molecular weight
thiols such as cysteine and glutathione. Conversely, the low molecular
weight thiols can act as NO acceptors in transnitrosation reactions
where NO is donated by S-nitrosated proteins (2, 3).
Hb-based NO transport via SNO-Hb is significant because it can greatly
extend the range of NO-dependent reactions. Unlike SNO-Hb,
free NO is a very reactive molecule, whose lifetime in the complex
cellular milieu would be expected to be very short. It is this
characteristic of NO that delayed the discovery of NO-dependent reactions in smooth muscle relaxation,
platelet inhibition, neurotransmission, and immune regulation (4-8).
What is learned about Hb-based NO transport will have far-ranging
applications in these disparate fields.
The studies reported here concern the oxygen binding properties of
variably S-nitrosated adult human hemoglobin (Hb
A0) and sickle cell hemoglobin (Hb S) that has a Glu Functional and crystallographic studies have shown that the Cys- Because the sulfhydryl groups at Cys- We previously showed that increasing anion levels can modulate Hb
function by decreasing the frequency and extent of conformational fluctuations that control the accessibility of the heme groups where
oxygen is bound (15). In this report we show that these same
considerations underlie the anion-dependence of oxygen binding to
SNO-Hb and its decreased stability in the presence of low molecular weight thiols.
Sample Preparation--
Samples of Hb A0 and Hb S
were prepared by using the ammonium sulfate method, stripped of organic
phosphate cofactors and purified by chromatography as described
previously (16). The amounts of oxidized Hb (metHb), oxygenated Hb
(oxyHb), and hemichrome were determined by spectral analysis by methods
published by Winterbourn and co-workers (17). Samples that contained
any detectable hemichrome or greater than 5% metHb were discarded. For
experiments with SNO-Hb, the stock Hb solutions, typically 1-3
mM in heme units (Fe porphyrin units), were made just
before use and were never frozen. Other experiments where metHb
formation was less critical used stock Hb solutions that were stored in
liquid nitrogen prior to use. To generate samples with progressively
higher levels of metHb, samples were repetitively deoxygenated and
re-oxygenated with intervening periods of exposure to low oxygen levels.
HEPES, 2,3-diphosphoglycerate, and KNO2 (from Sigma) and
KCl (Fisher; >99%), were dissolved and adjusted to pH values
indicated. The metal chelators used were 0.1 mM DTPA or
0.05 mM EDTA, which gave equivalent results in the studies
reported here.
Preparation of SNO-Hb--
Our protocol for generation of Hb
with varied levels of S-nitrosation used purified oxygenated
Hbs at 0.5-3 mM in heme in 2% borate buffer at pH 9.2 with 0.1 M DTPA or 0.05 M EDTA. These were kept
in the dark at 4 °C and exposed to CysNO at varied levels (ratios of
0.5-4 CysNO to heme) and incubated for time periods of 0-5 min prior
to chromatograhy. This range of conditions was found to be adequate for
generation of samples with a wide span of S-nitrosation. The
low molecular weight NO donor, CysNO, was removed by passage through a
22-cm Sephadex G25 column at 4 °C. The elution volumes for free Hb
and free CysNO were determined, and controls were run to ensure that
all low molecular weight materials were separated from the eluted
SNO-Hb sample. The purified SNO-Hb was dialyzed against selected
buffers for use in subsequent experiments. The variably
S-nitrosated Hb samples were spectrally evaluated after
dialysis to the desired pH and anion condition and rejected if they
contained detectable hemichromes or more than 5% oxidized Hb
A0 or 9% oxidized Hb S.
Analysis of Extent of S-Nitrosation of Hb Samples--
The
fraction of S-nitrosation was typically determined by
spectral deconvolution analysis as described here. Prior to spectral evaluation, the samples were subjected to Sephadex G-25 chromatography with 2% borate, 0.1 mM DTPA buffer, pH 9.2 (carried out at
4 °C) to standardize the sample pH and buffer conditions and to
remove any low molecular weight materials. The samples were degassed in
tonometers and subjected to spectral analysis before and after treatment with 4 mg/ml dithionite (sodium hydrosulfite, Tech, Acros
Chemical Co.). Dithionite addition rapidly removes any residual oxygen,
reduces any metHb present and releases NO from the SNO linkage. The NO
is effectively captured by the reduced heme, and the spectrum that
results is that of partially NO-Hb. NO gas (National Welders, CP
grade), further purified by passage through 5 M and then 1 M NaOH, is added to the degassed, dithionite-treated
sample, and the spectrum of fully NO-Hb is obtained after a 10-min
equilibration period. Comparisons of the partially and fully NO-Hb
spectra allow the fraction of S-nitrosation prior to
dithionite treatment to be determined. The degree to which the
dithionite-treated sample shows the presence of the NO-Hb spectrum is a
direct result of NO released from SNO-Hb, since there was no prior NO
ligation of the heme and all low molecular weight thiols and nitrogen
oxides that could contribute NO have been removed. Accordingly, a
sample that has 50% NO-Hb after dithionite treatment corresponds to
100% S-nitrosation of the two
Electrospray ionization mass spectrometry with some modifications of
the method reported by Ferranti et al. (19) confirmed that
the sole effect of the transnitrosation treatment was
S-nitrosation of Oxygen Binding Measurements--
Oxygen binding measurements
were performed tonometrically, with a modified spectrophotometric
method based on that of Riggs and Wolbach (20). The
S-nitrosated samples were kept in the dark except during
spectral analysis (<30 s for data collection with a Hewlett-Packard
model 8451A or model 8453 diode array spectrophotometer), and very few
data points were collected in each set to minimize the time under low
oxygen conditions where Hb oxidation was most pronounced. The total
curves presented are composites of data points from multiple
experiments, with each experiment limited to about 2 h. Since the
presence of metHb can influence the binding curves, experiments that
resulted in >12% metHb A0 or Hb S at the end of the
experiment were not used.
Formation of Variably S-Nitrosated Hb--
Modification of
previously published procedures was required for generation of
S-nitrosated Hb suitable for measurements of oxygen binding
and anaerobic redox reactions. We found that exposing high (>500
µM) concentrations of oxyHb at 4 °C with low ratios of
CysNO to Hb for short time intervals in the dark in 2% borate-EDTA or
DTPA buffer, pH 9.2, largely avoided metHb formation and nitrosation of
internal SH groups. These conditions also minimized metal or light-induced release of NO from CysNO and subsequent NO-induced Hb
oxidation. Inclusion of a metal chelator in all buffers was essential
to prevent the metal-catalyzed formation of disulfides (5). The short
half-life usually found for CysNO is due to artifactual contamination
of buffers with copper or other redox-active trace metals (21). Free
copper and iron in off-the-shelf buffers may frequently be in the
nanomolar range, levels sufficient to alter reactions from the pathways
they would take in the absence of redox metals.
Transnitrosation reactions to create SNO-Hb have the net result shown
simplistically in the equilibrium representation below, where R
represents a low molecular weight thiol.
The experimental conditions used in our experiments were established by
detailed studies of reactions between CysNO and Hb, in which we used an
innovative approach to electrospray ionization mass spectrometry that
avoided the use of organic
solvents.2 Using this
technique, we obtained spectra of Hb samples after varied periods of
exposure to CysNO and chromatographic removal of this low molecular
weight NO donor. A representative spectrum is shown in Fig.
2, in which the Oxygen Equilibria of Hb A0 with Varied Levels of
Nitrosation of Cys-
The results presented in Fig.
3A are for approximately 30%,
40%, and 80% S-nitrosated Hb A0 and 0%
S-nitrosated controls in chloride-free HEPES/EDTA buffer at
pH 7.5. Our estimates of the degree of S-nitrosation of a
sample varied by ±5%. The changes toward higher affinity are
progressive with increases in degree of S-nitrosation. Fig.
3B shows similar data for 0% and approximately 80%
S-nitrosated Hb A0 in the presence of
2,3-diphosphoglycerate (DPG) at 50-fold excess over tetramer
concentration. The data shown are representative of many similar
experiments and show shifts induced by S-nitrosation toward
higher oxygen affinity in both the presence and absence of
2,3-diphosphoglycerate. For approximately 80% S-nitrosated
samples, the S-Nitrosylation and MetHb Formation--
Some metHb is formed
during S-nitrosation reactions as a result of NO (liberated
from CysNO) interacting with oxygenated Hb. By limiting the levels of
CysNO used and time of exposure, it was possible to generate SNO-Hb
samples with low levels of metHb for use in oxygen binding experiments.
To estimate the effect of increased levels of metHb, we carried out
oxygen binding studies with unmodified Hb A0 with
progressively higher levels of metHb. The Hill plots of Fig.
3C show that the R-state shifts induced by metHb formation
are similar in character to those associated with
S-nitrosation. The shifts associated with
S-nitrosation are, however, much larger than can be
attributed to metHb formation in the SNO-Hb samples. Under our assay
conditions, where levels of metHb were minimized, the shifts in
logP50 attributable to the presence of metHb in
the S-nitrosated samples are less than 10% of the shifts observed.
Stability of SNO-Hb--
Prior studies have shown deoxy SNO-Hb to
be less stable than oxy SNO-Hb (1). Deoxygenation is a necessary step
in the oxygenation studies reported here, a condition that was expected
to cause some loss of NO due to instability of deoxy SNO-Hb. Our
preparations, however, showed no appreciable loss of NO during oxygen
binding experiments. As shown in Fig. 4,
the preparations of oxy SNO-Hb used in these studies were stable for
over a week without loss of NO when stored at 4 °C at high protein
concentration. Samples stored in the deoxygenated condition or at lower
protein concentration were less stable and had greater loss of NO and
more metHb formation. The data shown in Fig. 4 are for aliquots
withdrawn from oxy and deoxy samples of about 80%
S-nitrosated Hb, 1 mM in heme, held at 4 °C.
The aliquots were brought to standard conditions for spectral
deconvolution assays as described under "Experimental Procedures."
The stability of SNO-Hb as demonstrated in this figure is important in
allowing for its storage or shipment prior to use in functional
studies.
Low Molecular Weight Thiols Destabilize S-Nitrosated Hb--
In
contrast to the stability noted above, appreciable diminution of
S-nitrosation of Hb A0 occurs when experiments
are carried out in the presence of a 5-fold excess of reduced
glutathione (GSH). The bar graph of Fig.
5 illustrates the destabilizing influence of GSH at several levels of S-nitrosation (achieved by
exposure to different levels of CysNO for varied times). Previous work demonstrated that GSH can accept NO from SNO-Hb in transnitrosation reactions (1). Fig. 5 shows that SNO-Hb is partly but not completely destabilized when deoxygenation occurs in the presence of this high
level of GSH. In vivo conditions may result in more loss of
NO during the deoxygenation process. Fig. 5 also shows that the loss of
NO from SNO-Hb is roughly the same in the presence and absence of
2,3-diphosphoglycerate. Appreciable increases in metHb levels occur
when oxygenation of SNO-Hb is carried out in the presence of
glutathione, with metHb levels typically 25-27% after oxygen
equilibria, in contrast to <10% metHb in its absence. This is a
consequence of GSH interactions with SNO-Hb, since control experiments
showed no effect of GSH on oxygen affinity or metHb levels for
unmodified Hb A0 (data not shown). The increase in metHb
probably results from interactions between oxyHb and free NO that is
released from either SNO-Hb or S-nitrosated glutathione during oxygen equilibria.
Oxygen Equilibria of Hb S with Varied Levels of Nitrosation of
Cys-
The initial stages of oxygenation of unpolymerized Hb S are most
affected by S-nitrosation ( Understanding the properties of SNO-Hb is critical for better
understanding of the NO-dependent reactions moderated by
Hb. Results presented here show that S-nitrosation
stabilizes the high affinity R-state conformation of Hb, as previously
reported for Hbs reacted with N-ethylmaleimide,
iodoacetamide, and other SH group modifiers (11, 14, 24-27).
Thiosteric effects brought about by S-nitrosation of
Cys- The conformational sensitivity of SNO-Hb was shown in an earlier
publication to be involved in a dynamic cycle in which SNO-Hb was
formed in the lungs and decomposed when NO was delivered to the
tissues. The greater accessibility of the Cys- Differences in thiosteric and anionic mechanisms of control of Hb
function are indicated by the results presented here and by previous
studies on other SH-modified Hbs. Notably, S-nitrosation results in equivalent shifts of logP10 in the
presence or absence of 2,3-diphosphoglycerate, the responses of normal
and SNO-Hb to 2,3-diphosphoglycerate are equivalent, and
2,3-diphosphoglycerate does not significantly affect the release of NO
from SNO-Hb in a cycle of deoxygenation/re-oxygenation in the presence
of glutathione. These observations indicate the existence of regional
rather than global effects associated with thiosteric and anionic
effectors, a conclusion reached earlier by Perutz and co-workers in
regard to the anion sensitivity of Hb in which the SH groups at
Cys- The regional effects associated with SH group modification have been
shown to result, in large part, from disruption of the normal salt
bridge between His- Anion-induced shifts in the Hill plot asymptotes are not interpretable
as solely due to their preferential binding to the T-state, a feature
that led Minton and Imai (30) to suggest that a minimum of three states
was required to describe Hb function. We recently advanced a new
paradigm of Hb function in which anion-induced shifts in the apparent
T-state are explained by anion-dependent alterations in the
conformational fluctuations that expose "buried" sites (15). The
functional properties of SNO-Hb as described above are supportive of
this paradigm. This paradigm also rationalizes the absence of a
2,3-diphosphoglycerate effect on the loss of NO from SNO-Hb in the
presence of glutathione, since Cys- Differences in thiosteric and anionic mechanisms of control of Hb
function may be of considerable significance for understanding the
linkage between the oxygen affinity and polymerization of Hb S and for
designing better treatments to alleviate sickle cell disease.
Treatments that increase the oxygen affinity of Hb S generally decrease
polymerization and the associated red cell morphological changes
associated with sickle cell disease (9). Significantly, Hb S
derivatives with SH groups modified with glutathione (26) or other
thiol reagents (25, 27) have decreased tendencies to polymerize.
Accordingly, the results reported here lead us to anticipate that new
approaches to sickle cell therapies may involve ways to increase the
S-nitrosation of Hb S and thereby reduce its tendency to
form polymers.
At the relatively low protein concentrations used in the experiments
reported here, Hb A0 and Hb S exhibit similar oxygen affinities and similar responses to S-nitrosation. In
contrast, at protein concentrations like those in red cells, the
protein concentration-dependent formation of Hb S polymers
is accompanied by decreases in its oxygen affinity that may result in
distinctive responses to S-nitrosation. The lowering of
oxygen affinity associated with polymerization of Hb S (31) and with
crystallization of Hb A0 (32) may reflect decreases in the
conformational fluctuations that expose the sterically restricted
active sites. Readers will recognize this interpretation as another
facet of the conformational fluctuations paradigm described above.
Inhalation of low concentrations of NO was recently reported to
increase the oxygen affinity of sickle erythrocytes in vitro and in vivo (33). After treatment, the oxygen affinity of
the sickle erythrocytes was approximately equal to that of the normal controls, where polymerization does not occur. This result implies that
the NO treatment brought about a reduction in the polymerization of Hb
S, as would be expected to occur if the non-polymerizing R-state of Hb
S was partially stabilized, as it is in SNO-Hb. Partial heme ligation
by NO or heme oxidation induced by NO would also result in higher
oxygen affinity. Accordingly, we have initiated studies to determine if
Hb S gelation is significantly inhibited by NO ligation or
S-nitrosation at levels that might be achievable in
vivo.
The data thus far available on the vasodilatory effects of SNO-Hb show
that oxidation of the metal center affects NO release and thereby the
NO-dependent physiological reactions associated with SNO-Hb
(1). In the studies reported here, Hb S oxidation was much more
pronounced than that of Hb A0, suggestive of altered NO
uptake and delivery for this variant form of Hb even in its unpolymerized state. The greater tendency of Hb S to autoxidize has
been noted previously, and the resistance to malaria associated with Hb
S has been postulated to arise in part as a consequence of greater
oxidative events in red cells containing Hb S than for red cells
containing only Hb A0 (34, 35). Consequently, the R-state
shifts in Hb S induced by S-nitrosation and by oxidation may
be joint determinants of the role played by Hb S in malarial resistance, Hb-linked vasodilation, and sickle cell disease.
We thank the Duke University Sickle Cell
Center for providing blood samples for this project.
A paper by Patel et al. (28), published
subsequent to acceptance of this manuscript, supports our finding that
S-nitrosation of Hb A increases its oxygen affinity. A major
difference in results reported is that these researchers found
unaltered cooperativity for SNO-Hb A. This is in marked contrast to our
observation that cooperativity is reduced in SNO-Hb relative to
unmodified Hb and also in contrast to the pattern of behavioral
alterations commonly associated with *
This work was supported by National Institutes of Health
Grant R01 HL58248, National Institutes of Health NIEHS Grant No. ES0-1908, and North Carolina Biotechnology Center financial support of
the Duke University Mass Spectrometry Facility.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: Duke University Marine
Biomedical Center, NSOE Marine Laboratory, 135 Duke Marine Lab Rd.,
Beaufort, NC 28516. Tel.: 252-504-7591; Fax: 252-504-7648; E-mail:
bona@duke.edu.
2
C. Bonaventura, G. Ferruzzi, S. Tesh, and
R. D. Stevens, manuscript in preparation.
The abbreviations used are:
Hb, hemoglobin;
SNO-Hb, S-nitrosated Hb;
Hb A0, purified
hemoglobin of adult human;
Hb S, sickle cell hemoglobin;
CysNO, S-nitrosated cysteine;
DTPA, diethylenetriaminepentaacetic
acid;
thiosteric, stereochemical effects associated with SH group
modification;
DPG, 2,3-diphosphoglycerate.
Effects of S-Nitrosation on Oxygen Binding by
Normal and Sickle Cell Hemoglobin*
§,,, and Duke University Marine Biomedical Center,
Nicholas School of the Environment Marine Laboratory, Beaufort,
North Carolina 28516 and the ¶ Department of Pediatrics, Duke
University Medical Center, Durham, North Carolina 27708
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Val substitution at 
6. Although physiological levels of
S-nitrosation of Hb are too low for oxygen transport to be
significantly affected, the linkage between S-nitrosation
and oxygen binding by Hb can affect the stability and subsequent
vasoactivity of SNO-Hb. Understanding this linkage is critical for
understanding the NO-dependent reactions of Hb in normal
and sickle cell erythrocytes, in cell-free Hb-based blood substitutes,
or in pharmaceuticals. As will be shown, S-nitrosation of Hb
A and unpolymerized Hb S results in increased oxygen affinity via
partial stabilization of their high affinity, R-state, conformations. Since R-state-stabilized Hb S does not readily polymerize (9), this
finding prompts us to suggest that S-nitrosation of Hb S may
be viewed as a possible therapeutic approach to alleviating sickle cell disease.
93
residues at which NO is bound as NO+ in SNO-Hb are more
accessible in the high affinity conformation of oxy (R-state) Hb than
in deoxy (T-state) Hb (10-12). This conformational sensitivity results
in a rate dependence for SNO-Hb formation that mirrors the greater
relative exposure of Cys-93 in conditions that favor the R-state and
was invoked to explain the decreased stability of the deoxy form of
SNO-Hb (1). Although deoxy SNO-Hb is less stable than the oxy form, we
found that purified deoxy SNO-Hb is sufficiently stable to allow
oxygen-binding studies to be carried out over a period of several
hours. However, as will be documented, loss of NO from SNO-Hb during a
cycle of deoxygenation/re-oxygenation can occur under simulated
in vivo conditions where NO acceptors such as glutathione
are present.
93 are in a conformationally
sensitive position on the Hb tetramer, the S-nitrosation of
Hb would be expected to have heterotropic allosteric effects on ligand
binding by the heme groups at the four active sites of the tetramer.
The following results document the existence and nature of these
thiosteric effects. We show that S-nitrosation promotes
increased oxygen affinity and thus acts in opposition to anionic
allosteric effectors that decrease oxygen affinity. Hb A0
and Hb S show similar responses to S-nitrosation, as
expected based on their similar structures in the region of Cys-93
(13). The shift of SNO-Hb forms toward higher oxygen affinity probably involves a regional conformational alteration of the deoxyHb
tetramer that prevents His-146 from making its
normal contribution to T-state stability. This was previously
shown to be the case in Hb in which the SH groups at Cys-93 were
modified by N-ethylmaleimide (11, 14).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
93 SH groups. Samples with
internal SH groups nitrosated can give higher than 50% NO-Hb readings
after dithionite treatment. The dithionite-treated sample was analyzed 5 min after dithionite addition. Since the NO binding to deoxy heme is
very fast (diffusion limited) and the NO dissociation rate is extremely
slow, the fact that free NO liberated from the NO-Hb complex is slowly
reduced by dithionite is not a serious detriment to this spectral assay
(18). This somewhat cumbersome protocol avoids experimental
difficulties associated with troublesome dithionite reaction products
and the pH and anion-dependent variation of the Hb-NO spectrum.
93 SH groups. This methodology also
guided our choice of experimental conditions where internal SH groups
were neither nitrosated nor cystinylated. The mass spectrometry method
was done in aqueous solution using a Micromass Quattro LC triple
quadrupole mass spectrometer equipped with Z-spray ion source. The Hb
samples were electrosprayed in 0.02 M ammonium bicarbonate,
pH 9.04.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
The extent of S-nitrosation of Hb samples after removal
of the low molecular weight NO donor was typically determined by spectral deconvolution analysis under standard conditions as described under "Experimental Procedures." Fig.
1 shows representative spectra for
deoxygenated SNO-Hb, the mixture of deoxy and NO-Hb that results from
dithionite addition, and the final NO-Hb spectrum obtained after NO
addition to fully occupy the heme binding sites. The spectrum of
deoxygenated SNO-Hb indicates the presence of a low level of metHb that
exceeds that of the deoxygenated control sample, but shows no evidence
of NO ligation of the heme. The amount of NO liganded to the heme that
appears after the NO-linkage to Cys-
93 is disrupted by dithionite is
used to estimate the extent of S-nitrosation. For some
samples the extent of S-nitrosation was also quantified by
the Saville reaction (22, 23), with a standard curve generated with
precisely determined quantities of nitrite. The results obtained were
within 10% of the values obtained with our deconvolution assays.
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Fig. 1.
Spectra representative of those used in
deconvolution assays of S-nitrosation. Spectra
shown are for a deoxygenated SNO-Hb sample (dotted line) prepared as described under "Experimental
Procedures," the same after treatment with dithionite
(dashed and dotted line), and then
after exposure to saturating levels of NO (dashed line). The solid line is for a
deoxygenated Hb A0 control. The spectrum of deoxygenated
SNO-Hb (before dithionite addition) has a greater amount of metHb than
the deoxygenated control, which accounts for their peak differences at
556 nm.
chains are about 50%
S-nitrosated. Under our reaction conditions, the mass change
associated with NO addition to the chains is the only mass change
observed.
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Fig. 2.
A portion of the mass spectrum observed for
S-nitrosated Hb A0. The multiply
charged state of 
-globin + heme exhibits only cationic adduction,
whereas -globin shows 50% conversion to the S-nitrosated
form. The m/z assignments are: 1211.7 = [-globin + heme + 13H+]/13; 1213.3 = [-globin + heme + 12H+ + Na+]/13; 1214.9 = [-globin + heme + 12H+ + K+]/13; 1221.3 = [-globin + 13H+ ]/13; 1223.5 = [-globin-SNO + 13H+]/13; and 1225.1 = [-globin + 12H+ + K+]/13. The Hb was electrosprayed in
0.02 M ammonium bicarbonate, pH 9.04, without use of
organic solvents.
93--
We determined the effects of
S-nitrosation on oxygen binding by Hb A0 in
chloride-free HEPES buffer and in the presence of a 50-fold excess of
2,3-diphosphoglycerate over tetramer concentration. The extent of
S-nitrosation of Hb A0 was measured before and
after oxygen equilibria and found to be equivalent in these
experiments. Readers are reminded that care was taken to avoid
S-nitrosation of buried residues, which can result in shifts
in logP50 larger than those shown. As noted
under "Experimental Procedures," these experiments were designed to
minimize exposure to light and to time at low O2. In the
results presented here, the Hb samples had low (<5%) initial levels
of metHb and less than 10% metHb after completion of oxygen
equilibria, even at high levels of S-nitrosation.
logP50 values in the absence and
presence of 2,3-diphosphoglycerate were 0.211 and 0.262, respectively.
The initial stages of oxygenation are most affected by
S-nitrosation (logP10 > logP50), resulting in asymmetric shifts away
from the control data. The corresponding values of
logP10 were 0.408 and 0.365, again
approximately the same for Hb in HEPES/DTPA and in the presence of
2,3-diphosphoglycerate. The asymmetric shifts in oxygen affinity
associated with S-nitrosation result in decreases in
cooperativity of oxygen binding as measured by the slope of Hill plots
at the mid-point of the binding curves (n50).
For samples in HEPES/DTPA, n50 decreased from
3.16 to 2.10, while for samples in 2,3-diphosphoglycerate,
n50 decreased from 3.33 to 2.54. These
alterations of oxygen binding brought about by S-nitrosation
are very similar to alterations reported for Hb in which the SH groups
are modified with N-ethylmaleimide (24).
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Fig. 3.
Progressive effects of
S-nitrosation and metHb formation on Hill plots of
oxygen binding by Hb A0. Hill plots shown are
composites of several experiments. Purified 60 µM Hb
samples were in 0.05 M HEPES buffer containing 0.5 mM EDTA or 0.1 mM DTPA, pH 7.5, 20 °C. In
A, data are shown for unmodified Hb A0
(closed circles), and for Hb A0 with
about 30% (open circles), 40%
(closed triangles), and 80% (open triangles) of the Cys- 93 groups nitrosated. MetHb levels,
measured before and after oxygen binding, were 1.5-3%, 2.9-4.2%,
5.1-9%, and 4.8-9.4%, respectively. In B, data obtained
with a 50-fold excess of DPG over Hb tetramer are shown for unmodified
Hb A0 (closed circles) and for about
80% S-nitrosated Hb A0 (open triangles). MetHb levels, measured before and after oxygen
binding, were 4.7-6.7% and 7.2-9.7%, respectively. Fig.
3C shows effects of increasing levels of metHb in Hb
A0. MetHb levels, measured before and after oxygen binding,
were 1.5-3% (closed circles), 5.1-6.9%
(open circles), 5.4-7.7% (closed triangles), 8.6-9.8% (open triangles), and 12.8-15.6% (closed squares).
View larger version (10K):
[in a new window]
Fig. 4.
Stability of oxygenated and deoxygenated
samples of SNO-Hb. Spectral deconvolution assays to determine the
extent of S-nitrosation of Hb A0 were done with
aliquots withdrawn from oxygenated (closed circles) and deoxygenated (open circles) samples of partially S-nitrosated Hb
A0, 1 mM in heme, held at 4 °C in the dark.
The aliquots were brought to standard conditions for spectral
deconvolution assays as described under "Experimental Procedures"
and illustrated in Fig. 1. Squares denote samples subjected
to a second chromatography through Sephadex G-25 prior to spectral
analysis.
View larger version (26K):
[in a new window]
Fig. 5.
Effects of reduced glutathione (GSH) on
stability of SNO-Hb. The bar graphs show the
results of spectral deconvolution assays of the degree of
S-nitrosation of Hb samples before and after oxygen binding
studies in 0.05 M HEPES/DTPA buffer, pH 7.5, 20 °C with
5× GSH over heme and, in the third pair, in the same buffer with the
addition of DPG. Samples were assayed as in Fig. 4. The ratio of CysNO
relative to 2 mM Hb (in heme) and time of incubation
required to generate the varied levels of S-nitrosation are
indicated on the x axis (ratio, min).
93--
We determined the effects of S-nitrosation
on oxygen binding by purified Hb S in HEPES buffer containing EDTA or
DTPA and in the presence of a 50-fold excess of 2,3-diphosphoglycerate over tetramer concentration. No aggregation of Hb S was expected or
observed upon deoxygenation at the concentrations (60 µM
in heme) used in these experiments. Aggregation-dependent
effects that are evident at much higher protein concentration were not studied. The representative results presented in Fig.
6A are for approximately 80%
S-nitrosated Hb S and 0% S-nitrosated controls in chloride-free HEPES/EDTA buffer at pH 7.5. Fig. 6B shows
a similar pair in the presence of DPG. In these studies, both control and S-nitrosated samples of Hb S were more prone to
autoxidation than comparable samples of Hb A0. The sample
handling required for our studies made it difficult to obtain data with
low levels of metHb S, and many data sets were not useful due to high
metHb levels. The results shown are for control and
S-nitrosated forms of Hb S samples with relatively low
levels of metHb measured before (
9%) and after (15%) oxygen
binding.
View larger version (19K):
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Fig. 6.
Effects of S-nitrosation on
Hill plots of oxygen binding by Hb S. Purified 60 µM
samples of Hb S were in 0.05 M HEPES buffer containing 0.05 mM EDTA or 0.1 mM DTPA, pH 7.5, 20 °C. In
A, data are shown for unmodified Hb S (closed circles), and for Hb S with about 80% of the 93SH groups
nitrosated (open circles). MetHb levels, measured
before and after oxygen binding, were 5.9-8.3% and 8.3-15.3%,
respectively. In B, data obtained in the presence of a
50-fold excess of DPG over Hb tetramer are shown for unmodified Hb S
(closed circles) and for Hb S about 80%
S-nitrosated (open circles). MetHb
levels, measured before and after oxygen binding, were 5-6.2% and
8.4-12.2%, respectively.
logP10 > logP50), resulting in asymmetric shifts
away from the control data. The leftward shift of
logP10 is approximately the same in the presence
and absence of 2,3-diphosphoglycerate, resulting in equivalent
decreases in cooperativity to about two thirds of the normal values.
These results are very similar to those described above for
S-nitrosation of Hb A0.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
93 thus act in opposition to the well known allosteric effectors
of Hb function (protons, chloride, organic and inorganic phosphates,
bicarbonate, and carbon dioxide) that typically decrease oxygen
affinity and thereby enhance oxygen delivery to respiring tissues. For
SNO-Hb, as found for other SH-modified Hbs, it is probable that the
Cys-93 residues do not assume their normal position in the
deoxygenated molecule, with the consequence that formation of the salt
bridge between His-146 and Asp-94 is inhibited. This critical
salt bridge is oxygen-linked, and its pH-dependent
formation accounts for about half of the normal Bohr effect (11,
12).
93 residues in the oxy
(R-state) conformation compared with the deoxy (T-state) conformation
of Hb was invoked to account for the more facile S-nitrosation of liganded (R-state) Hb and the greater
stability of SNO-Hb in the liganded form (1). The results presented
here expand upon these earlier findings and illustrate that
deoxygenation alone is not sufficient to cause release of NO from
SNO-Hb. NO is accommodated in the altered deoxy structure of SNO-Hb,
making it possible to deoxygenate the protein without significant loss of NO. However, we show that the presence of glutathione destabilizes SNO-Hb and allows for NO transfer away from SNO-Hb during a cycle of
deoxygenation and re-oxygenation. In red cells, where thiols such as
GSH can serve as NO acceptors, the conformational sensitivity of SNO-Hb
would facilitate NO release upon deoxygenation. These findings are
relevant to the increased vasodilatory action reported for oxy SNO-Hb
in the presence of glutathione (1).
93 were modified by N-ethylmaleimide (11). This
regional control of oxygen affinity makes it possible for SNO-Hb forms
to exhibit varied oxygen affinities, dependent on the nature of the
parent Hb and its environment.
146 and Asp-94, which in turn decreases the
proximal-side pull on the heme-iron that normally confers T-state
character on deoxygenated Hb (11, 12, 29). It does not appear that SH
group modification by S-nitrosation appreciably alters the
chain anion-binding site in Hb A0 or in Hb S, since in
both proteins the magnitude of the 2,3-diphosphoglycerate effect is
equivalent for the normal and S-nitrosated forms. The similarity of responses of Hb A0 and Hb S to
S-nitrosation mirrors their structural similarities as shown
by x-ray crystallography (13).
93 in the altered deoxy state of
S-nitrosated Hb is relatively free of steric hindrance and
is thereby insensitive to anion-induced decreases in conformational fluctuations.
ACKNOWLEDGEMENT
Addendum
93 modification by other
SH-specific reagents. A difference in methodology is that we overcame
problems of metHb formation during exposure of Hb to CysNO, while they
compensated for this problem by use of a metHb reductase system after
SNO-Hb formation. There is, however, agreement that the SH group
modification with NO does not abolish the quaternary conformational
changes associated with oxygenation and deoxygenation that underlie
allosteric control of NO delivery from SNO-Hb.
FOOTNOTES
ABBREVIATIONS
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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