|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 277, Issue 31, 27818-27828, August 2, 2002
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From the
Received for publication, April 4, 2002, and in revised form, May 17, 2002
Our previous results run counter to the
hypothesis that S-nitrosohemoglobin (SNO-Hb) serves as an
in vivo reservoir for NO from which NO release is
allosterically linked to oxygen release. We show here that SNO-Hb
undergoes reductive decomposition in erythrocytes, whereas it is stable
in purified solutions and in erythrocyte lysates treated with an
oxidant such as ferricyanide. Using an extensively validated
methodology that eliminates background nitrite and stabilizes
erythrocyte S-nitrosothiols, we find the levels of SNO-Hb
in the basal human circulation, including red cell membrane fractions,
were 46 ± 17 nM in human arterial erythrocytes and
69 ± 11 nM in venous erythrocytes, incompatible with
the postulated reservoir function of SNO-Hb. Moreover, we performed
experiments on human red blood cells in which we elevated the levels of
SNO-Hb to 10,000 times the normal in vivo levels. The
elevated levels of intra-erythrocytic SNO-Hb fell rapidly, independent
of oxygen tension and hemoglobin saturation. Most of the NO released
during this process was oxidized to nitrate. A fraction (25%) was
exported as S-nitrosothiol, but this fraction was not
increased at low oxygen tensions that favor the deoxy (T-state)
conformation of Hb. Results of these studies show that, within the
redox-active erythrocyte environment, the Nitric oxide (NO) is a soluble gas that is continuously
synthesized in endothelial cells and is a critical endogenous
vasodilator (1-3). Although it is generally believed that
endothelium-derived NO is the primary determinant of NO-mediated
control of basal blood flow in humans (4, 5), considerable recent
interest and controversy have focused on the role of intravascular
NO-derived molecules that could stabilize NO bioactivity and contribute
to blood flow and oxygen delivery. Such molecules include high and low
molecular weight S-nitrosothiols in plasma (6-10) and
nitrite (4, 11). In addition, NO reacts reversibly with hemoglobin to
form an NO-heme adduct, iron-nitrosyl-hemoglobin
(HbFeIINO),1 and
can also nitrosate a surface thiol on cysteine 93 of the However, the evidence for a dynamic cycle is brought into question by
widely varying reports for the basal levels of intracellular SNO-Hb in
arterial and venous blood. The reported levels vary from 200 nM to 5 µM. These levels were determined
using a variety of different assays, mostly based on photolysis or
chemically mediated release of NO gas and subsequent detection by the
ozone-based chemiluminescent analyzer (8, 9, 12, 15). We have developed methodologies to selectively oxidize the NO first from
iron-nitrosyl-hemoglobin followed by reduction of the S-NO
bond from hemoglobin in solutions of I3 In the present studies we present a methodology for the stabilization
SNO-Hb in red cell lysates, the elimination of all background nitrite
signal, and the specific detection of SNO-Hb in human blood down to 5 nM concentration (0.00005% SNO/heme). Very low levels of
SNO-Hb were found in both arterial and venous erythrocytes with this
improved detection method, and no significant arterial-venous gradients
were observed. To evaluate the allosteric properties of SNO-Hb within
intact erythrocytes, we modified human red cells so that they contained
up to 10,000 times the normal in vivo levels of SNO-Hb and
evaluated the effects of hemoglobin oxygen saturation on its levels and
on NO export. In these experiments we found that, within the
redox-active erythrocyte environment, the Human Subjects--
All protocols were approved by the
Institutional Review Board of the NHLBI (National Institutes of Health,
Bethesda, MD) following formal scientific review. All subjects signed
an informed consent.
Materials--
All chemicals and NO gas, unless otherwise
stated, were obtained from Sigma-Aldrich. Hemolysates of cells
containing native human hemoglobin (Hb A0) were used to
prepare purified Hb by the ammonium sulfate method with chromatographic
purification with a fast protein liquid chromatography system. Pure red
cell lysates from freshly obtained venous or arterial blood were used
when indicated.
Synthesis of S-Nitrosothiol NO
Donors--
S-Nitrosothiol reagents were prepared by
reaction of the acidified amino acid with nitrite, neutralized to pH
7.4 using 5 N NaOH and measured by both the
I3 Synthesis of SNO-Hb--
Oxyhemoglobin was equilibrated in 2%
borate buffer at pH 9.2 or in PBS at pH 7.4, temperature 4 °C, and
then treated with S-nitrosocysteine at a ratio of 10:1
(CysNO to heme) for 30 min, followed by extensive dialysis or
Sephadex G25 desalting (8, 18, 23).
Synthesis of SNO-Albumin--
Human and bovine albumin (Sigma;
40 mg/ml with 0.5 µM EDTA in PBS) was reduced with
dithiothreitol (3-5 mM); passed through Sephadex G25
columns to remove dithiothreitol, nitrite, and small thiols; and
incubated with S-nitrosocysteine (final concentration at 2 mM) for 30 min at room temperature in the dark to form
SNO-albumin. This solution was then dialyzed at 4 °C against 3 × 3 liters of 0.5 µM diethylenetriaminepentaacetic acid
(DTPA) in PBS for 24 h.
Synthesis of SNO-Hb-containing Erythrocytes--
Whole blood was
collected from normal volunteers in EDTA followed by an overnight
incubation in room air with a 10% volume of 100 ml of
phosphate-buffered saline with citrate, dextrose, and adenine (CPD-A
solution; Baxter, Covina, CA). Blood samples were centrifuged at
750 × g for 5 min and adjusted to a 50% hematocrit by
removal of small amounts of plasma. To elevate intra-erythrocytic SNO-Hb levels, S-nitrosocysteine (10 mM final
concentration) was incubated with these whole blood samples for 3 h at 4 °C (on ice). To ensure the removal of all of the
S-nitrosocysteine, the cells were centrifuged, plasma and
buffy coat discarded, and cells then washed with a 25-fold excess
4 °C PBS for 5 min. The PBS was removed and the wash repeated for a
total of five 5-min washes. The final wash PBS was collected, and the
residual S-nitrosothiol in the wash was less than 50 nM. Red blood cells prepared by this methodology contained
1.1 ± 0.2% SNO/heme (220 µM intracellular SNO-Hb),
0% iron-nitrosyl-hemoglobin, and 8.7% methemoglobin.
Griess-Saville Assay for SNO-Hb and SNO-Albumin--
To measure
S-nitrosothiol and nitrite levels, standards of SNO-Hb or
SNO-albumin (prepared as described above) were added to 1.7-ml reaction
tubes cooled on ice. After cooling, 100 µl of
sulfanilamide/HgCl2 solution (16% sulfanilamide, 0.2%
HgCl2 in 2 M HCl), or sulfanilamide alone, was
added to the reaction mixtures followed by a balance of PBS/DTPA (100 µM) to make a final volume of 1100 µl. After 5 min of
incubation in the dark at room temperature, 100 µl of 1.6%
N-(1-naphthyl)ethenediamine in 2 M HCl was added
to the reaction mixture and again the sample was allowed to incubate in
the dark for 5 min. Following the second incubation, the reaction
mixtures were centrifuged at 17,900 × g for 5 min, and
1 ml of supernatants were transferred to optical cuvettes to measure
absorbance at 540 nm. Subtraction of appropriate blanks and of values
for samples treated without HgCl2 determined the levels of
S-nitrosothiol without nitrite. The concentration of
S-nitrosothiol or nitrite was calculated using the
extinction coefficient of 50,000 M Ozone-based Chemiluminescent Assays--
Different chemical
reagents are mixed in a glass purge vessel. Helium gas is bubbled
sequentially through the purge vessel, through 15 ml of 1 M
NaOH, and then into the chemiluminescent nitric oxide analyzer (model
280 NO analyzer; Sievers, Boulder, CO), which can detect
upward from 0.3 pmol of NO gas. The following chemicals were placed in
the purge vessel for the experiments described herein.
I3 Vanadium HCl Reagent--
Nitrate was measured by reduction in
vanadium(III) at 90 °C (24). To reduce foaming during the analysis
of nitrate levels in plasma, the samples were treated with a 2:1 volume
of cold ethanol and centrifuged at 17,900 × g for 5 min. The value for nitrate was determined by subtraction of the nitrite
and S-nitrosothiol values, derived by
I3 Reaction of NO-modified Hemoglobin with Acidified Sulfanilamide
to Eliminate Nitrite Contamination--
To eliminate nitrite
contamination from human hemoglobin samples, Sephadex G25 desalted
samples were reacted with and without 5 mM
HgCl2 and then treated with a 10% volume of 5%
sulfanilamide in 1 N HCl as described under "Results"
(10). These solutions completely abolished contaminating nitrite
signal, whereas purified SNO-Hb was completely stable. The levels of
SNO-Hb after acidified sulfanilamide treatment as measured by the
I3 Stabilization and Measurement of SNO-Hb in Erythrocytes with
Elimination of Background Nitrite Signal--
Red blood cell pellet
samples (100 µl) were lysed in 900 µl of PBS with
KCN/K3FeIII(CN)6 (4 mM), NEM (10 mM), DTPA (100 µM),
and 1% Nonidet P-40 (to solubilize membrane, which may contain
S-nitrosated anion exchange protein 1 (Ref. 25)). The pH of
this solution was 7.2. Lysed red blood cell samples were incubated in
this solution at room temperature for 5 min and 500 µl passed through
a prerinsed 9.5-ml bed volume Sephadex G25 column. The hemoglobin
aliquots from 3-3.7 ml were collected, separated into two fractions
(270 µl each), and reacted with and without HgCl2 (final
concentration 5 mM) for 2 min. 10% volume of 5%
sulfanilamide in 1 N HCl was added to the hemoglobin
solutions and incubated for 5 min (final concentration of 0.5%
sulfanilamide in 0.1 N HCl). 300-µl volumes of the
hemoglobin solutions, with and without HgCl2 treatment (to
specify S-nitrosothiol), were injected into 8 ml of
I3 Measurement of the Basal Levels of S-Nitrosohemoglobin in the
Human Circulation--
For the determination of the in vivo
levels of red blood cell SNO, whole blood was drawn from 8 normal
volunteers from the artery and vein into vacuum containers with sodium
heparin (to preserve physiological arterial and venous hemoglobin
oxygen saturations). Samples were spun at 750 × g for
5 min and plasma discarded. 100 µl of the red cell pellet below the
buffy coat was removed and immediately lysed in 900 µl of PBS with
1% Nonidet P-40, KCN/K3FeIII(CN)6
(4 mM), NEM (10 mM), and DTPA (100 µM). The pH of this solution was 7.2. Following Sephadex
G25 desalting, hemoglobin concentration was measured in Drabkin's
reagent, and samples were reacted with and without 5.0 mM
HgCl2 for 2 min, then with 10% volume of 5% sulfanilamide
in 1 N HCl for 3 min, followed by NO measurement by
I3 Stability and Oxygenation Studies of SNO-Hb-containing Red Blood
Cells--
Erythrocytes containing elevated levels of SNO-Hb were
prepared as described above. Only samples with less than 10%
methemoglobin were used. One ml of pre-agitated (to ensure mixing)
erythrocyte suspension was added to 9 ml of buffer in two tonometers.
As described under "Results," the tonometers alternatively
contained human plasma collected from the same individual who donated
the red cells, PBS with 24 mM NaHCO3 (distilled
water added to keep sodium concentration 140 mM), and PBS
with 24 mM NaHCO3 and 400 µM GSH. GSH was added as a thiol acceptor for potential erythrocyte-derived trans-nitrosation reactions (26). One tonometer was pre-equilibrated for 60 min with 21% oxygen and 5% CO2 gases and the
second tonometer with 0% oxygen and 5% CO2. The
tonometers were protected from light and agitated on a rocker platform.
Following the addition of cells, 1-ml samples were withdrawn every 5 min using Hamilton syringes. Samples were added to 8 mM NEM
and 100 µM DTPA to stabilize S-nitrosothiols
(10) and centrifuged at 750 × g for 5 min. The cells
and buffer were separated and flash-frozen on dry ice. The buffer
nitrite, nitrate, and S-nitrosothiol content and red cell SNO-Hb was subsequently measured by chemiluminescence (as described above). Samples were also withdrawn at the same 5-min intervals for
determination of hemoglobin saturation (by co-oximetry),
pO2, pCO2, and pH (i-STAT Corp., East Windsor,
NJ). Methemoglobin concentrations were measured by absorption
spectroscopy at 700, 630, 576, and 560 nm using the Winterbourn
relationship (27). Oxygen dissociation curve measurements of
SNO-Hb-containing red blood cells were performed using the
Hemox-Analyzer (TCS Scientific Corp., New Hope, PA) (28).
Statistical Analysis--
We have found that the Sievers
analytical software (model 280 NO analyzer) produces some error when
measuring the area under the curve for samples with 0.3-5 pmol of NO.
We therefore transferred raw data from the Sievers program to Origin
(Microcal Software, Inc., Northampton, MA). The data were smoothed
using the Savitzky-Golay filter method provided with the software
(symmetric, 21-point window; polynomial degree = 2). Two-tailed
paired t tests were used to analyze differences in the
values of paired samples before, during, and after experiments. For
time-course experiments, the data were analyzed by repeated measures
analysis of variance (ANOVA). Data are reported as the mean ± the
standard error of the mean.
Reduction of S-Nitrosothiols Results in Rapid and Complete Release
of NO from SNO-Hb in Solutions of I3 Stability of SNO-Hb in KCN and
K3FeIII(CN)6--
We have used
solutions of KCN and K3FeIII(CN)6
to selectively oxidize the NO from the heme while preserving the
S-nitrosothiol linkage. This sample treatment allows us to
first remove the NO bound to the heme group so that we can specifically
measure the NO release from cysteine 93 in the
I3 Stabilization and Protection of SNO-Hb in Red Cell Lysates and
under denaturing Conditions by Reaction with Solutions of
K3FeIII(CN)6 with and without
KCN--
Not only is SNO-Hb not degraded in solutions of
KCN/K3FeIII(CN)6, it appears that
the oxidation of hemoglobin and red blood cell lysates protects SNO-Hb
from degradation in red blood cell lysates and under denaturing
conditions. To illustrate the effects of denaturing conditions, such as
detergent treatment, on SNO-Hb stability, samples of purified SNO-Hb
were incubated in 1% SDS for 30 min with and without
KCN/K3FeIII(CN)6 pretreatment and
measured by the I3
Fig. 3 (B and C) shows results of additional
studies carried out to determine the stability of SNO-Hb in erythrocyte
lysates and the effects of oxidation (with
K3FeIII(CN)6), thiol sequestration
(with NEM), and copper chelation (with DTPA) on this stability. In
these studies purified SNO-Hb was added to fresh red blood cells lysed
with 1% Nonidet P-40 to give a final concentration of 0.02% SNO/heme.
This solution was incubated with 1)
KCN/K3FeIII(CN)6 with NEM and DTPA,
2) KCN/K3FeIII(CN)6 with NEM, 3)
KCN/K3FeIII(CN)6 alone, 4) NEM
alone, 5) DTPA alone, or 6) NEM and DTPA. The concentration of KCN (4 mM) used in these experiments had minimal effect on the pH
of the phosphate-buffered solution (see "Experimental Procedures").
Similar experiments were performed with
K3FeIII(CN)6 without KCN, as well
as with red blood cell lysates without added Nonidet P-40 (data not
shown). As shown in Fig. 3 (B and C), the
critical inclusion of
KCN/K3FeIII(CN)6 protects the
SNO-Hb from decomposition. Similar results were obtained using only
K3FeIII(CN)6 without KCN. Although
the critical reagent appears to be ferricyanide, an additional small
albeit nonsignificant effect of KCN, NEM, and DTPA on SNO-Hb
stabilization was observed consistent with the further inactivation of
redox active methemoglobin and CuI/thiol. The stability of
SNO-Hb in the presence of oxidants suggests that it is the reductive
environment of the red blood cell cytoplasm that renders SNO-Hb
unstable in vivo. Clearly, oxidation with ferricyanide of
materials in the cell lysate stabilizes added SNO-Hb. We conclude from
these studies that a reagent mixture containing
K3FeIII(CN)6 as a primary
ingredient, fine-tuned by addition of KCN, NEM, and DTPA, allows for
the preservation and measurement of SNO-Hb within the complicated
redox-active red cell environment.
Treatment of SNO-Hb with Acidified Sulfanilamide to Eliminate
Background Nitrite Signal--
It should be noted that the
concentrations of SNO-Hb in human erythrocytes as previously measured
by our laboratory are at the limits of detection (<200 nM
in whole blood) of our published protocol (5, 8). To precisely measure
concentrations lower than 100-200 nM (equivalent to
0.001-0.002% SNO/heme in whole blood), a technique was needed to
reduce the background signal, likely derived from nitrite. We
introduced such a technique by employing the first step of the classic
Griess assay, in which nitrite reacts with acidified sulfanilamide to
form a diazonium complex. Marley and colleagues (10) have demonstrated
that this reaction product has no signal in
I3
As shown in Fig. 4A, SNO-Hb is
stable in acidified sulfanilamide. Interestingly, even though our
purified SNO-Hb standards have been extensively Sephadex desalted and
dialyzed, these samples still have some nitrite contamination as shown
by a reduction in signal with acidified sulfanilamide treatment,
suggesting an anionic interaction of nitrite with hemoglobin (Fig.
4A). The levels of SNO-Hb measured by the
I3 Processing of Human Blood, Stabilization of SNO-Hb, and Elimination
of Background Nitrite for Ozone-based Chemiluminescent Detection of
S-Nitrosohemoglobin in the Human Circulation--
To specifically
measure the low concentrations of SNO-Hb present in the human
circulation, including membrane-bound S-nitrosothiols, we
developed and validated an assay to rapidly stabilize and selectively measure SNO-Hb in red blood cell lysates. For validation, standards of
purified SNO-Hb were mixed to a ratio of 0.0% to 0.02% mol of
SNO/heme (0-2,000 nM SNO in whole blood). These samples
were made with a freshly obtained, washed red blood cell pellet from human venous blood (which contains negligible levels of SNO-Hb (Ref.
12)). The cell pellet samples (100 µl) were lysed in 900 µl of PBS
with 1% Nonidet P-40,
KCN/K3FeIII(CN)6 (4 mM), NEM (10 mM), and DTPA (100 µM) and incubated in this solution for 5 min. As
described under "Experimental Procedures," the hemoglobin aliquots
containing varied levels of SNO-Hb were passed through a sizing column
and reacted with and without HgCl2, then with acidified
sulfanilamide. The data shown in Fig. 4 (B and C)
indicate that this assay procedure is specific for
S-nitrosothiol, has no nitrite-derived background signal,
and achieves a stoichiometric conversion of SNO-Hb to NO gas. The
improved assay is sensitive down to 0.00005% SNO/heme (5 nM SNO in whole blood).
For the determination of the in vivo levels of red blood
cell SNO, whole blood was drawn from 8 normal volunteers from the artery and vein directly into vacuum containers with sodium heparin (filling the vacuum containers ensures that the physiological hemoglobin oxygen saturations are preserved). The freshly drawn blood
was centrifuged and the red blood cell pellets were lysed directly in
the Nonidet
P-40/KCN/K3FeIII(CN)6/NEM/DTPA
mixture described above and under "Experimental Procedures."
Following Sephadex G25 desalting, samples were reacted with and without
5.0 mM HgCl2 (where the reduction in signal
with HgCl2treatment is that of authentic SNO-Hb) and with
acidified sulfanilamide (to eliminate nitrite-derived background), and
then analyzed by I3 Temperature Dependence of SNO-Hb Stability in Red Cells--
Red
blood cells (with less than 10% methemoglobin) with significantly
elevated SNO-Hb levels were synthesized as described under
"Experimental Procedures," extensively washed in PBS, and then
incubated in PBS for 4 h at either 4 or 37 °C. SNO-Hb levels were then measured by the I3 Effects of Oxygen Tension on Stability of Intra-erythrocytic SNO-Hb
and on NO Export--
To determine the effects of oxygen tension on
SNO-Hb stability in red blood cells and on NO export from these cells,
erythrocytes containing elevated SNO-Hb levels (prepared as described
previously) were incubated in a variety of normoxic and deoxygenated
buffers at 25 °C (Fig. 6). The levels
of S-nitrosated species in sample aliquots within and
outside of cells were periodically assayed. The overall oxygen affinity
of the SNO-Hb-containing erythrocytes was normal (~27 mm Hg at pH
7.4), consistent with the low overall percentage of
S-nitrosation. Similar rates of SNO-Hb decomposition occurred during the incubation of these SNO-Hb-containing cells (10%
hematocrit in PBS with 24 mM NaHCO3 and 400 µM GSH) during exposure to either 21% O2,
5% CO2, 74% N2 or 0% O2, 5%
CO2, 95% N2. Hemoglobin saturations in the
hypoxic tonometers dropped to 30 ± 0.04% (Fig. 6A)
and the mean pO2 at each time point was 48.7, 36.3, 27.5, and 19.3 mm Hg, consistent with a P50 of these red cells of
~30 mm Hg (the pH of the buffers equilibrated with 5% CO2 was 7.2, explaining the slightly higher P50
in this system). Consistent with the instability of SNO-Hb within the
red cell, described above, intra-erythrocytic SNO-Hb levels decreased
in a time-dependent fashion with similar rates of decrease
in both oxygenated and deoxygenated conditions (Fig. 6B).
Furthermore, the decomposition of SNO-Hb began immediately in the cells
exposed to deoxygenated buffers, prior to 50% hemoglobin
desaturation.
Nitric oxide export was assessed by measuring the extra-erythrocytic
NEM stabilized S-nitrosothiol, nitrite, and nitrate levels at selected time points during the process of SNO-Hb decomposition. S-Nitrosothiols were readily detectable in both cases, with
no greater export of NO for cells in hypoxic conditions as compared with normoxic conditions (Fig. 6C). Moreover, no significant
differences in extracellular nitrite and nitrate levels between
normoxic and hypoxic conditions were observed (Fig. 6, D and
E, respectively). The net NOx outside of
the erythrocytes was only slightly higher than the
measured decrease in SNO-Hb concentration, indicating that most but not
all of the NOx was derived from intracellular SNO-Hb. No significant
net effect of hypoxia on NOx export was observed (Fig. 6F).
Similar results to those shown in Fig. 6 were obtained with red blood
cells incubated in normal human plasma and in PBS without GSH
(n = 5 and n = 2, respectively) and at pH 6.5 (n = 3, data not shown).
Measured levels of S-nitrosated hemoglobin and plasma
albumin in the human circulation vary enormously in the published
literature from 50 nM to 2.5 µM and from 30 nM to 7 µM, respectively (4, 6, 8-10, 12,
29, 30). An understanding of the true levels of S-nitrosated
proteins in human blood will help clarify their role in blood flow
regulation. There exist three significant obstacles to the accurate
measurement of these species: 1) the levels of nitrite in blood and in
reagent solutions and labware (4, 10, 31), 2) the instability of SNO-Hb
in the presence of millimolar glutathione (which will be present within
the erythrocyte) (16, 18, 19, 21), and 3) the difficulty of
distinguishing NO derived from two distinct sites on hemoglobin, the
heme group and We have previously reported (and further extensively validate here)
that I3 The analysis of SNO-Hb in red blood cells lysates, and presumably its
stability in red cells under physiological conditions, is further
limited by the rapid decomposition of the S-nitroso linkage
by intracellular reductants, such as glutathione, which is normally
present at 5 mM concentration in red blood cells. As is
clearly demonstrated in the experiments presented here, the decrease of
SNO-Hb levels occurs in oxygenated red blood cell lysates (equilibrated
with room air) as rapidly as in hypoxic conditions. Although this
observation is consistent with the published oxygen-independent
decrease of SNO-Hb levels and associated vasoactivity in the presence
of millimolar concentrations of glutathione (16, 20, 21), there had
been no consideration of how this would effect intra-erythrocytic
SNO-Hb viability. In fact, studies of the cytosolic thiol-disulfide
equilibrium in all cells, and in particular the erythrocyte, show this
environment to be reducing with rare formation of disulfide bonds
(32-34). Such conditions should prohibit the oxidation of We therefore hypothesized that oxidation of the heme and ligation of
intracellular thiols might stabilize the S-nitrosothiol linkage in the red blood cell. Indeed, we found that SNO-Hb can be
stabilized by reaction with ferricyanide (with or without added cyanide) in both SDS, in red blood cell lysates, and in the mass spectrometry fragmentor (the latter data not shown). Treatment of red
blood cell lysates with ferricyanide was the critical step for SNO-Hb
stabilization; treatment with copper chelators and thiol blocking
reagents alone did not prevent the rapid decomposition of the
S-nitroso linkage observed in erythrocyte lysates. Although ferricyanide is the critical reagent necessary for SNO-Hb
stabilization, we include CN to form redox-inactive cyanomethemoglobin,
NEM to block free thiols and prevent ex vivo SNO-Hb
formation, DTPA to chelate copper that may potentially contaminate
labware and catalyze decomposition of S-nitroso linkages,
and 1% Nonidet P-40 to solubilize the red cell membrane to include the
detection of S-nitrosated membrane proteins. We provide
extensive validation that these reagents stabilize SNO-Hb within red
blood cells and lysates. In fact, with the hemoglobin converted to
cyanomethemoglobin, SNO-Hb can be denatured, digested for fragment
analysis, acidified, or alkalinized with minimal disruption of the
S-nitrosothiol linkage.
The basal levels of red cell S-nitrosothiol (SNO-Hb and/or
other S-nitrosated proteins) in arterial and venous blood
(carefully collected to control the physiological hemoglobin oxygen
saturations) were found to be 46 and 69 nM, respectively,
and are substantially lower than those previously published (8, 12). We
believe this reflects the elimination of nitrite contamination, which produces a signal with both I3 The low levels of SNO-Hb found in vivo and the lack of
dynamic artery-to-vein gradients of red blood cell
S-nitrosothiol are consistent with low level NO
auto-oxidation reactions rather than complex intramolecular
redox-dependent NO transfers. The NO auto-oxidation reaction (forming N2O3) will produce both
nitrite and S-nitrosothiols, particularly in lipid membrane
compartments where the relative hydrophobicity of NO would increase its
local concentration (35). Slowed diffusion of NO across the erythrocyte
membrane and the submembrane protein matrix reduces the rate of
reaction between Hb(O2) and NO, possibly promoting
S-nitrosation reactions within the "protected" red blood
cell membrane (36). Such formation of membrane
S-nitrosothiols does not require a
hemoglobin-derived allosteric function but rather a temporal and
spatial promotion of the NO-autooxidation reaction, which could be
driven by regional NO and oxygen concentrations.
In unmodified human hemoglobin, the environment of Allosteric coupled release of NO and oxygen from SNO-Hb has been
suggested based on the thermodynamic linkage between hemoglobin (heme)
oxygenation and S-nitrosation of Previous experiments designed to test the role of SNO-Hb as a reservoir
for bioactive NO have evaluated the effects of cell-free SNO-Hb
solutions on aortic ring vasodilation (12). Such experiments are
difficult to perform with isolated hemoglobin, particularly S-nitrosated hemoglobin, because of the very high oxygen
affinity of cell-free hemoglobin (P50 of 5-7 mm Hg and <4
mm Hg for SNO-Hb; Refs. 16, 18, and 19) and its tendency to dissociate
into two NO released from In conclusion, we show herein that SNO-Hb is intrinsically unstable in
the reductive erythrocytic environment, but that ferricyanide treatment
of lysates offsets this reductive decomposition of SNO-Hb. Consistent
with the observed instability and lack of allosteric NO/O2-linked behavior of SNO-Hb in red blood cells, we
found that SNO-Hb levels in human blood are in the low nanomolar range,
much lower than previously reported, without significant
arterial-venous gradients. These results suggest that, within the
redox-active erythrocyte environment, We gratefully acknowledge Drs. Rakesh Patel,
Neil Hogg, Daniel Kim-Shapiro, and Jose Tanus-Santos for critical
review and helpful suggestions.
*
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 all correspondence and reprint requests
should be addressed: Warren G. Magnuson Clinical Center, Critical Care
Medicine Dept., Bldg. 10, Rm. 7D43, 10 Center Dr., MSC 1662, Bethesda, MD 20892-1662. Tel.: 301-496-9320; Fax: 301-402-1213; E-mail: mgladwin@nih.gov.
Published, JBC Papers in Press, May 21, 2002, DOI 10.1074/jbc.M203236200
2
C. Ho, personal communication.
3
R. P. Patel, personal communication.
The abbreviations used are:
HbFeIINO, iron-nitrosyl-hemoglobin;
SNO, S-nitroso;
Hb, hemoglobin;
NEM, N-ethylmaleimide;
PBS, phosphate-buffered saline;
ANOVA, analysis of variance;
NOx, nitrite and nitrate;
DTPA, diethylenetriaminepentaacetic
acid.
S-Nitrosohemoglobin Is Unstable in the Reductive
Erythrocyte Environment and Lacks O2/NO-linked Allosteric
Function*
§¶,§,§,,§,
, and Critical Care Medicine Department, Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda,
Maryland 20892, the § Laboratory of Chemical Biology,
NIDDK, National Institutes of Health, Bethesda, Maryland 20892, and the Marine/Freshwater Biomedical Center, Duke University,
Beaufort, North Carolina 28516
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-globin cysteine 93 is
maintained in a reduced state, necessary for normal oxygen
affinity, and incapable of oxygen-linked NO storage and delivery.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-globin
chain to form S-nitrosohemoglobin (SNO-Hb). The potential role of S-nitrosated hemoglobin as an NO transporter is
particularly appealing, because the environment of -cysteine 93 is
sensitive to the R 
T conformational equilibrium of hemoglobin. The
conformational transition from the R- to T-state could thus promote the
allosteric delivery of both oxygen and NO to regions with low oxygen
tension. Two central observations supporting this SNO-Hb hypothesis are reports of observed arterial-venous gradients of SNO-Hb in the rat
(suggesting a dynamic cycle) and evidence that delivery of oxygen and
NO are allosterically coupled events (12-14).
,
releasing NO gas from the cysteine for ozone-based chemiluminescent detection (4, 5, 8), whereas other laboratories first cleave the
S-NO linkage with mercury and then measure the NO levels (with and without mercury) using ultraviolet light photolysis (12, 16).
In addition to liberating NO gas from both S-nitrosothiols and iron-nitrosyls, both systems reduce nitrite to NO, requiring extensive treatment of samples through molecular sizing columns. However, hemoglobin possesses anion binding sites that may retain nitrite, raising concerns that the measured NO levels may be
overestimated as a result of conversion of hemoglobin-bound nitrite to
NO (17). In addition, there have been a number of challenges to the
second core principle of the SNO-Hb hypothesis, that NO is released
during the oxygen-linked conformational shift of hemoglobin from its R-
to T-state. Although both kinetic and thermodynamic arguments have been
made to support the allosterically mediated release of NO from SNO-Hb
(12, 14, 16), the physiological relevance of this possible linkage has
been challenged on the basis of the very high oxygen affinity of
SNO-Hb, potentially limiting its role in basal regulation of NO/oxygen
delivery (18-20), and the oxygen-independent kinetics of the reaction
of SNO-Hb with millimolar levels of glutathione, present at such
concentration in erythrocytes (16, 19-21).
-globin cysteine 93 is
maintained in a reduced state, which may be necessary for normal oxygen
affinity, and that SNO-Hb is rapidly degraded independent of hemoglobin
oxygen saturation. The reductive decomposition of SNO-Hb in the
erythrocytes provides an explanation for why SNO-Hb levels in human
blood are low. Because the lifetime of SNO-Hb in erythrocytes is both
transient and insensitive to oxygen tension, its participation in NO
storage, delivery, and regulation of human blood flow under normal
physiological conditions appears unlikely. It remains possible that
SNO-Hb may participate in NO-dependent events in
vivo during pathological conditions associated with red cell
oxidation or under circumstances where NO generation is increased in
response to infection or pharmacological NO treatment.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(22) and
Cu+/L-cysteine (7) chemiluminescence assays,
and compared with SNO-glutathione (Calbiochem) and nitrite standards.
1
cm1 and with standards of known concentrations of GSNO.
Reagent--
The method for the
measurement of nitrite and S-nitrosothiols by reaction with
I3 to release NO gas (22) was applied to
hemoglobin (8). In brief, 7 ml of glacial acetic acid and 2 ml of
distilled water were mixed with 100 mg of KI. A crystal of
I2 was added to yield a concentration of ~20-30
mM. Alternatively, to allow for the injection of large
volumes of solutions with high protein concentration, a stock solution
of I3 was prepared each day. Serial
injections of S-nitrosoglutathione and nitrite were used
over 4 h to document the stability of the I3 stock solution exposed to air. There was
no decrease in NO release from S-nitrosothiols over this
time period. This allowed us to change the reagent prior to each
injection of hemoglobin solutions and removed variability produced by
increasing protein concentrations and foaming. To specifically identify
nitrite versus S-nitrosothiol, samples were
incubated with and without acidified sulfanilamide (as described below)
and reacted in the I3 reductant. Specific
treatment of red cell and hemoglobin samples prior to reaction in the
I3 reductant is described below.
assay, from the result derived from
vanadium assay.
chemiluminescent assay were the same as
the levels measured by the Griess-Saville assay after subtraction of
the nitrite background, suggesting that some nitrite is associated with
preparations of SNO-Hb even after extensive dialysis or column separation.
solution in the reaction chamber (see
"I3 Reagent"). The
I3 was replaced for each injection from a
stock solution prepared fresh each day and no antifoam agent was used.
The quantity of NO released was determined by calculation of the area
under the curve (see "Statistical Analysis"), corrected for the
dilution of added HgCl2 and acidified sulfanilamide, and
the value divided by the concentration of heme measured
spectrally in Drabkin's reagent (prior to addition of
HgCl2 and acidified sulfanilamide).
chemiluminescent assay.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
--
To
validate our standards and improved chemiluminescent methodologies,
preparations of SNO-Hb and SNO-albumin were analyzed by both the
I3 chemiluminescent assay and by the
Griess-Saville assay. As shown in Fig. 1
(A and B), the I3
chemiluminescent assay compared favorably with the well established Griess-Saville assay over a wide range of concentrations for both SNO-Hb and SNO-albumin. To determine the limits of sensitivity, purified samples of synthesized SNO-Hb were added to freshly drawn, washed (in PBS), lysed erythrocyte pellets at concentrations from 200 nM (0.002% S-NO/heme) to 3,200 nM
(0.032% S-NO/heme). Following treatment with ferricyanide
and cyanide (described below) to eliminate iron-nitrosyl signal and to
stabilize SNO-Hb in red blood cell lysates, the hemoglobin samples were
passed through a prerinsed G25 Sephadex sizing column and injected into
the I3 reagent. The NO recovery was
stoichiometric with an r2 value of 0.996 (Fig.
1, C and D; p < 0.001;
n = 4). Levels of SNO-Hb measured by other laboratories
using UV photolysis have been reported to be as high as
0.001/hemoglobin tetramer or 2.5 µM in whole blood. As
can be seen in Fig. 1 (C and D), such levels would be readily measured using our assay.
View larger version (18K):
[in a new window]
Fig. 1.
Reduction of S-nitrosothiols
result in rapid and complete release of NO from
S-nitrosohemoglobin in solutions of
I3 . Preparations
of SNO-Hb (panel A) and SNO-albumin
(panel B) analyzed by
I3 chemiluminescent assay (closed
circles) and by the Griess-Saville assay (open
squares). Panel C, SNO-Hb standards in
lysed red cell pellet at concentrations from 200 nM
(0.002% S-NO/heme) to 3,200 nM (0.032%
S-NO/heme); n = 4. Panel
D, a representative set of nitric oxide analyzer readouts
from one experiment is shown.
chemiluminescence-based assay. The
standards of SNO-Hb shown in Fig. 1 (C and D)
were pretreated with
KCN/K3FeIII(CN)6 prior to reaction
in the I3 chemiluminescence-based assay (8).
SNO-Hb is stable in a large molar excess of KCN and
K3FeIII(CN)6. This was shown using
three approaches. In the first experiment (Fig.
2A), 10-µl volumes of
solutions containing SNO-Hb (0.9 mM SNO), nitrite (10 mM), nitrate (10 mM), and HbFeIINO
(0.67 mM) were injected into a purge vessel containing 0.2 M KCN/K3FeIII(CN)6 in
PBS. The reaction chamber was purged with helium in-line with the
chemiluminescent NO analyzer to detect released NO gas. As shown in
Fig. 2A, SNO-Hb, nitrite, and nitrate are stable in this
solution (i.e. do not release appreciable amounts of NO), whereas NO gas is released from HbFeIINO. The small NO
signal from the injection of SNO-Hb derives from small amounts of
iron-nitrosyl-hemoglobin formed in the synthesis of SNO-Hb (see small
mercury-stable peak in Fig. 2B). In the second set of
experiments, SNO-Hb was measured using the I3
chemiluminescence-based assay with and without cyanide/ferricyanide and
HgCl2 pretreatments. Briefly, 10 µl of 0.9 mM
SNO-Hb was incubated for 30 min in 90 µl of PBS, 90 µl of 0.2 M KCN/K3FeIII(CN)6 in
PBS, or 90 µl of 0.5 mM HgCl2 and then passed
through a Sephadex G25 sizing column. The samples were then injected
into a reaction vessel containing the I3
reagent. The reaction chamber was purged with helium in-line with the
chemiluminescent NO analyzer to detect released NO gas. As shown in
Fig. 2B, there is no loss of SNO-Hb that has been incubated
in a large molar excess of
KCN/K3FeIII(CN)6, in the absence of
HgCl2, whereas it is completely decomposed by incubation
with HgCl2. In the final experiment, SNO-Hb was measured
using the Griess-Saville assay, incubated for 2 h in PBS, 0.2 M K3FeIII(CN)6 or 0.2 M KCN/K3FeIII(CN)6,
passed through a Sephadex G25 sizing column to remove CN (which
interferes with the Saville assay), and then measured again by the
Saville assay (Fig. 2C). The slight reduction in SNO-Hb signal (5.7%) with KCN is a result of KCN interference with the Saville methodology, which is largely prevented by passing samples through sizing columns and is not observed with the
I3 chemiluminescence-based assay.
View larger version (11K):
[in a new window]
Fig. 2.
Stability of SNO-Hb in
KCN/K3FeIII(CN)6.
Panel A, standard samples of SNO-Hb, nitrite,
nitrate, and HbFeIINO were injected into a purge vessel with 0.2 M
KCN/K3FeIII(CN)6 in PBS. The
reaction chamber was purged with helium in-line with the
chemiluminescent NO analyzer to detect released NO gas.
Panel B, SNO-Hb was incubated for 30 min in PBS,
0.2 M KCN/K3FeIII(CN)6
in PBS, or 0.5 mM HgCl2 and then passed through
a Sephadex G25 sizing column. The samples were then injected into a
reaction vessel containing the I3 reagent
in-line with the chemiluminescent NO analyzer to detect released NO
gas. Panel C, SNO-Hb was measured using the
Saville assay, incubated for 2 h in 0.2 M
K3FeIII(CN)6 and 0.2 M
KCN/K3FeIII(CN)6, passed through a
Sephadex G25 sizing column to remove
KCN/K3FeIII(CN)6, and then measured
again by the Saville assay.
chemiluminescent assay. As
shown in Fig. 3A, SNO-Hb
levels are decreased by exposure to the denaturing effects of SDS, an
effect that is prevented by oxidation of the hemoglobin. Although we have used a mixture of K3FeIII(CN)6
and KCN to discriminate between NO at heme and SH groups (by
eliminating signals from NO liganded to the heme group), it is apparent
from these experiments that the critical reagent required to stabilize
the S-nitrosothiol bond on hemoglobin is
K3FeIII(CN)6. Similar results are
observed in the mass spectrometry fragmentor, where SNO-Hb is more
stable in this oxidizing environment than in its absence (spectra
obtained at 30 HPLC electrospray ionization mass spectrometry; data not
shown).
View larger version (13K):
[in a new window]
Fig. 3.
Stability of SNO-Hb under denaturing
(detergent treatment) conditions and in red cell lysates.
Panel A, standards of SNO-Hb were incubated for 30 min in 1% SDS and SDS/NEM with and
without K3FeIII(CN)6 or
KCN/K3FeIII(CN)6 pretreatment.
Panel B, electrical signal from NO analyzer when
SNO-Hb was incubated in fresh red blood cell lysates to a final
concentration of 0.02% SNO/heme with 1)
KCN/K3FeIII(CN)6 with NEM and DTPA,
2) KCN/K3FeIII(CN)6 with NEM, 3)
KCN/K3FeIII(CN)6 alone, 4) NEM
alone, 5) DTPA alone, or 6) NEM and DTPA. The decrease in signal with
HgCl2 pretreatment indicates SNO-Hb specificity.
Panel C, mean values and S.E. for three
experiments as shown in panel B.
-based chemiluminescent assays (10). To
determine whether acidified sulfanilamide can be used to eliminate
background nitrite signal from SNO-Hb, solutions of purified SNO-Hb
were incubated with and without a molar excess of HgCl2,
treated with NEM to block free thiol groups, reacted with acidified
sulfanilamide; the SNO-Hb in these treatments was then measured by
I3 chemiluminescent assay.
methodology, after treatment with
acidified sulfanilamide, are in agreement with measured levels by the
Griess-Saville methodology after nitrite subtraction. Although our
reagent mixture includes NEM to block free thiols, we found that the
reaction of acidified nitrite with sulfanilamide is sufficiently fast
that no S-nitrosothiol formation occurs in this reaction
even without NEM pretreatment (Fig. 4A). In additional
experiments purified SNO-Hb was incubated with a millimolar excess of
nitrite and then treated with acidified sulfanilamide. The nitrite
signal was completely eliminated, whereas the SNO-Hb signal was intact
as measured by I3 chemiluminescent assay,
suggesting that no artifactual S-nitrosation occurs
following the addition of acid in the presence of sulfanilamide (data
not shown). Finally, pretreatment of SNO-Hb with a molar excess of
HgCl2 converts the SNO-Hb to nitrite, which is completely eliminated by treatment with acidified sulfanilamide. The elimination of nitrite from SNO-Hb, with and without mercury pretreatment, thus
creates a zero-background assay for maximal sensitivity and specificity
in measurements of SNO-Hb in vivo.
View larger version (11K):
[in a new window]
Fig. 4.
Stability of SNO-Hb in solutions of acidified
sulfanilamide and/or mercury allow for the specific and sensitive
detection of SNO-Hb in red cells with no background.
Panel A, SNO-Hb standards were incubated with
acidified sulfanilamide, with and with- out NEM, HgCl2 or both, and measured by the
I3 chemiluminescent assay. Panel
B, the electrical signal from the NO analyzer obtained from
standards of SNO-Hb mixed in red cell lysates. The red cell pellet
samples were lysed in PBS with 1% Nonidet P-40,
KCN/K3FeIII(CN)6, NEM, and DTPA (pH
7.2), passed through a sizing column, reacted with and without
HgCl2 for 2 min, followed by reaction with acidified
sulfanilamide. 300 µl of the desalted hemoglobin solution was then
injected into I3 solution in the reaction
chamber in-line with the chemiluminescent NO analyzer to detect
released NO gas. Panel C, this assay had no
background signal following mercury treatment, achieved a
stoichiometric conversion of SNO-Hb to NO gas, and was sensitive down
to 0.00005% SNO/heme (5 nM SNO/heme in whole blood).
chemiluminescent assay.
The results are shown in Fig. 5. The levels of SNO-Hb in the basal human circulation, including red cell
membrane fractions, were 46 ± 17 nM (0.00046%
SNO/heme) in human arterial erythrocytes and 69 ± 11 nM (0.00069% SNO/heme) in venous erythrocytes.
View larger version (17K):
[in a new window]
Fig. 5.
Arterial and venous red blood cell
S-nitrosothiol levels (SNO-Hb and membrane SNO
compounds) in the human circulation. Panel
A, NO release (in mV) following the injection of 300 µl of
red cell lysates treated with Nonidet P-40,
KCN/K3FeIII(CN)6, NEM, and DTPA;
lysates were from arterial and venous blood obtained from 8 normal
volunteers as described under "Experimental Procedures."
Panel B, mean of data ± S.E. for arterial
and venous red blood cell total S-nitrosothiol levels in 8 normal volunteers.
-based
chemiluminescent assay (using the protocols described above to
stabilize and measure the SNO-Hb). SNO-Hb levels in the erythrocytes in
this series of experiments (2.72 ± 1.72% SNO/heme) were stable at 4 °C for the entire 4 h, whereas levels dropped to 0.17 ± 0.01% SNO/heme after 1 h and to zero at 2, 3, and 4 h at
37 °C (n = 3; p < 0.01 for 4 °C
versus 37 °C by repeated measures ANOVA). The stability
of SNO-Hb in these cells was then evaluated at 30 and 60 min at 25 and
37 °C. At 25 °C the SNO-Hb levels dropped from 1.48 ± 0.17% to 1.17 ± 0.20% at 30 min and 0.87 ± 0.30% SNO/heme at 60 min. SNO-Hb was less stable at 37 °C,
dropping to 0.22 ± 0.04% SNO/heme at 60 min (n = 5; p < 0.05 for 25 °C versus 37 °C at
60 min by repeated measures ANOVA). The instability at 37 °C is
consistent with reductive decomposition of S-nitrosothiol in
erythrocyte lysates and the low levels found in vivo.
Purified SNO-Hb is stable outside the cell at similar temperatures, or in lysates containing our oxidative mixture, strongly suggesting that
intracellular reductants are responsible for decomposing the
S-nitroso linkage at physiological temperatures.
Additionally, solution studies revealed that the red blood cell
reductant NADPH greatly decreases the lifetime of both
S-nitrosocysteine and SNO-Hb (data not shown). The stability
of SNO-Hb in red blood cells maintained at 4 °C allows for extensive
washing of the cells at low temperature, to remove excess
S-nitrosocysteine, for subsequent oxygenation studies of red
blood cells containing SNO-Hb presented in the following section.
View larger version (28K):
[in a new window]
Fig. 6.
Effects of hypoxia on the stability of
chemically produced SNO-Hb-containing human erythrocytes and on NO
export. Red cells were incubated in tonometers at 10% hematocrit
with 21% oxygen and 0% oxygen as described under "Experimental
Procedures." Panel A, hemoglobin saturations in
normoxia (solid line) and hypoxia
(dashed line). Panel B,
SNO-Hb levels in normoxia (solid line) and
hypoxia (dashed line). Panels
C-E, S-nitrosothiol, nitrite, and nitrate export
in normoxia (solid lines) and hypoxia
(dashed lines). Panel F,
total SNO-Hb degradation versus NOx formation during
normoxia (open bars) and hypoxia
(hatched bars).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cysteine 93. In the present work, we have addressed
all three obstacles and present an improved strategy for SNO-Hb
measurement, its validation, and results of the methodologies employed,
which help establish the true levels of S-nitrosated species
in human erythrocytes.
will rapidly release NO gas from both
SNO-Hb and HbFeIINO (8). I3 will
also reduce nitrite (but not nitrate) to NO gas. A similar assay has
been developed for the measurement of plasma S-nitrosothiols using KI/CuII/acetic acid to generate I3 and
has similar sensitivity and specificity (10). To eliminate the
micromolar nitrite background present in plasma, which has contributed
to an overestimation of the levels of SNO-albumin, these authors first
treated plasma samples with acidified sulfanilamide, recapitulating the
first step of the classic Griess reaction to form a diazonium complex,
which does not release NO gas in the I3
reductant. Akin to removing the haystack to find the needle, such
pretreatment eliminates the micromolar nitrite background and allows
for accurate measurement of the low nanomolar concentrations of plasma
S-nitrosothiols. Nitrite, which is reduced to NO by both the
I3- and UV photolysis-based chemiluminescent
methodologies, can contaminate hemoglobin by binding to anionic binding
cavities, which prevents its separation from hemoglobin on Sephadex G25 sizing columns (17). Therefore, we hypothesized that published levels
of SNO-Hb might be overestimated, and evaluated the effects of nitrite
elimination by acid-sulfanilamide treatment of SNO-Hb and of red blood
cell solutions containing SNO-Hb. We found that SNO-Hb was stable in
this solution and that nitrite-derived signals were completely
eliminated, with no artifactual S-nitrosothiol formation.
Because HgCl2 converts S-nitrosothiol, but not
iron-nitrosyl derivatives, to nitrite, this treatment can be used prior
to addition of acidified sulfanilamide to discriminate between
S-nitrosothiols and other sources of NO (i.e. for
specific determination of whether the NO measured by
I3 is derived from SNO-Hb or from
iron-nitrosylhemoglobin).
-cysteine
93 by NO+ to form SNO-Hb and limit the storage of NO in this form.
reduction and
with ultraviolet light photolysis. The lower levels of SNO-Hb reported
here are consistent with 1) a reappraisal of the levels of plasma
S-nitroso-albumin (4, 6, 9, 10, 29, 30) 2) data that show
SNO-Hb to be unstable in solutions of millimolar reduced glutathione
and in red blood cell lysates (16, 19-21), and 3) data showing that
SNO-albumin is unstable in the presence of reduced thiols (9, 10).
-cysteine 93 is
greatly altered by the transition between the oxy (R-state) conformation and the deoxy (T-state) conformation. This oxygen-linked conformational change is the basis for the large difference in rate of
SNO-Hb formation in oxy and deoxy conditions (12). This oxygen-linked
conformational change has other consequences, such as the increase in
oxygen affinity that occurs when ligands (sulfhydryl reagents) bind to
-cysteine 93. The increased oxygen affinity results from disruption
of the important salt bridge from -aspartate 94 to -histidine 146 that normally stabilizes the T-state (37-39). Such ligands include
many thiol compounds, NEM, and NO (16, 18, 19, 40, 41). Similarly,
amino acid substitutions at -cysteine 93 can lead to increased
oxygen affinity, suggesting that a reduced cysteine residue at this
position is critical for normal allosteric control of oxygen binding
and release from hemoglobin (42,
43).2 Such a conserved
function would not invoke an NO transport mechanism. In fact, robust
in vivo mechanisms to remove -cysteine 93 ligands by
reduction would be necessary to maintain normalcy of physiological oxygen binding and release. The existence of such mechanisms is supported by our observations of rapid SNO-Hb degradation in
erythrocytes and the limited S-nitrosation and
S-thiolation (44) of this residue we observe in
vivo.
-cysteine 93 (12-14, 16, 18, 19). A kinetic mechanism to allow physiological linkage was
initially postulated to involve an O2-sensitive
trans-nitrosation reaction between SNO-Hb and glutathione forming
S-nitrosoglutathione (12, 16). However, such mechanisms do
not consider all the reactions between SNO-Hb and intra-erythrocytic
reductants such as NADPH, glutathione (in mM
concentration), glutathione reductase, superoxide dismutase, and other
reducing components. Additionally, the kinetics of trans-nitrosation
between SNO-Hb and GSH apparently conform to a second order reversible
reaction with a rate constant that is not sensitive to the oxygenation
state of SNO-Hb (19). We hypothesize that intra-erythrocytic reductants
greatly limit the build-up of SNO-Hb in red blood cells. This
hypothesis and the data supporting it reported herein run counter to
the postulated role of SNO-Hb as a reservoir for bioactive NO.
chain dimers, which lack cooperative behavior. The low oxygen tensions required to deoxygenate cell free hemoglobin are known
to alter the vasodilatory profile of S-nitrosothiols on vessel preparations, potentially creating artifactual evidence for the
oxygen-linked delivery of NO from SNO-Hb
(20).3 To circumvent these
problems, we developed a methodology to elevate the levels of SNO-Hb in
red blood cells without formation of nitrosylated heme groups or large
concentrations of methemoglobin. We made use of these erythrocytes with
appreciably elevated SNO-Hb levels as physiological models for the
measurement of SNO-Hb stability at normal and reduced oxygen tensions.
These models are appealing because intracellular hemoglobin is almost
entirely tetrameric and in equilibrium with 2,3-diphosphoglycerate,
resulting in a normal oxygen affinity and facile deoxygenation under
hypoxia. To avoid the confounding effects of oxygen tension on blood
vessel preparations, we directly measured SNO-Hb decay and NO export in
these model red blood cells. In these experiments the concentrations of
intra-erythrocytic SNO-Hb decrease independent of oxygen tension and
can be successfully fit to a single exponential decay equation. We
believe this is consistent with pseudo-first order kinetics controlled
by limiting amounts of SNO-Hb and excess reductant, with reduced
S-nitrosothiol forming NO that is largely oxidized to
nitrate. As we have not measured the reaction kinetics at multiple SNO-Hb concentrations and cell lysate concentrations, we cannot distinguish between mechanisms.
-cysteine 93 would form four potential
reaction products that can be measured: iron-nitrosyl-hemoglobin, S-nitrosothiol, nitrate, and nitrite. No significant
formation of iron-nitrosyl-hemoglobin was observable spectrally or by
chemiluminescence, and ~75% of the NO released from SNO-Hb was
oxidized to nitrate, without appreciable oxygen dependence, consistent
with a reaction of NO with residual oxyhemoglobin within the red blood
cell. The second reaction would require trans-nitrosation reactions
with reduced glutathione or membrane protein thiols. Such an event has
been proposed by Gaston and colleagues (26), who described an increase
in S-nitrosoglutathione following incubation of rat venous
blood at low oxygen tensions with 400 µM glutathione and no increase with incubations with arterial blood at higher oxygen tensions. We evaluated this mode of release by incubating the SNO-Hb-containing red blood cells with 400 µM
glutathione. Although significant extracellular accumulation of
S-nitrosothiol was observed, there was no observed oxygen
dependence. In fact the levels were slightly higher under oxygenated
conditions, suggesting that some of the S-nitrosothiol
formed is via the NO auto-oxidation reaction (N2O3 pathway) rather than pure
trans-nitrosation. These experiments are consistent with effective
trans-nitrosation-mediated and low molecular weight
S-nitrosothiol diffusion-mediated transport of NO into and
out of erythrocytes. However, the appearance of extracellular S-nitrosothiols and the decomposition of intracellular
SNO-Hb have time courses that are largely independent of oxygen
tension. The observed export of S-nitrosothiols, which are
known to be vasoactive, is consistent with observations that NO-treated
red blood cells can dilate blood vessels (25). However, evidence reported herein suggests that the oxygen dependent vasoactivity reported by other laboratories is likely secondary to hypoxic effects
on vessel responsiveness, rather than on the proposed allosteric
linkage of oxygen and NO release from SNO-Hb.
-cysteine 93 is maintained in
a reduced state, allowing for normal allosteric control of oxygen
binding and release. Although trans-nitrosation reactions can pass NO into and out of erythrocytes, the reductive intra-erythrocytic environment decomposes SNO-Hb and prevents its accumulation as a
reservoir of bioactive NO. Such data suggest that SNO-Hb does not play
a role in the regulation of blood flow during normal physiology. The
observations that SNO-Hb can be stabilized in oxidized erythrocytes in
which SNO-Hb levels are elevated, and that S-nitrosothiols
can be readily exported from these cells, suggests a potential role for
SNO-Hb in NO-dependent events in vivo during
pathological conditions associated with red blood cell oxidation or
under circumstances where NO generation is increased in response to
infection or pharmacological NO treatment.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Furchgott, R. F.,
and Zawadzki, J. V.
(1980)
Nature
288,
373-376[CrossRef][Medline]
[Order article via Infotrieve]
2.
Ignarro, L. J.,
Buga, G. M.,
Wood, K. S.,
Byrns, R. E.,
and Chaudhuri, G.
(1987)
Proc. Natl. Acad. Sci. U. S. A.
84,
9265-92659 3.
Palmer, R. M.,
Ferrige, A. G.,
and Moncada, S.
(1987)
Nature
327,
524-526[CrossRef][Medline]
[Order article via Infotrieve]
4.
Gladwin, M. T.,
Shelhamer, J. H.,
Schechter, A. N.,
Pease-Fye, M. E.,
Waclawiw, M. A.,
Panza, J. A.,
Ognibene, F. P.,
and Cannon, R. O., III
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
11482-11487 5.
Cannon, R. O., III,
Schechter, A. N.,
Panza, J. A.,
Ognibene, F. P.,
Pease-Fye, M. E.,
Waclawiw, M. A.,
Shelhamer, J. H.,
and Gladwin, M. T.
(2001)
J. Clin. Invest.
108,
279-287[CrossRef][Medline]
[Order article via Infotrieve]
6.
Stamler, J. S.,
Jaraki, O.,
Osborne, J.,
Simon, D. I.,
Keaney, J.,
Vita, J.,
Singel, D.,
Valeri, C. R.,
and Loscalzo, J.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
7674-7677 7.
Fang, K.,
Ragsdale, N. V.,
Carey, R. M.,
MacDonald, T.,
and Gaston, B.
(1998)
Biochem. Biophys. Res. Commun.
252,
535-540[CrossRef][Medline]
[Order article via Infotrieve]
8.
Gladwin, M. T.,
Ognibene, F. P.,
Pannell, L. K.,
Nichols, J. S.,
Pease-Fye, M. E.,
Shelhamer, J. H.,
and Schechter, A. N.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
9943-9948 9.
Jourd'heuil, D.,
Gray, L.,
and Grisham, M. B.
(2000)
Biochem. Biophys. Res. Commun.
273,
22-26[CrossRef][Medline]
[Order article via Infotrieve]
10.
Marley, R.,
Feelisch, M.,
Holt, S.,
and Moore, K.
(2000)
Free Radical Res.
32,
1-9[Medline]
[Order article via Infotrieve]
11.
Li, H.,
Samouilov, A.,
Liu, X.,
and Zweier, J. L.
(2001)
J. Biol. Chem.
276,
24482-24489 12.
Jia, L.,
Bonaventura, C.,
Bonaventura, J.,
and Stamler, J. S.
(1996)
Nature
380,
221-226[CrossRef][Medline]
[Order article via Infotrieve]
13.
Stamler, J. S.,
Jia, L., Eu, J. P.,
McMahon, T. J.,
Demchenko, I. T.,
Bonaventura, J.,
Gernert, K.,
and Piantadosi, C. A.
(1997)
Science
276,
2034-2037 14.
Gow, A. J.,
and Stamler, J. S.
(1998)
Nature
391,
169-173[CrossRef][Medline]
[Order article via Infotrieve]
15.
Funai, E. F.,
Davidson, A.,
Seligman, S. P.,
and Finlay, T. H.
(1997)
Biochem. Biophys. Res. Commun.
239,
875-877[CrossRef][Medline]
[Order article via Infotrieve]
16.
McMahon, T. J.,
Exton Stone, A.,
Bonaventura, J.,
Singel, D. J.,
and Stamler, J.
(2000)
J. Biol. Chem.
275,
16738-16745 17.
Imai, K.
(1982)
in
Allosteric Effects in Haemoglobin
(Imai, K., ed)
, pp. 39-45, Cambridge University Press, Cambridge, United Kingdom
18.
Bonaventura, C.,
Ferruzzi, G.,
Tesh, S.,
and Stevens, R. D.
(1999)
J. Biol. Chem.
274,
24742-24748 19.
Patel, R. P.,
Hogg, N.,
Spencer, N. Y.,
Kalyanaraman, B.,
Matalon, S.,
and Darley-Usmar, V. M.
(1999)
J. Biol. Chem.
274,
15487-15492 20.
Wolzt, M.,
MacAllister, R. J.,
Davis, D.,
Feelisch, M.,
Moncada, S.,
Vallance, P.,
and Hobbs, A. J.
(1999)
J. Biol. Chem.
274,
28983-28990 21.
Deem, S.,
Gladwin, M. T.,
Berg, J. T.,
Kerr, M. E.,
and Swenson, E. R.
(2001)
Am. J. Respir. Crit. Care Med.
163,
1164-1170 22.
Samouilov, A.,
and Zweier, J. L.
(1998)
Anal. Biochem.
258,
322-330[CrossRef][Medline]
[Order article via Infotrieve]
23.
McMahon, T. J.,
and Stamler, J. S.
(1999)
Methods Enzymol.
301,
99-114[Medline]
[Order article via Infotrieve]
24.
Ewing, J. F.,
and Janero, D. R.
(1998)
Free Radical Biol. Med.
25,
621-628[CrossRef][Medline]
[Order article via Infotrieve]
25.
Pawloski, J. R.,
Hess, D. T.,
and Stamler, J. S.
(2001)
Nature
409,
622-626[CrossRef][Medline]
[Order article via Infotrieve]
26.
Lipton, A. J.,
Johnson, M. A.,
Macdonald, T.,
Lieberman, M. W.,
Gozal, D.,
and Gaston, B.
(2001)
Nature
413,
171-174[CrossRef][Medline]
[Order article via Infotrieve]
27.
Winterbourn, C. C.
(1990)
Methods Enzymol.
186,
265-272[Medline]
[Order article via Infotrieve]
28.
Gladwin, M. T.,
Schechter, A. N.,
Shelhamer, J. H.,
Pannell, L. K.,
Conway, D. A.,
Hrinczenko, B. W.,
Nichols, J. S.,
Pease-Fye, M. E.,
Noguchi, C. T.,
Rodgers, G. P.,
and Ognibene, F. P.
(1999)
J. Clin. Invest.
104,
937-945[Medline]
[Order article via Infotrieve]
29.
Gaston, B.,
Reilly, J.,
Drazen, J. M.,
Fackler, J.,
Ramdev, P.,
Arnelle, D.,
Mullins, M. E.,
Sugarbaker, D. J.,
Chee, C.,
Singel, D. J.,
Loscalzo, J.,
and Stamler, J. S.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
10957-10961 30.
Rossi, R.,
Giustarini, D.,
Milzani, A.,
Colombo, R.,
Dalle-Donne, I.,
and Di Simplicio, P.
(2001)
Circ. Res.
89,
E47-E47 31.
Kelm, M.,
Preik-Steinhoff, H.,
Preik, M.,
and Strauer, B. E.
(1999)
Cardiovasc. Res.
41,
765-772 32.
Hwang, C.,
Lodish, H. F.,
and Sinskey, A. J.
(1995)
Methods Enzymol.
251,
212-221[Medline]
[Order article via Infotrieve]
33.
Hwang, C.,
Sinskey, A. J.,
and Lodish, H. F.
(1992)
Science
257,
1496-1502 34.
Zheng, M.,
Aslund, F.,
and Storz, G.
(1998)
Science
279,
1718-1721 35.
Nedospasov, A.,
Rafikov, R.,
Beda, N.,
and Nudler, E.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
13543-13548 36.
Huang, K. T.,
Han, T. H.,
Hyduke, D. R.,
Vaughn, M. W.,
Van Herle, H.,
Hein, T. W.,
Zhang, C.,
Kuo, L.,
and Liao, J. C.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
11771-11776 37.
Kister, J.,
Kiger, L.,
Francina, A.,
Hanny, P.,
Szymanowicz, A.,
Blouquit, Y.,
Prome, D.,
Galacteros, F.,
Delaunay, J.,
and Wajcman, H.
(1995)
Biochim. Biophys. Acta
1246,
34-38[CrossRef][Medline]
[Order article via Infotrieve]
38.
Shih, D. T.,
Luisi, B. F.,
Miyazaki, G.,
Perutz, M. F.,
and Nagai, K.
(1993)
J. Mol. Biol.
230,
1291-1296[CrossRef][Medline]
[Order article via Infotrieve]
39.
Imai, K.,
Tsuneshige, A.,
Harano, T.,
and Harano, K.
(1989)
J. Biol. Chem.
264,
11174-11180 40.
Garel, M. C.,
Domenget, C.,
Caburi-Martin, J.,
Prehu, C.,
Galacteros, F.,
and Beuzard, Y.
(1986)
J. Biol. Chem.
261,
14704-14709 41.
Garel, M. C.,
Domenget, C.,
Galacteros, F.,
Martin-Caburi, J.,
and Beuzard, Y.
(1984)
Mol. Pharmacol.
26,
559-565[Abstract]
42.
Gattoni, M.,
Piro, M. C.,
Boffi, A.,
Brinigar, W. S.,
Fronticelli, C.,
and Chiancone, E.
(2001)
Arch. Biochem. Biophys.
386,
172-178[CrossRef][Medline]
[Order article via Infotrieve]
43.
Luisi, B. F.,
and Nagai, K.
(1986)
Nature
320,
555-556[CrossRef][Medline]
[Order article via Infotrieve]
44.
Gladwin, M. T.,
Shelhamer, J. H.,
Ognibene, F. P.,
Pease-Fye, M. E.,
Nichols, J. S.,
Link, B.,
Patel, D. B.,
Jankowski, M. A.,
Pannell, L. K.,
Schechter, A. N.,
and Rodgers, G. P.
(2002)
Br. J. Haematol.
116,
436-444[CrossRef][Medline]
[Order article via Infotrieve]
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  M. T. Salgado, E. Nagababu, and J. M. Rifkind Quantification of Intermediates Formed during the Reduction of Nitrite by Deoxyhemoglobin J. Biol. Chem., May 8, 2009; 284(19): 12710 - 12718. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Feelisch, B. O. Fernandez, N. S. Bryan, M. F. Garcia-Saura, S. Bauer, D. R. Whitlock, P. C. Ford, D. R. Janero, J. Rodriguez, and H. Ashrafian Tissue Processing of Nitrite in Hypoxia: AN INTRICATE INTERPLAY OF NITRIC OXIDE-GENERATING AND -SCAVENGING SYSTEMS J. Biol. Chem., December 5, 2008; 283(49): 33927 - 33934. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. T. Gladwin and D. B. Kim-Shapiro The functional nitrite reductase activity of the heme-globins Blood, October 1, 2008; 112(7): 2636 - 2647. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. R. Jarboe, D. R. Hyduke, L. M. Tran, K. J. Y. Chou, and J. C. Liao Determination of the Escherichia coli S-Nitrosoglutathione Response Network Using Integrated Biochemical and Systems Analysis J. Biol. Chem., February 22, 2008; 283(8): 5148 - 5157. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. C. Rogers, A. Khalatbari, B. N. Datta, S. Ellery, V. Paul, M. P. Frenneaux, and P. E. James NO metabolite flux across the human coronary circulation Cardiovasc Res, July 15, 2007; 75(2): 434 - 441. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Grubina, Z. Huang, S. Shiva, M. S. Joshi, I. Azarov, S. Basu, L. A. Ringwood, A. Jiang, N. Hogg, D. B. Kim-Shapiro, et al. Concerted Nitric Oxide Formation and Release from the Simultaneous Reactions of Nitrite with Deoxy- and Oxyhemoglobin J. Biol. Chem., April 27, 2007; 282(17): 12916 - 12927. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Sonveaux, I. I. Lobysheva, O. Feron, and T. J. McMahon Transport and Peripheral Bioactivities of Nitrogen Oxides Carried by Red Blood Cell Hemoglobin: Role in Oxygen Delivery Physiology, April 1, 2007; 22(2): 97 - 112. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Hausladen, R. Rafikov, M. Angelo, D. J. Singel, E. Nudler, and J. S. Stamler Assessment of nitric oxide signals by triiodide chemiluminescence PNAS, February 13, 2007; 104(7): 2157 - 2162. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Doctor, B. Gaston, and D. B. Kim-Shapiro Detecting physiologic fluctuations in the S-nitrosohemoglobin micropopulation: triiodide versus 3C. Blood, November 1, 2006; 108(9): 3225 - 3227. [Full Text] [PDF]
|
|
|
|
 |
|
 M. T. Gladwin, N. J. H. Raat, S. Shiva, C. Dezfulian, N. Hogg, D. B. Kim-Shapiro, and R. P. Patel Nitrite as a vascular endocrine nitric oxide reservoir that contributes to hypoxic signaling, cytoprotection, and vasodilation Am J Physiol Heart Circ Physiol, November 1, 2006; 291(5): H2026 - H2035. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Wang, N. S. Bryan, P. H. MacArthur, J. Rodriguez, M. T. Gladwin, and M. Feelisch Measurement of Nitric Oxide Levels in the Red Cell: VALIDATION OF TRI-IODIDE-BASED CHEMILUMINESCENCE WITH ACID-SULFANILAMIDE PRETREATMENT J. Biol. Chem., September 15, 2006; 281(37): 26994 - 27002. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. B. Manukhina, H. F. Downey, and R. T. Mallet Role of nitric oxide in cardiovascular adaptation to intermittent hypoxia. Experimental Biology and Medicine, April 1, 2006; 231(4): 343 - 365. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. B. Kim-Shapiro, A. N. Schechter, and M. T. Gladwin Unraveling the Reactions of Nitric Oxide, Nitrite, and Hemoglobin in Physiology and Therapeutics Arterioscler. Thromb. Vasc. Biol., April 1, 2006; 26(4): 697 - 705. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. T. Huang, I. Azarov, S. Basu, J. Huang, and D. B. Kim-Shapiro Lack of allosterically controlled intramolecular transfer of nitric oxide from the heme to cysteine in the beta subunit of hemoglobin Blood, April 1, 2006; 107(7): 2602 - 2604. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Piknova, M. T. Gladwin, A. N. Schechter, and N. Hogg Electron Paramagnetic Resonance Analysis of Nitrosylhemoglobin in Humans during NO Inhalation J. Biol. Chem., December 9, 2005; 280(49): 40583 - 40588. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. C. Rogers, A. Khalatbari, P. W. Gapper, M. P. Frenneaux, and P. E. James Detection of Human Red Blood Cell-bound Nitric Oxide J. Biol. Chem., July 22, 2005; 280(29): 26720 - 26728. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Dejam, C. J. Hunter, M. M. Pelletier, L. L. Hsu, R. F. Machado, S. Shiva, G. G. Power, M. Kelm, M. T. Gladwin, and A. N. Schechter Erythrocytes are the major intravascular storage sites of nitrite in human blood Blood, July 15, 2005; 106(2): 734 - 739. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. J. Gow Nitric Oxide, Hemoglobin, and Hypoxic Vasodilation Am. J. Respir. Cell Mol. Biol., June 1, 2005; 32(6): 479 - 482. [Full Text] [PDF]
|
|
|
|
|
|
F. J. Schopfer, P. R. S. Baker, G. Giles, P. Chumley, C. Batthyany, J. Crawford, R. P. Patel, N. Hogg, B. P. Branchaud, J. R. Lancaster Jr., et al. Fatty Acid Transduction of Nitric Oxide Signaling: NITROLINOLEIC ACID IS A HYDROPHOBICALLY STABILIZED NITRIC OXIDE DONOR J. Biol. Chem., May 13, 2005; 280(19): 19289 - 19297. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Doctor, R. Platt, M. L. Sheram, A. Eischeid, T. McMahon, T. Maxey, J. Doherty, M. Axelrod, J. Kline, M. Gurka, et al. Hemoglobin conformation couples erythrocyte S-nitrosothiol content to O2 gradients PNAS, April 19, 2005; 102(16): 5709 - 5714. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. M. Pluta, A. Dejam, G. Grimes, M. T. Gladwin, and E. H. Oldfield Nitrite Infusions to Prevent Delayed Cerebral Vasospasm in a Primate Model of Subarachnoid Hemorrhage JAMA, March 23, 2005; 293(12): 1477 - 1484. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Yang and J. Loscalzo S-nitrosoprotein formation and localization in endothelial cells PNAS, January 4, 2005; 102(1): 117 - 122. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Deem, S. S. Kim, J.-H. Min, R. Eveland, J. Moulding, S. Martyr, X. Wang, E. R. Swenson, and M. T. Gladwin Pulmonary vascular effects of red blood cells containing S-nitrosated hemoglobin Am J Physiol Heart Circ Physiol, December 1, 2004; 287(6): H2561 - H2568. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Fago, C. Hundahl, S. Dewilde, K. Gilany, L. Moens, and R. E. Weber Allosteric Regulation and Temperature Dependence of Oxygen Binding in Human Neuroglobin and Cytoglobin: MOLECULAR MECHANISMS AND PHYSIOLOGICAL SIGNIFICANCE J. Biol. Chem., October 22, 2004; 279(43): 44417 - 44426. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. H. Crawford, B. K. Chacko, H. M. Pruitt, B. Piknova, N. Hogg, and R. P. Patel Transduction of NO-bioactivity by the red blood cell in sepsis: novel mechanisms of vasodilation during acute inflammatory disease Blood, September 1, 2004; 104(5): 1375 - 1382. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. T. Gladwin and A. N. Schechter NO Contest: Nitrite Versus S-Nitroso-Hemoglobin Circ. Res., April 16, 2004; 94(7): 851 - 855. [Full Text] [PDF]
|
|
|
|
|
|
N. S. Bryan, T. Rassaf, R. E. Maloney, C. M. Rodriguez, F. Saijo, J. R. Rodriguez, and M. Feelisch Cellular targets and mechanisms of nitros(yl)ation: An insight into their nature and kinetics in vivo PNAS, March 23, 2004; 101(12): 4308 - 4313. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. H. Han, E. Qamirani, A. G. Nelson, D. R. Hyduke, G. Chaudhuri, L. Kuo, and J. C. Liao Regulation of nitric oxide consumption by hypoxic red blood cells PNAS, October 14, 2003; 100(21): 12504 - 12509. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Xu, M. Cho, N. Y. Spencer, N. Patel, Z. Huang, H. Shields, S. B. King, M. T. Gladwin, N. Hogg, and D. B. Kim-Shapiro Measurements of nitric oxide on the heme iron and {beta}-93 thiol of human hemoglobin during cycles of oxygenation and deoxygenation PNAS, September 30, 2003; 100(20): 11303 - 11308. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. S. Stamler, B. M. Gaston, J. M. Hare, T. J. McMahon, J. R. Pawloski, D. J. Singel, A. N. Schechter, and M. T. Gladwin Hemoglobin and Nitric Oxide N. Engl. J. Med., July 24, 2003; 349(4): 402 - 405. [Full Text] [PDF]
|
|
|
|
 |
|
 S. Luckhart, A. L. Crampton, R. Zamora, M. J. Lieber, P. C. Dos Santos, T. M. L. Peterson, N. Emmith, J. Lim, D. A. Wink, and Y. Vodovotz Mammalian Transforming Growth Factor {beta}1 Activated after Ingestion by Anopheles stephensi Modulates Mosquito Immunity Infect. Immun., June 1, 2003; 71(6): 3000 - 3009. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. H. Crawford, C. R. White, and R. P. Patel Vasoactivity of S-nitrosohemoglobin: role of oxygen, heme, and NO oxidation states Blood, June 1, 2003; 101(11): 4408 - 4415. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. N. Schechter and M. T. Gladwin Hemoglobin and the Paracrine and Endocrine Functions of Nitric Oxide N. Engl. J. Med., April 10, 2003; 348(15): 1483 - 1485. [Full Text] [PDF]
|
|
|
|
|
|
S. Herold and G. Rock Reactions of Deoxy-, Oxy-, and Methemoglobin with Nitrogen Monoxide. MECHANISTIC STUDIES OF THE S-NITROSOTHIOL FORMATION UNDER DIFFERENT MIXING CONDITIONS J. Biol. Chem., February 21, 2003; 278(9): 6623 - 6634. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Rodriguez, R. E. Maloney, T. Rassaf, N. S. Bryan, and M. Feelisch Chemical nature of nitric oxide storage forms in rat vascular tissue PNAS, January 7, 2003; 100(1): 336 - 341. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Rassaf, P. Kleinbongard, M. Preik, A. Dejam, P. Gharini, T. Lauer, J. Erckenbrecht, A. Duschin, R. Schulz, G. Heusch, et al. Plasma Nitrosothiols Contribute to the Systemic Vasodilator Effects of Intravenously Applied NO: Experimental and Clinical Study on the Fate of NO in Human Blood Circ. Res., September 20, 2002; 91(6): 470 - 477. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |