 |
INTRODUCTION |
The interaction of nitrogen monoxide with hemoproteins, in
particular with myoglobin and hemoglobin, is currently an area of
intense research (1-15). The reaction between NO· and
oxyhemoglobin (oxyHb)1 has
repeatedly been suggested to represent the main route for NO·
depletion in the blood vessels (16-22) and the cause for an increase in blood pressure observed when extracellular hemoglobin-based blood
substitutes are administered (18, 23). However, the in vivo
relevance of this reaction has recently been questioned (11, 15, 24).
Indeed, it has been proposed that in vivo NO·
preferentially binds to the very small amount of deoxygenated heme of
hemoglobin that is present under physiological conditions (about 1%)
to yield an iron(II)-nitrosyl complex (HbFe(II)NO) (15). This
proposal is based on the hypothesis that NO· binds R-state
hemoglobin at least 100 times faster than T-state hemoglobin (25). It
has further been suggested that the "NO group" can be transferred
intramolecularly from the heme (HbFe(II)NO) to the conserved cysteine
residue 
93, producing S-nitrosohemoglobin (SNO-Hb) (26).
This process has been proposed to be driven thermodynamically, since
the S-nitrosocysteine of SNO-Hb is significantly more stable when Hb is in the R-state. When the red blood cells reach hypoxic tissues, O2 is delivered from SNO-Hb, triggering the
conversion of Hb to the T-state (26). Because of steric interactions
with nearby amino acid residues, the S-nitrosocysteine of
SNO-Hb is highly destabilized in the deoxyHb T-state (27). Thus,
transnitrosation from SNO-Hb to other thiols within the red blood cell
is favored. In particular, it has been suggested that the "NO
group" is transferred to a cysteine residue of the band 3 anion
exchange membrane protein and finally transported out of the red blood
cell by a still unidentified route (11, 24). One of the central points
of this hypothetical mechanism is that the delivery of both
O2 and NO· to tissues is regulated allosterically;
cooperative liberation of NO· and O2, transported
under physiological conditions by hemoglobin, should thus allow for
very efficient delivery of dioxygen to peripheral respiring tissues
(11, 24).
An increasing amount of evidence has recently been published that
challenges this fascinating hypothesis. Huang et al. (28) have shown that there is a linear correlation between the oxygen saturation of Hb and the yield of HbFe(II)NO, indicating that the rate
of NO· binding to deoxyHb is independent of oxygen saturation.
In another work, the same group has shown that the rate constant for
NO· binding to R-state hemoglobin is (2.1 ± 0.1) × 107 M
1 s1, nearly
identical to that reported for NO· binding to T-state Hb
(2.6 × 107 M1
s1) (29). Gladwin et al. (12) found a strong
correlation between metHb and plasma nitrate in the venous blood from
volunteers inhaling 80 ppm of NO· gas and concluded that the
main reaction of NO· with Hb in the red blood cells of arterial
blood leads to NO· depletion, not conservation of its
bioactivity. In addition, the same group determined that SNO-Hb is
present in the low nm range in the blood, but they did not find a
significant arterial-venous gradient (30-32). Such a gradient should
be found if NO· was released in the capillaries after
O2 dissociation. Finally, in a recent paper Gladwin
et al. (32) showed that SNO-Hb is intrinsically unstable in
the reductive environment of the red blood cells and thus concluded
that SNO-Hb cannot accumulate in the red blood cells to form a
reservoir of bioactive NO·. Taken together, these results
suggest that SNO-Hb cannot play a role in the regulation of blood flow
under normal physiological conditions.
In addition, while this work was in progress, three papers have
appeared that address the problem of mixing artifacts from the addition
of an NO· bolus to oxyHb (33, 34) and oxymyoglobin (oxyMb) (35)
solutions. The common conclusion of the authors of the three mentioned
papers (33-35) is that the addition of a small volume of a saturated
(2 mM) NO· solution to a diluted oxyHb or oxyMb/GSH
solution may lead to artifactual generation of high SNO-Hb/HbFe(II)NO
or S-nitrosoglutathione (GSNO)/MbFe(II)NO yields,
respectively. Thus, some of the high SNO-Hb yields reported in previous
in vitro studies (15, 26) may be artifacts arising from the
chosen experimental conditions. However, the data reported in these
three papers (33-35) are not always consistent, and contrasting
mechanisms have been proposed to explain similar observations. In
particular, from their studies of the reaction between oxyHb and
NO· in the presence of cyanide, Han et al. (33)
concluded that, when an NO· bolus is added to an oxyHb solution,
SNO-Hb is formed through a pathway that does not include
NO+ generated from oxidation of NO· by metHb. In
contrast, they suggest that SNO-Hb is possibly formed by reaction of
Cys-93 with N2O3. According to their
mathematical simulations, formation of N2O3
after the addition of NO· to oxyHb under aerobic conditions is
not possible kinetically, and thus they suggested that
N2O3 must be present in the NO· stock
solution. This suggestion is highly unlikely, since
N2O3 rapidly hydrolyzes to nitrite in aqueous
solutions. Indeed, it has been shown that the addition of unpurified
gaseous NO· (directly from the gas tank) to GSH under anaerobic
conditions leads to the formation of GSNO (36). In contrast, GSNO was
not detected when a solution of unpurified NO· was added to the
GSH solution (under anaerobic conditions) (36). A final observation of
Han et al. (33) is that if NO· is delivered with the
NO· donor 2-(N,N-diethylamino)diazenolate
2-oxide (DEA), oxyHb is converted exclusively to metHb and nitrate, and
no detectable amounts of SNO-Hb are formed.
Joshi et al. (34) also studied the reaction of NO·
with oxyHb. From their results, the authors of this paper also
concluded that SNO-Hb is generated from the reaction of Cys-93 with
N2O3. However, Joshi et al. (34)
have proposed that, when NO· is added as a bolus, it is possible
that substantial amounts of N2O3 are formed
from the reaction of NO· with O2. Nevertheless, this
conclusion contrasts with their further observation that the SNO-Hb
yields were nearly identical when NO· was added as a bolus or
with the NO· donor
(Z)-1-{N-methyl-N-[6-(N-methylammoniohexyl)amino]}diazenium 1,2-diolate (NHMA).
Finally, from their experiments with oxyMb and NO· in the
presence of GSH, Zhang et al. (35) also concluded that the
addition of a bolus of NO· causes mixing artifacts that
facilitate the NO·/O2 reaction. Thus, analogously to
Joshi et al. (34), they suggest that GSNO is generated from
the reaction of the nitrosating species N2O3
with GSH. This conclusion is supported by the observation that, in
contrast to the data reported by Joshi et al. (34) but in
analogy to those described in Han et al. (33), the slow addition of NO· with the NO·-donor
2-(N,N-diethylamino)diazenolate 2-oxide to an
oxyMb/GSH solution did not result in detectable amounts of GSNO.
In the present work, we carried out a systematic study of the influence
of the following factors on the percentage of SNO-Hb generated relative
to the amount of NO· added to oxy-, deoxy-, and metHb solutions:
1) the volumetric ratio of the protein and the NO· solutions; 2)
the rate of addition of the NO· solution to the protein
solution; 3) the amount of NO· added; and 4) the concentration
of the phosphate buffer (0.1 versus 0.01 M). For comparison, in some of the reactions, we
substituted Hb with equimolar amounts of Mb mixed with 0.5 eq of GSH,
to mimic the heme/Cys-93 ratio in Hb. To be able to directly compare
our results, we chose to keep the protein concentration constant in most of our experiments (final concentration 50 µM) and
generally added 1 or 0.1 eq of NO·. Three different techniques
were used to add the NO· solution to the protein solution: the
addition of a small volume of a saturated (2 mM) NO·
solution (Method 1), the addition of an equivalent volume of a diluted NO· solution (Method 2), either as fast as
possible or very slowly, within 1-2 min.
| |
EXPERIMENTAL PROCEDURES |
Reagents--
Buffer solutions (0.1 or 0.01 M) were
prepared from
K2HPO4/KH2PO4 (Fluka)
with deionized Milli-Q water and always contained 0.1 mM diethylenetriaminepentaacetic acid (DTPA; Sigma).
Sodium nitrite, sodium nitrate, sodium dithionite, potassium
hexacyanoferrate(III), potassium cyanide, potassium permanganate,
sulfanilamide,
N-(1-naphthyl)-ethylenediaminedihydrochloride, ammonium
sulfamate, and glutathione were obtained from Fluka. 4,4'-Dithiopyridine (4-PDS) was purchased from Aldrich. GSNO was prepared by the method of Hart (37). Briefly, reduced glutathione was
allowed to react with sodium nitrite under acidic conditions at
0 °C, followed by the addition of cold acetone. The resulting precipitate was filtered off, washed, and dried under vacuum in the
dark. The pink solid of GSNO, which had similar visible absorption maxima and extinction coefficients as reported by Hart (37), was stored
at 
80 °C in the dark.
Nitrogen Monoxide Solutions--
Nitrogen monoxide was obtained
from Linde and passed through a NaOH solution as well as a column of
NaOH pellets to remove higher nitrogen oxides before use. A saturated
NO· solution was prepared by degassing water for at least 1 h with argon and then saturating it with NO· for at least 1 h. When needed, the obtained stock solution (~2 mM) was
diluted with degassed buffer in gas-tight SampleLock Hamilton syringes.
The final NO· concentrations were measured with an ANTEK
Instruments nitrogen monoxide analyzer, with a chemiluminescence detector.
Myoglobin Solutions--
Horse heart myoglobin was purchased
from Sigma. metMb was prepared by oxidizing the protein with a small
amount of potassium hexacyanoferrate(III). The solution was then
purified over a Sephadex G-25 column by using a 0.1 M
phosphate buffer solution (pH 7.0) as the eluant. The concentration of
the metMb solutions was determined by measuring the absorbances at 408, 502, and/or 630 nm (
408 = 188 mM1 cm1, 502 = 10.2 mM1 cm1, and
630 = 3.9 mM1
cm1) (38). oxyMb was prepared by reducing metMb with a
slight excess of sodium dithionite. The solution was purified
chromatographically on a Sephadex G-25 column by using a 0.1 M phosphate buffer solution (pH 7.0) as the eluant. The
concentration of the oxyMb solutions was determined by measuring the
absorbances at 417, 542, and/or 580 nm (417 = 128 mM1 cm1, 542 = 13.9 mM1 cm1, and
580 = 14.4 mM1
cm1) (38).
Hemoglobin Solutions--
Purified human oxyHb stock solution
(57 mg/ml solution of HbA0 with ~1.1% metHb) was
a kind gift from APEX Bioscience, Inc. The obtained solution was frozen
in small aliquots (0.5-1 ml) and stored at 80 °C. oxyHb solutions
were prepared by diluting the stock solution with buffer, and
concentrations (always expressed per heme) were determined by measuring
the absorbance at 415, 541, and/or 577 nm (415 = 125 mM1 cm1, 541 = 13.8 mM1 cm1, and
577 = 14.6 mM1
cm1) (38). metHb solutions were prepared by oxidizing
oxyHb with a slight excess of potassium hexacyanoferrate(III). The
solution was purified chromatographically on a Sephadex G-25 column by using a 0.1 M phosphate buffer solution (pH 7.0) as the
eluant. The concentration of the metHb solutions (always expressed per heme) was determined by measuring the absorbances at 405, 500, and/or 631 nm (405 = 179 mM1 cm1, 500 = 10.0 mM1 cm1, and
631 = 4.4 mM1
cm1) (38). HbFe(II) (deoxyHb) solutions were prepared by
thoroughly degassing oxyHb solutions with argon for at least 1 h
without causing foaming of the solution. The concentration of the
HbFe(II) solutions was determined by measuring the absorbance at 430 and/or 555 nm (430 = 133 mM1
cm1 and 555 = 12.5 mM1 cm1) (38). metHbCN
solutions were prepared by treating metHb with a slight excess of KCN.
The solution was purified chromatographically on a Sephadex G-25 column
by using a 0.1 M phosphate buffer solution (pH 7.0) as the
eluant. The concentration of the metHbCN solutions (always expressed
per heme) was determined by measuring the absorbances at 419 and/or 540 nm (419 = 124 mM1
cm1 and 540 = 12.5 mM1 cm1) (38). SNO-Hb (36 mg/ml
with an S-nitroso content of 850 µM; i.e. about 74% of Cys-93 and metHb content of 10.7%)
was a kind gift from APEX Bioscience, Inc.
UV-visible Spectroscopy--
Absorption spectra were collected
in 1 cm cells on a UVIKON 820 or on an Analytik Jena Specord 200.
General Procedures for the Reactions of Different Forms of
Hemoglobin and Myoglobin with NO·--
All reactions were
carried out in phosphate buffer (0.1 or 0.01 M), pH 7.2, at
room temperature. For Method 1, 2 ml of a protein solution
(mostly ~50 µM in 0.1 M phosphate buffer,
pH 7.2, containing 0.1 mM DTPA) were placed in a 5-ml round
bottom flask, which was then closed with a rubber septum. If anaerobic
conditions were required, the solutions were thoroughly degassed with
argon for 45-60 min. Depending on the equivalents of NO·
required (mostly 1, 0.5, or 0.1 eq) 5-50 µl of a
NO·-saturated solution were rapidly added to the
oxyHb solution under constant stirring by using a Hamilton gas-tight
syringe. For the experiments under aerobic conditions, particular care
was taken to add the NO· solution at the bottom of the flask to
avoid any contact of NO· with the oxygen present in the head
space of the flask. Alternatively, the reaction was carried out in a
sealable cell for anaerobic applications (Hellma). In this case, the
required amount of a saturated NO· solution (5-50 µl) was
added to 2 ml of a protein solution while vortex-mixing. Method
2 used the same procedure as described for Method 1 with the difference that the volumetric ratio of the protein and the
NO· solutions was always 1. In a typical experiment, 2 ml of a
100 µM protein solution (in 0.1 M phosphate
buffer, pH 7.2, containing 0.1 mM DTPA) were mixed under
constant stirring with 2 ml of a diluted NO· solution (10-100
µM depending on the required equivalents). The diluted
NO· solutions were prepared by mixing in a gas-tight SampleLock
Hamilton syringe the NO·-saturated solution with the required
amounts of degassed buffer. In most cases, each experiment was carried
out by adding the NO· solution in two different ways: fast
(in one shot as fast as possible) or slow (as slowly as
possible, within 1-2 min).
Reaction of oxyHb with NO·--
The reactions were
carried out under aerobic conditions according to Method 1 or 2. After the addition of the NO· solution, the
reaction mixtures were stirred for 10 min and then analyzed for
S-nitrosated Cys-93 and free Cys-93, and, in
some cases, a UV-visible spectrum was recorded. A control experiment was carried out to check that no artifactual S-nitrosation
was taking place under the acidic conditions of the Saville assay. For
this purpose, after treating the oxyHb solution with NO·, 100 µl of a 10 mM N-ethylmaleimide (NEM) solution
in H2O were added and allowed to react for 20 min. Finally,
the amount of S-nitrosated Cys-93 was quantified.
A series of experiments was carried out in the presence of an excess
KCN. For this purpose, a solution containing oxyHb and either 10 or 100 eq of KCN (relative to the oxyHb concentration) was treated with 1 eq
of NO· according to Method 1 exactly as described
above for the reaction in the absence of KCN.
Reaction of deoxyHb with NO·--
Most of the reactions
were carried out in a sealable cell for anaerobic applications. The
oxyHb solutions were thoroughly degassed for about 30 min, until the
recorded UV-visible spectrum indicated the complete formation of
deoxyHb. Alternatively, the reactions were carried out in round bottom
flasks under anaerobic conditions according to Method 1 or
2. After the addition of the NO· solution, the
reaction mixtures were stirred for 10 min. Then the flasks or the cells
were opened, and the solutions were analyzed for
S-nitrosated Cys-93 (under aerobic conditions)
immediately and/or after 10 min. In a control experiment, the
S-nitrosated Cys-93 content was determined under
anaerobic conditions by carrying out the analysis in a sealable cell
with thoroughly degassed Saville reagents. A series of experiments was
carried out by adding an excess KCN before exposing the reaction
mixtures to air. For this purpose, to 2 ml of deoxyHb (50 µM in 0.1 M phosphate buffer) we first added
100 µl of a NO·-saturated solution and, immediately after, 100 µl of a 10 mM KCN solution in water. The reaction mixture
was stirred for 10 min, exposed to air, and, after 10 more min,
analyzed with the Saville assay.
Reaction of Mixtures of oxyHb and deoxyHb (1:1 and 3:1) with
NO·--
An oxyHb solution (2 ml, 50 µM in 0.1 M phosphate buffer, pH 7.2, containing 0.1 mM
DTPA) was placed in a sealable cell and degassed for ~30 min, until
the recorded UV-visible spectrum indicated the formation of deoxyHb.
Then 110 or 130 µl of an O2-saturated H2O
solution were added until the recorded UV-visible spectrum matched a
spectrum calculated for a 1:1 or a 3:1 mixture, respectively. The
concentration of the two hemoglobin species present in solution was
also controlled by using the equation given in Ref. 39. The solutions
were then treated with a concentrated NO· solution according to
Method 1. The reaction mixtures were stirred for 10 min.
Finally, the cell was opened, and the solution was immediately analyzed
for S-nitrosated Cys-93 under aerobic conditions.
Reaction of metHb with NO·--
The reactions were
carried out both under aerobic and under anaerobic conditions according
to Method 1 or 2. After the addition of the
NO· solution, the reaction mixtures were stirred for 30 min,
degassed for 30-60 min (only for the anaerobic experiments), and
analyzed for S-nitrosated Cys-93 under aerobic conditions.
Reaction of a Mixture of metHb and oxyHb with
NO·--
Solutions containing twice the amount of Hb (50 µM oxyHb and 50 µM metHb) were mixed under
aerobic conditions with 50 or 5 µM NO· according
to either Method 1 or Method 2 (fast and slow).
The reaction mixtures were stirred for 30 min and then analyzed for S-nitrosated Cys-93.
Reaction of a Mixture of metMb and oxyHb with NO·--
To
2 ml of a solution containing 50 µM oxyHb and 50 µM metMb, 1 or 0.1 eq of NO· were added from a
saturated solution (Method 1) under aerobic conditions. The
reaction mixture was stirred for 30 min and then analyzed for
S-nitrosated Cys-93.
Reaction of metHbCN with NO·--
The addition of
NO· was performed under strictly anaerobic conditions according
to Method 1 or 2. After 10 min, the solution was
thoroughly degassed for 1 h and analyzed for
S-nitrosated Cys-93 under aerobic conditions.
Reaction of metMb with NO· in the Presence of
GSH--
metMb was mixed with 0.5 eq of GSH (relative to the metMb
concentration), treated with NO·, and analyzed exactly as
described above for the reaction of metHb with NO·.
Reaction of oxyMb with NO· in the Presence of
GSH--
oxyMb was mixed with 0.5 eq of GSH (relative to the oxyMb
concentration), treated with NO·, and analyzed exactly as
described above for the reaction of oxyHb with NO·.
Analysis of the S-Nitrosothiol Content with the Saville
Assay--
The S-nitrosothiol content was determined by the
method of Saville (40). Our NO· solutions always contained
variable amounts of nitrite (variable amounts between 0.1 and 1 eq,
relative to the NO· concentration). Thus, since the analysis of
the S-nitrosothiol content is carried out under acidic
conditions, we first eliminated excess nitrite by the addition of
ammonium sulfamate (40). For the analysis of the
S-nitrosothiol content, two different sets of reagents were
prepared. Diluted reagents include solution A (1 mM
ammonium sulfamate in 0.5 M HCl), solution B (1%
sulfanilamide in 0.5 M HCl), solution C (1% sulfanilamide
and 0.2% HgCl2 in 0.5 M HCl), and solution D
(0.02% N-(1-naphthyl)-ethylendiaminedihydrochloride in
0.5 M HCl. Concentrated reagents include solution A
(10 mM ammonium sulfamate in 0.5 M HCl),
solution B (7% sulfanilamide in 0.5 M HCl), solution C
(7% sulfanilamide and 1% HgCl2 in 0.5 M HCl),
and solution D (0.2%
N-(1-naphthyl)-ethylendiaminedihydrochloride in 0.5 M HCl). Analyses with the diluted reagents were carried out
as follows. Two protein samples (500 µl) were mixed with an equivalent volume of solution A and allowed to react for 10 min to
destroy the nitrite present in the reaction mixture. Solution B (500 µl) was then added to one of the samples, and the same amount of
solution C was added to the other. After 5 min, when the formation of
the diazonium salt was complete, 500 µl of solution D were added to
each of the two samples. After 5 min, when color formation indicative
of the azo dye was complete, the absorbance of 540 nm was read
spectrophotometrically. Analyses with the concentrated reagents were
carried out analogously by mixing 1 ml of two protein solutions with
0.1-0.2 ml of solution A, 0.1 ml of solution B or C, respectively, and
0.1 ml of solution D. The amount of S-nitrosothiol was
quantified as the difference in absorbance between the samples containing solution C and solution B. The extinction coefficient was
determined by measuring a series of calibration curves by using either
the diluted or the concentrated reagents.
S-Nitrosoglutathione and SNO-Hb standard solutions gave an
average value of 51 mM1 cm1 for
the extinction coefficient at 540 nm. In general, the diluted reagents
were used for samples containing S-nitrosothiol
concentrations higher than 0.3 µM, whereas the
concentrated reagents were utilized when the amounts of
S-nitrosothiols were lower. However, several SNO-Hb
concentrations (higher than 0.3 µM) were determined with both the diluted and concentrated reagents, and the results were always
in very good agreement.
Analysis of the Sulfhydryl Content--
The amount of free
sulfhydryl groups was measured by reaction with 4,4'-dithiodipyridine
(PDS) (41). This analytical method allows for the determination of the
amount of free Cys-93, the only reactive cysteine residue of
hemoglobin. In brief, the protein sample to be analyzed (500 µl) was
mixed with an equal volume of 4-PDS (10 mM in 0.1 M phosphate buffer, pH 7.2, containing 0.1 mM
DTPA). After incubation for 20 min at room temperature, when the
product 4-thiopyridone had completely formed, the absorbance of the
sample was measured spectrophotometrically at its maximum (324 nm). The
free Cys-93 content was quantified as the difference in
absorbance between this solution and that of a protein sample (500 µl) mixed with an equivalent volume of buffer. The extinction coefficient of 4-thiopyridone was determined by measuring a calibration curve of 5-10 glutathione standard solutions (324 = 21 mM1 cm1) and was comparable
with that described in the literature (324 = 19.8 mM1 cm1 (41)). To measure the
total amount of thiols in hemoglobin (six cysteine residues/tetramer),
the analysis was carried out with 1% SDS added to the buffer (42).
Statistics--
The experiments reported here were carried out
at least in triplicate on independent days. The results are given as
mean values of at least three experiments plus or minus the
corresponding standard deviation.
| |
RESULTS |
Detection Limits of SNO-Hb with the Saville Method--
To
validate our methodology used to quantify low amounts of SNO-Hb with
the Saville assay, a series of calibration curves were measured to
determine the limit of detection of S-nitrosothiols under
our experimental conditions. With the concentrated Saville reagents
(see "Experimental Procedures" for details), it was possible to
measure accurately only standard solutions containing more than 0.5 µM GSNO. This result is in agreement with a recent report of Gladwin et al. (32), who showed that quantification of
SNO-Hb and SNO-albumin within the concentration range 0.5-7
µM gave comparable results with the Saville and the
I
chemiluminescence
assays.2 This detection limit
is set by the absorbance values obtained with very low
S-nitrosothiol concentration, which are lower than 0.1 and,
thus, cannot be measured accurately. However, under our experimental
conditions, the reaction solutions to be analyzed with the Saville
assay always also contained ~50 µM hemoglobin. Thus, at
540 nm, the wavelength corresponding to the absorbance maximum of the
azo dye quantified in the assay, our solutions had a basal absorbance
of about 0.3-0.4. Consequently, in the presence of 50 µM
hemoglobin, GSNO concentrations as low as 0.05 µM could
still be measured accurately. Indeed, as shown in Fig. 1, an excellent correlation was found in
the range of 0.05-5 µM GSNO by measuring the lower
concentrations (0.05-0.1 µM) in the presence and the
higher concentrations (2.5-5 µM) in the absence of 50 µM oxyHb.
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|
Fig. 1.
Validation of the Saville assay for the
quantification of very low amounts of S-nitrosothiols
in the presence of 50 µM
oxyHb. Standard GSNO solutions were analyzed in the absence
(2.5-5 µM) or in the presence (0.05-1 µM)
of 50 µM oxyHb. Inset, enlargement of the
lowest GSNO concentrations, all measured in the presence of 50 µM oxyHb.
|
|
Our NO· solutions were usually contaminated with nitrite
(variable amounts between 0.1 and 1 eq, relative to the NO·
concentration). Thus, before carrying out the Saville assay, we always
eliminated excess nitrite by the addition of ammonium sulfamate (40) to
avoid a high background absorbance arising from the reaction of nitrite
with the Saville reagents and to prevent nitrite-mediated
acid-catalyzed formation of S-nitrosothiol compounds.
Ammonium sulfamate reduces nitrite to dinitrogen, but it has recently
been observed (43) that nitrite cannot be removed completely with this
reagent. However, since the Saville assay is based on the measurement
of an absorbance difference, incomplete removal of nitrite does not
interfere with our measurements. In addition, it has recently been
argued that the reaction of ammonium sulfamate with nitrite, which also
takes place under acidic conditions, might lead to artifactual
formation of S-nitrosated thiols (44). As a control, we thus
mixed glutathione (50 µM) with nitrite (10 mM) and then destroyed it by the addition of a 5-fold
excess of ammonium sulfamate (equivalent volume of a 50 mM
solution in 0.5 M HCl). In our hands, this reaction does
not lead to detectable amounts of S-nitrosoglutathione, and
analysis of the free sulfhydryl group of glutathione confirmed that no
reaction had taken place. Finally, as a further control, we determined
the SNO-Hb yield of the reaction between oxyHb and NO· after the
addition of ~500 µM N-ethylmaleimide (NEM)
prior to the Saville assay. Also in this case, no difference was
observed between the samples analyzed with or without prior blockage of the free thiols with N-ethylmaleimide.
Since a large excess of ammonium sulfamate was used to remove the
nitrite contaminations, it is likely that part of it was still present
during the Saville assay. It has been reported that the reaction of
nitrite with sulfanilamide proceeds at a significantly faster rate,
and, thus, nitrite liberated from S-nitrosothiols can be
analyzed quantitatively also in the presence of ammonium sulfamate
(40). Nevertheless, we checked that this observation was true also
under our experimental conditions. For this purpose, we prepared
S-nitrosoglutathione solutions (~4 µM)
containing different amounts of nitrite (0, 1, 10, and 100 µM) and analyzed them with the Saville assay after the
addition of the usual excess of ammonium sulfamate. In all cases, the
amount of S-nitrosothiol found corresponded to the GSNO
content of the original solution.
Reaction of oxyHb with NO·--
It has been proposed that
the reaction of oxyhemoglobin with NO· leads to significant
nitrosation of its cysteine residue 93 (~40% relative to the
amount of added NO·) and thus to the formation of considerable
amounts of SNO-hemoglobin (15). This hypothesis does not fit with our
observation that nitrate is formed quantitatively from the reaction of
oxyHb with 1 eq of NO· (45). Thus, we determined the amount of
SNO-Hb generated after the addition of 1, 0.5, and 0.1 eq of NO·
to an oxyHb solution. The reactions were carried out in a closed vial
under aerobic conditions at room temperature in 0.1 M
phosphate buffer, pH 7.2, in the presence of 0.1 mM of the
metal chelator DTPA. As summarized in Table
I, when 1, 0.5, or 0.1 eq of NO·
from a saturated (2 mM) solution were added to a 50 µM oxyHb solution (Method 1) and allowed to
react for 10 min, as expected, the yield of nitrosated Cys-93
decreased continuously. However, when the SNO-Hb yields are expressed
relative to the NO· concentration, it becomes apparent that
these relative yields of SNO-Hb increase with decreasing amounts of
NO· added. The relative SNO-Hb yields are very low and span from 1.5 to 5% of NO· added (1 or 0.1 eq), respectively. The sum of
the concentration of the nitrosated Cys-93 and that of
unreacted Cys-93, determined separately by reaction with 4-PDS
(41), was always in good agreement with the total amount of
Cys-93. In a typical experiment, when 53 µM
NO· were allowed to react with 53 µM oxyHb (total
Cys-93 concentration 26.5 µM), we found 0.8 µM nitrosated Cys-93 and about 26 µM free Cys-93. Taken together, these data
suggest that only the cysteine residue Cys-93 is nitrosated and
that the other two less exposed cysteine residues of Hb, Cys-
104 and
Cys-112, are not modified.
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Table I
Amount of nitrosated Cys-93 formed from the reaction of oxyHb with
different amounts of NO· in 0.1 M and (in
parenthesis) in 0.01 M phosphate buffer, pH 7.2: Influence
on the concentration of the added NO· solution
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To avoid artifacts derived from inhomogeneous mixing of different
volumes of two solutions (33-35) with significantly different concentrations (2 ml of a 50 µM oxyHb solution and 5-50
µl of a 2 mM NO· solution), we repeated the same
reactions by mixing equivalent volumes of oxyHb and NO·
solutions having similar concentrations (2 ml of a 100 µM
oxyHb solution and 2 ml of a 10-100 µM NO·
solution; Method 2). Surprisingly, as shown in Table I, the yields of nitrosated Cys-93 (relative to the amount of
NO· added) were almost identical to those obtained by adding
NO· directly from the saturated solution.
Interestingly, higher yields of nitrosated Cys-93 were obtained
when the reaction was carried out by adding very slowly (within 1-2
min) a diluted NO· solution to a oxyHb solution at a volume
ratio of 1:1. As summarized in Table I, for all three amounts of
NO· added (1, 0.5, or 0.1 eq) the yields of SNO-Hb were always
larger than those obtained by the fast addition of a diluted NO·
solution. The highest yield of SNO-Hb obtained, expressed relative to
the amount of NO· added, was 14 ± 1%, for the slow
addition of 0.1 eq of NO·.
As a control, to check that the dilution and/or slow addition of the
NO· solution did not lead to artifactual SNO-Hb generation due
to O2 contamination of the NO· solution, we carried
out a series of reactions between GSH and NO· under anaerobic
conditions. In the absence of O2, it is known that thiols
and NO· do not generate nitrosothiols (46-48). Thus, this
reaction can be utilized to confirm that we do not have O2
contamination in the system under our experimental conditions. 1 eq of
NO· was added to a thoroughly degassed 50 µM
(final concentration) GSH solution either directly from a saturated
solution (Method 1) or after dilution at a volumetric ratio
of 1:1 fast or slow (Method 2). After 30 min, the reaction
mixtures were degassed to remove the unreacted NO· and finally
analyzed with the Saville assay under aerobic conditions, in the
absence or in the presence of 50 µM oxyHb. As expected, independently of the way the NO· solution was added, no
detectable amounts of GSNO were formed. Thus, we can exclude that the
differences between the SNO-Hb yields obtained when the NO·
solution was added fast or slowly are due to O2
contamination during the addition of the NO· solution.
Since it has recently been argued that the phosphate concentration
influences the nature of the reaction between oxyHb and NO·
(15), we determined the yields of SNO-Hb generated by the fast and slow
addition of a diluted NO· solution to oxyHb in a 0.01 M phosphate buffer at pH 7.2. As summarized in Table I
(data in parenthesis), when the reactions were carried out in the
diluted buffer, the nitrosation yields were higher than those obtained
in 0.1 M phosphate buffer under the same experimental conditions.
The reaction between oxyHb and 1 eq of NO· (in 0.1 M
phosphate buffer, pH 7.2), added with the three different methods
described above, was also followed by UV-visible spectroscopy. As shown in Fig. 2A, the slow addition
of a diluted NO· solution led to the formation of ~90-95%
metHb. In contrast, the fast addition of either a saturated or a
diluted NO· solution gave a slightly lower metHb yield, about
80%.
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Fig. 2.
UV-visible spectra of the products of the
reaction of 1 eq of NO· with oxyHb, deoxyHb, and oxyHb/deoxyHb
mixtures. A, the addition of NO· (50 µM, final concentration) to oxyHb (50 µM,
final concentration) from a saturated solution (Method 1),
and fast or slow from a diluted solution (Method 2).
B, the addition of NO· (50 µM, final
concentration) to a 3:1 mixture of oxyHb/deoxyHb (50 µM,
final protein concentration) from a saturated solution (Method
1). C, the addition of NO· (50 µM,
final concentration) to a 1:1 mixture of oxyHb/deoxyHb (50 µM, final protein concentration) from a saturated
solution (Method 1). D, a deoxyHb solution (46 µM in 0.1 M phosphate buffer
(thick line 1) was treated under
anaerobic conditions with 1 eq of NO· (from a saturated
solution, Method 1) to generate HbFe(II)NO (thick
dotted line 2). The cell was exposed
to air, and a spectrum was recorded immediately (spectrum
3). Spectra 4-10 were measured at
time intervals of 10 min, for a total of 70 min.
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Finally, to find out whether metHb played a role for the generation of
SNO-Hb from oxyHb and NO·, we carried out a series of
experiments in the presence of CN. For this purpose, we
added 1 eq of NO· from a saturated solution (Method
1) to a solution containing 50 µM oxyHb and 500 µM KCN. Under these conditions, the SNO-Hb yield was
1.1 ± 0.1% (expressed relative to the amount of NO·
added), not significantly lower than that observed under the same
conditions in the absence of added CN (1.5 ± 0.1%). The slow addition of NO· from a diluted stock solution
(Method 2, slow) to 50 µM oxyHb in the
presence of 50 µM KCN led to the formation of 5 ± 1% SNO-Hb, approximately the same amount generated under the same
conditions in the absence of added CN (5.9 ± 0.4%). For both methods, the addition of 10 times more CN (5 mM) did not lead to a significant
change in the relative SNO-Hb yield. Since it has recently been noted
that CN interferes with the Saville assay (32), we mixed
GSNO (5 µm) with 0, 1, and 10 eq of KCN and measured the amount of
S-nitrosothiols. In our hands, no interference was observed;
indeed, the values were within 4% error (5.0 ± 0.2 µM) without showing any clear dependence on the amount of
cyanide added.
Reaction of deoxyHb with NO·--
Since it has been
proposed that in vivo NO· reacts with the small
amount of deoxygenated hemoglobin present in the red blood cells to
generate SNO-Hb (15), we also determined the yield of SNO-Hb obtained
when deoxyHb was allowed to react with NO· both in 0.1 and 0.01 M phosphate buffer, pH 7.2. The deoxyHb solutions were
incubated for 10 min with NO· (added in different ways),
oxygenated, and immediately subjected to the Saville assay under
aerobic conditions. The results summarized in Table
II show that the rapid addition of 1 eq
of NO· (from a concentrated solution, Method 1) to a
50 µM deoxyHb solution (in 0.1 M phosphate
buffer) yielded approximately the same amount of SNO-Hb as that
obtained by the analogous addition of NO· to oxyHb. In contrast,
we found about 1.5-2 times higher relative SNO-Hb yields (relative to
the amount of NO· added) when we added 0.1 eq of NO·.
Almost identical yields were obtained by the fast addition of a diluted
NO· solution (Method 2). Interestingly, in contrast
to the reaction with oxyHb, no significant increase in the SNO-Hb
yields was observed when the diluted NO· solution
was added very slowly (Table II). Finally, for the reactions with
deoxyHb, no significant differences were detected when all of the
reactions were carried out in 0.01 M phosphate buffer
(Table II; data in parenthesis). The observation that the method of
NO· addition does not influence the SNO-Hb yields suggests that
formation of SNO-Hb takes place only after oxygenation of the reaction
solutions, which is from the reaction of HbFe(II)NO with
O2. Indeed, the rapid addition of 1 eq of NO· to a
deoxyHb solution, followed by Saville analysis under strictly anaerobic
conditions yielded only ~0.2% SNO-Hb.
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Table II
Amount of nitrosated Cys-93 formed from the reaction of deoxyHb with
different amounts of NO· in 0.1 M and (in
parenthesis) in 0.01 M phosphate buffer. pH 7.2: Influence
on the concentration of the added NO· solution and on the time
elapsed before analysis
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We thus investigated whether allowing HbFe(II)NO to react with
O2 for longer times (10 min or 1 h) had an influence
on the amount of SNO-Hb produced. As summarized in Table II (10 min), when we carried out the Saville analysis 10 min after oxygenation of
the reaction solutions, we found ~10-20% higher SNO-Hb yields, both
in 0.1 and 0.01 M (data in parenthesis) phosphate buffer. However, because of the large errors associated with some of these data
(up to 20%), this small increase cannot be considered significant. When 1 h elapsed prior to the Saville assay, no significant
further increase in the SNO-Hb yields was observed. In a separate
experiment, we determined that no loss of nitrosated Cys-93 in
SNO-Hb is observed within 1 h under the conditions of our
experiment. Finally, if the solution was allowed to stand overnight,
the amount of SNO-Hb slightly decreased, probably because of the
instability of the S-nitrosocysteine.
The nitrosyl complex HbFe(II)NO is known to slowly oxidize to metHb
(49). To find out the extent of oxidation that takes place under our
experimental conditions, we also carried out a parallel UV-visible
spectroscopic analysis of the reaction solution. As expected, the
addition of 1 eq of NO· to a deoxyHb solution (thick
line 1 in Fig. 2D) led to the
quantitative formation of HbFe(II)NO (thick
dotted line 2 in Fig. 2D).
Oxygenation of this solution causes the slow decay of HbFe(II)NO to
metHb (spectra 3-10 in Fig. 2D). The
solution measured immediately after opening the vial
(spectrum 3 in Fig. 2D) contained
~90% HbFe(II)NO and 10% metHb. After 10 min (spectrum
4 in Fig. 2D), the reaction mixture consisted of
~85% HbFe(II)NO and 15% metHb. After 60 min (spectrum
9 in Fig. 2D) ~50% of HbFe(II)NO had been
oxidized to metHb.
To find out whether metHb played a role in the reaction that leads to
nitrosation of Cys-93 when HbFe(II)NO is oxygenated, we added
KCN prior to opening the reaction flask. Thus, we prepared a deoxyHb
solution, added 1 eq of NO· from a saturated solution, added 10 eq KCN from a thoroughly degassed solution, and allowed it to react for
10 min. Then the reaction mixture was exposed to air and analyzed
immediately with the Saville assay. Interestingly, the relative SNO-Hb
yield (expressed relative to the amount of NO· added) was
1.7 ± 0.2%, almost identical to the corresponding relative yield
obtained in the absence of CN (1.5 ± 0.1%).
Reaction of Mixtures of oxy- and deoxyHb (1:1 and 3:1) with
NO·--
Since in vivo hemoglobin is present as a
mixture of the oxy and deoxy forms, depending on the dioxygen
concentration, we also measured the SNO-Hb yields obtained by addition
of a NO· solution to mixtures of deoxy- and oxyHb. The
percentages of oxygenated molecules were determined by measuring a
UV-visible spectrum immediately prior to the addition of the NO·
solution and by comparing it to a spectrum calculated from those of
pure deoxy- and oxyHb. In addition, the relative concentration of the
two hemoglobin species was controlled by using the equations described
by Benesh et al. (39). Thus, since it was essential to
measure a spectrum of the reaction mixture, the reactions had to be
carried out in sealable cells, and consequently it was only possible to
add NO· from a concentrated saturated solution (Method
1). The results of the experiments in 0.1 M buffer
show that the rapid addition of 1 or 0.1 eq of NO· to a 3:1
mixture of oxy- and deoxyHb (total protein concentration 50 µM) yielded 1.8 ± 0.3 and 5.6 ± 0.8% SNO-Hb
(relative to NO·), respectively. In 0.01 M phosphate
buffer, the yields were almost identical, 1.9 ± 0.5 and 6.0 ± 0.4% (relative to NO·), respectively. As for the reactions
only with deoxyHb, slightly, but not significantly, larger yields were
obtained when after the addition of 1 or 0.1 eq of NO· the
solutions were allowed to react for 10 min with air prior to the
S-nitrosothiol content analysis (2.1 ± 0.2 and
7.0 ± 0.1% in 0.1 M phosphate buffer and 2.3 ± 0.3 and 8.0 ± 0.8% in 0.01 M phosphate buffer, respectively).
Analogous experiments were carried out in 0.1 M phosphate
buffer with a 1:1 mixture of oxy- and deoxyHb. The rapid addition of 1 or 0.1 eq of NO· (from a concentrated solution, Method
1) to a 1:1 mixture of oxy- and deoxyHb (total protein
concentration 50 µM) yielded 1.5 ± 0.2 and
10.0 ± 0.6% SNO-Hb (relative to NO·), respectively. Only
slightly larger yields (2.0 ± 0.3 and 12.4 ± 0.7% SNO-Hb,
respectively) were obtained when the solutions were allowed to react
for 10 min with air prior to the S-nitrosothiol content analysis.
The reactions between the 3:1 or the 1:1 oxyHb/deoxyHb mixtures and 1 eq of NO· were followed by UV-visible spectroscopy. As shown in
Fig. 2B and 2C, the products of these reactions
corresponded approximately to a 3:1 and a 1:1 mixture of
metHb/HbFe(III)NO, respectively.
Reaction of metHb with NO·--
In contrast to
O2 and CO, NO· binds not only to deoxyHb but also to
the oxidized iron(III) form of hemoglobin. Fourier transform infrared
spectroscopy and resonance Raman data indicate that the resulting
nitrosyl complex, HbFe(III)NO, formally has a linear HbFe(II)(NO+) character, which means that partial charge
transfer from NO· to the iron(III) occurs during the
bonding process (50-52). HbFe(III)NO is not very stable, and
in the presence of an excess of NO· it undergoes reductive
nitrosylation to finally generate the reduced hemoglobin nitrosyl
complex, HbFe(II)NO (Equation 1). This reaction has recently
been shown to take place also within the red blood cells (33). In the
presence of substrates such as thiols, amines, and phenols, it has been
reported that the corresponding myoglobin complex MbFe(III)NO can act
as a nitrosating species (53).
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(Eq. 1)
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To find out whether HbFe(III)NO also acts as a
nitrosating agent, we mixed NO· with metHb both under anaerobic
and aerobic conditions and determined the amount of SNO-Hb generated.
The addition of 1 or 0.1 eq of NO· to 50 µM metHb
(in 0.1 M phosphate buffer) under argon led to the
formation of SNO-Hb yields significantly larger than those obtained
under the same conditions with oxy- and deoxyHb (Table III). No significant difference was
observed when NO· was added fast either from a saturated
(Method 1) of from a diluted solution (Method 2,
fast). Interestingly, in analogy to the reactions with oxy- and
deoxyHb, the addition of 0.1 eq of NO· to metHb led to
significantly larger relative SNO-Hb yields (expressed relative to the
amount of NO· added) than those generated by the addition of 1 eq of NO·. In addition, similarly to the reactions with oxyHb,
significantly larger amounts of SNO-Hb (approximately 2 times larger)
were generated when a diluted NO· solution was added slowly
(Method 2, slow). Surprisingly, when 1 eq of NO· was
added to metHb under aerobic conditions, only slightly larger SNO-Hb
amounts were generated (relative to the amount formed under anaerobic
conditions), independently of the method of NO· addition (Table
III, data in parenthesis). In contrast, the addition of 0.1 eq of
NO· under aerobic conditions generated approximately twice the
amount of SNO-Hb formed under anaerobic conditions, independently of the method of NO· addition (Table III, data in parenthesis).
Thus, the highest relative SNO-Hb yields (expressed relative to the
amount of NO· added) were obtained when 0.1 eq of a diluted
NO· solution were added slowly to the metHb solution (23 ± 3 and 42 ± 6% under anaerobic and aerobic conditions,
respectively).
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Table III
Amount of nitrosated Cys-93 formed from the reaction of metHb with
different amounts of NO· in 0.1 M phosphate buffer,
pH 7.2, under anaerobic and (in parenthesis) under aerobic conditions:
Influence on the concentration of the added NO· solution
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Reaction of oxyHb with NO· in the Presence of Added metHb or
metMb--
In a further set of experiments, we carried out the
reaction of oxyHb with NO· under aerobic conditions in the
presence of 1 eq metHb (relative to the oxyHb concentration). In all
cases, that is when 1 or 0.1 eq of NO· were added according to
Method 1 or Method 2 (fast and slow) the SNO-Hb
yields (expressed relative to the amount of NO· added) were
higher than those obtained from the corresponding experiments in the
absence of metHb (Table IV). The
difference between the yield obtained in the presence and in the
absence of added metHb was more significant in the experiment with 0.1 eq of NO·. In analogy to the reaction between oxyHb and
NO·, the SNO-Hb yields did not depend on the concentration of
the added NO· solution when it was added fast (Method
1 and Method 2 fast). In contrast, when the diluted
NO· solution was added slowly, the SNO-Hb yields were
significantly larger.
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Table IV
Amount of nitrosated Cys-93 formed from the reaction of a mixture of
oxyHb and metHb (50 µM each) with different amounts of
NO· in 0.1 M phosphate buffer, pH 7.2, under aerobic
conditions: Influence on the concentration of the added NO·
solution
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A similar reaction was carried out between oxyHb and NO· in the
presence of 1 eq of metMb (relative to the oxyHb concentration). Interestingly, when NO· was added from a saturated solution
(Method 1) we also obtained a significant increase in the
relative SNO-Hb yields, compared with that obtained in the absence of
added metMb. Indeed, the addition of 1 or 0.1 eq of NO·
(relative to the oxyHb concentration) to a mixture of 50 µM oxyHb and 50 µM metMb under aerobic
conditions led to the formation of 2.6 ± 0.1 and 10 ± 2%
SNO-Hb (expressed relative to the amount of NO· added).
Reaction of metHb with NO· in the Presence of Added
oxyMb--
Finally, we carried out the "inverse" reaction and
added under aerobic conditions 1 or 0.1 eq (relative to the metHb
concentration) of NO· from a saturated solution (Method
1) to a solution of 50 µM metHb and 50 µM oxyMb. In this case, we obtained 5 ± 1 and
15 ± 3% SNO-Hb (relative to the amount of NO· added) for
the addition of 1 or 0.1 eq of NO·, respectively. Interestingly,
these relative yields are only 30-40% lower than those obtained under
the same (aerobic) conditions in the absence of added oxyMb.
Reaction of oxyMb and metMb with NO· in the Presence of
GSH--
To determine the efficiency of oxyMb and metMb (and possibly
oxyHb and metHb) to nitrosate an external thiol, we carried out a set
of experiments analogous to those described above between NO·
and oxyHb or metHb, with the only difference being that Hb was replaced by an equimolar amount of Mb plus 0.5 eq of GSH. This ratio
was chosen to mirror the Cys-93/heme ratio in hemoglobin. As summarized in Table V, when
oxyHb was replaced by oxyMb/GSH, we observed the same trends and
similar GSNO yields (relative to the amount of added NO·) as
those discussed above for the reaction of NO· with oxyHb. In
particular, higher yields were obtained when NO· was added
slowly from a diluted solution, and higher relative GSNO (expressed
relative to the amount of added NO·) was obtained from the
reaction with 0.1 eq of NO·. Thus, the highest relative GSNO
yield, 16 ± 6% expressed relative to the amount of NO·
added, was obtained when 0.1 eq of a diluted NO· solution were
added slowly to the oxyMb/GSH solution. When metHb was replaced by
metMb/GSH, again the same trends in the GSNO yields were observed,
depending on the way the NO· solution was added. However, most
GSNO yields were ~2-3 times lower than the corresponding SNO-Hb
yields (Table V, data in parenthesis). Thus, the highest
relative GSNO yield, 13 ± 4% expressed relative to the amount of
NO· added, was obtained when 0.1 eq of a diluted NO·
solution were added slowly to the metMb/GSH solution.
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Table V
Amount of GSNO formed from the reaction of oxyMb (50 µM)
or (in parenthesis) from the reaction of metMb (50 µM)
with different amounts of NO· in the presence of GSH (25 µM) in 0.1 M phosphate buffer pH 7.2: The
experiments with oxyMb were carried out under aerobic conditions and
those with metMb under anaerobic conditions
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Reaction of metHbCN with NO·--
It has recently been
proposed that beside interacting with the heme and with Cys-93,
NO· can occupy hydrophobic sites within hemoglobin (8). To test whether noncovalent bonding of NO· played a role in our
reactions, we prepared a thoroughly degassed metHbCN solution, added
either 1 or 0.1 eq of NO· from a saturated solution
(Method 1), and allowed it to interact for 10 min. No
changes were observed in the UV-visible spectrum of metHbCN, suggesting
that, as expected, NO· cannot displace the strong cyanide ligand
from metHb (data not shown). Then we thoroughly degassed the reaction
mixture, opened the reaction vial, and finally immediately carried out
the Saville assay under aerobic conditions. Interestingly, we found
0.9 ± 0.2% and 4.0 ± 0.3% SNO-Hb (expressed relative to
the amount of NO· added) for the experiments with 1 and 0.1 eq
of NO·, respectively. Waiting 10 min or 1 h after opening
the reaction vials prior the Saville assay did not lead to a
significant increase in the measured relative SNO-Hb yields (~1.1%
after 1 h). Moreover, when 1 h elapsed after the addition of
NO·, prior to degassing and immediate analysis of the reaction
mixture, no significant change was detected in the SNO-Hb yields
(~1% relative to the amount of NO· added). The slow addition
of 1 eq of NO· from a diluted solution (Method 2,
slow) under the same conditions also led to approximately the same
SNO-Hb yield (1% relative to the amount of NO· added). Finally,
when 50 eq of NO· were added from a saturated solution
(Method 1), we obtained 1.2 and 1.3% SNO-Hb (relative to
the amount of NO· added) after 10 min or 1 h, respectively.
Taken together, our results suggest that in all of these experiments it
is conceivable that NO· is trapped in a hydrophobic pocket of
the protein and is not displaced by degassing. Interestingly,
Cys-93 is surrounded by hydrophobic residues. Thus, after
opening the reaction vials, O2 could also rapidly diffuse
into this pocket, react with NO·, and lead to the formation of
SNO-Hb.
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DISCUSSION |
Reactions of NO· with metHb or
metMb/GSH--
Among the three Hb forms studied, metHb is
the species most likely to be capable of nitrosating a thiol by
interaction with NO·. It has been shown that metHb binds
NO· and generates HbFe(III)NO, a nitrosyl complex that has
formally HbFe(II)(NO+) character (50-52), and could thus
act as a nitrosating agent. The corresponding myoglobin complex
MbFe(II)(NO+) has been shown to undergo NO+
transfer and, among others, react with thiols to generate
S-nitrosothiols (53, 54). Here we show that reaction of
metHb with NO· leads to nitrosation of Cys-93. Our
results indicate that the percentage of SNO-Hb generated relative to
the amount of NO· mixed with metHb depends on how the NO·
solution is added and is higher when substoichiometric amounts of
NO· are added. Maximal relative yields of SNO-Hb are obtained
when 0.1 eq of NO· are added very slowly to metHb from a diluted
solution (Method 2, slow). The higher relative SNO-Hb yields
obtained when 0.1 eq of NO· were mixed with metHb, compared with
the amount of SNO-Hb generated by the addition of 1 eq of NO·,
can be rationalized as follows. The association rates of NO· to
the and subunits of metHb are 6.4 × 103
M1 s1 and 1.71 × 103 M1 s1,
respectively (55). The dissociation rates of NO· from
HbFe(III)NO are 1.5 s1 and 0.65 s1, for the
and the subunits of Hb, respectively (55). Thus, under the
conditions of our experiments, all of these reactions have a half-life
on the order of seconds, and the system reaches equilibrium within a
few seconds. The equilibrium constants, calculated from the given
association and dissociation rates, indicate that the affinity for
NO· of the subunit is ~1.6 times larger than that of the
subunit. In addition, the percentage of the associated species
HbFe(III)NO generated by mixing 50 µM metHb with 50 or 5 µM NO·, expressed relative to the total amount of
NO· added, is 13 and 16%, respectively. Thus, when smaller
amounts of NO· are added to metHb, a larger fraction of
NO· is present as HbFe(II)(NO+) and may consequently
lead to higher relative SNO-Hb yields. However, since the difference in
the relative SNO-Hb yields obtained from the reaction with 50 or 5 µM NO· is about 10%, a value significantly larger
than the difference between the HbFe(II)(NO+)
concentrations (3%), this mechanism may represent only one of the
pathways responsible for the higher SNO-Hb yields.
The higher SNO-Hb yields obtained when the diluted NO· solution
is added slowly to the metHb solution, compared with the amount of
SNO-Hb generated by rapid addition, is difficult to explain. The
argumentation outlined above suggests that when NO· is added
slowly as a diluted solution, at the beginning there will be a large
excess of protein versus NO· and thus a large
relative amount of NO· bound to the protein. The fraction of
NO· present as HbFe(II)(NO+) will decrease
continuously by further addition of the NO· solution. However,
since the time needed to add the NO· solution is only about 1-2
min but the half-life of the hydrolysis of HbFe(II)(NO+) is
about 10 min (1.1 × 103 s1) (56),
this reasoning cannot explain the higher yields obtained when
NO· solutions are added slowly.
Interestingly, when NO· is added to metHb under aerobic
conditions the relative SNO-Hb yields are significantly higher only in
the experiments with 0.1 eq of NO·, whereas they are
approximately unchanged in those with 1 eq of NO·. The reaction
of NO· with dioxygen in aqueous solution is believed to proceed
according to the following mechanism (Equations 2-4) (57).
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(Eq. 2)
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(Eq. 3)
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and
TOP
ABSTRACT
INTRODUCTION
