Clinical Chemistry 43: 1442-1447, 1997;
(Clinical Chemistry. 1997;43:1442-1447.)
© 1997 American Association for Clinical Chemistry, Inc.
Binding of nitric oxide to thiols and hemes in hemoglobin H: implications for 
-thalassemia and hypertension
Poluru L. Reddy1,
Lemuel J. Bowie1,a and
Steven Callistein2
1
Clinical Laboratories, Evanston Hospital, 2650 Ridge Ave., Evanston, IL 60201-1783.
2
1323 W. Henderson, Chicago, IL 60657.
a Author for correspondence. Fax 847-570-2969; e-mail lem{at}merle.acns.nwu.edu
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Abstract
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Our earlier studies suggested an association between 
-thalassemia and
hypertension. We postulated that this association might involve
trapping of the vasodilator nitric oxide (NO) by hemoglobin (Hb). Hb A
has recently been shown to carry NO on its sulfhydryl groups in
addition to its hemes. In this report we studied the interaction of
purified Hb H as well as Hb A with NO. The number of reactive
sulfhydryls were determined spectrophotometrically with
bis-dithionitrobenzoate. Spectral studies and nitrosothiol
measurements after treatment with NO or nitrosothiols indicated that
all eight reactive sulfhydryls of Hb H were capable of binding NO. Hb
A, however, was only able to bind and transfer two molecules of NO per
tetramer. These findings support the biochemical basis for the
association between -thalassemia and hypertension.
Key Words: indexing terms: hemoglobin • thalassemia • sulfhydryl compounds • nitroso compounds • methemoglobin
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Introduction
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-Thalassemia is the most prevalent genetic trait in the world
(1), yet little is known about its impact on other
clinical conditions. This is partially due to the difficulty of testing
for this condition in clinical laboratories. Recently developed DNA
methods make it possible to detect not only the most prevalent
deletions that give rise to -thalassemia (2)(3)(4), but
also to determine the number and types of deletions present
(4). On the basis of the chronic mild to significant
hemolysis seen in various -thalassemia syndromes, we hypothesized
that -thalassemia may be a cause of hypertension, since hemoglobin
(Hb) is known to cause transient hypertension when introduced into the
bloodstream (5).1
The
mechanism for this effect is believed to be due to the essentially
irreversible binding of the vasodilator, nitric oxide (NO), by the
hemes of Hb.
We used a multiplex PCR method (4) to test samples for the
presence of the -3.7 deletion, the most prevalent -thalassemia
deletion in the US. These data demonstrate a significant correlation
between the presence of -thalassemia deletion(s) and the prevalence
of hypertension (5)(6) in hospitalized
African-American adults; we have proposed a model incorporating the
mechanisms described above (6). Recently, Jia et al.
[7] demonstrated that NO has an important function in the
normal regulation of blood pressure. Although these data support the
mechanism we propose, they do not address the potential impact of the
ability of Hb H (a tetramer of Hb ß-chains present in
-thalassemias) to trap NO. In this study we provide biochemical data
that support the involvement of Hb H in the trapping of NO. We propose
a model to explain the possible impact of the trapping of NO by Hb H in
-thalassemia.
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Materials and Methods
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specimens
Aliquots of EDTA-anticoagulated whole blood were obtained from
blood specimens that remained after diagnostic testing had been
performed in the clinical laboratories at Evanston Hospital. This
protocol was approved through the Institutional Review Board of
Evanston Hospital.
hb preparation
Hb A was prepared by centrifuging whole blood from healthy
individuals (no -3.7 deletion) at 1500g, removing the
plasma, and washing the erythrocyte pellet three times with isotonic
saline. The erythrocytes were then hemolyzed by adding an equal volume
of deionized water. The hemolysate was adjusted to a Hb concentration
of 130 g/L with deionized water and passed through a
SephadexTM G25 gel filtration column (2.5 x 30 cm;
Pharmacia Biotech, Uppsala, Sweden) that had been equilibrated with 0.1
mol/L NaCl. This procedure removes ~98% of 2,3-diphosphoglyceric
acid, an allosteric effector for Hb (8).
Hb H was prepared by separating the - and ß-chains of Hb A and
allowing the isolated ß-chains to spontaneously form tetrameric Hb H
(9). In this method p-mercuribenzoate (PMB) is
allowed to react with the sulfhydryls of Hb A. The modified chains have
sufficiently different charges and can be easily separated by
ion-exchange chromatography on CM cellulose (Sigma Chemical Co., St.
Louis, MO). After chain separation on a 2.5 x 30 cm column, the
free sulfhydryls were regenerated by passing the free ß-chains
through a 2.5 x 30 cm Sephadex G10 gel filtration column
equilibrated with 0.1 mol/L ß-mercaptoethanol. The tetrameric Hb H
was eluted and checked for purity by electrophoresis on agarose pH 8.6
(Beckman ParagonTM, Brea, CA) and by globin chain
electrophoresis.
sulfhydryl determinations
The number of reactive sulfhydryl groups on Hb A and Hb H were
determined with bis-dithionitrobenzoate (DTNB)
(10). The molar concentration of reactive sulfhydryls was
determined by measuring the corrected absorbance at 412 nm after
reaction with a minimum 10-fold molar excess of DTNB over total Hb
sulfhydryls available. The corrected absorbance at 412 nm was
determined by subtracting the absorbance contributed by Hb and
unreacted DTNB (obtained by measuring the absorbance of the respective
blanks) from the total absorbance at 412 nm.
nitrosylation of hemoglobins
Nitrosylation of Hb A and Hb H was achieved by in situ generation
of NO with sodium nitrite (10 mg) and buffered ascorbate (30 g/L
ascorbic acid in 0.1 mol/L phosphate, pH 7.4) (11).
Nitrosylation of Hb with NO gas was accomplished by generating NO gas
from sodium nitrite/buffered ascorbate in a closed container (2 x
5 cm screw-capped vial). A small centrifuge tube (1.5-mL capacity)
containing the Hb solution, along with a separate centrifuge tube
containing buffered ascorbate, were then placed in the vial. The
generation of NO gas was initiated by adding sodium nitrite crystals to
the tube containing buffered ascorbate. The vial was quickly capped and
the reaction was allowed to proceed for 10 min. Selective modification
of only the reactive sulfhydryls of Hb A and Hb H was achieved with
S-nitroso-N-acetylpenicillamine (SNAP)
(12). Nitrosylated Hbs formed by this method were analyzed
spectrophotometrically for the presence of heme-bound NO
(7) and chemically for the presence of sulfhydryl-bound NO
with the Saville reaction (13).
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Results
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To compare the binding properties of NO with Hb A and Hb H, Hb A
was "stripped" of the allosteric effector, 2,3-diphophoglyceric
acid, by gel filtration chromatography. This procedure was not
necessary for Hb H because the preparation of Hb H involved
dissociating the globin chains, which simultaneously removed
diphosphoglycerate.
Figure 1
shows the migration patterns of PMB-treated Hb obtained after
electrophoresis at pH 8.6 on agarose. Lane 4 shows the separation of
PMB-derivatized - and ß-chains. Note that the more negatively
charged ß-chains move faster toward the anode (left) while the
-chains do not migrate significantly and remain near the application
point (shown by arrow). Lanes 5–7 show the purity of products eluted
from the ion-exchange column at different pH values. Fractions that
eluted between pH 6.2 and 6.6 were essentially free of any -chains
and were used for subsequent analyses.
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Figure 1. Agarose electrophoresis of Hbs and modified Hbs.
Electrophoresis was carried out on agarose gels at pH 8.6 with a
Paragon electrophoresis cell and power supply. Lanes 1,
2, and 3 contain Hb controls for A and F; A, S, and C;
and A, respectively. Lane 4 contains the products from the
treatment of Hb A with PMB before ion-exchange chromatography on CM
cellulose. Lane 6 contains the eluate obtained at pH 6.2 as
the peak begins to elute from the column. Lane 5 contains
the pH 6.2 eluate after concentration by membrane ultrafiltration.
Lane 7 contains the eluate obtained at pH 6.6. The anode is
to the left and the cathode is to the right. The application point is
indicated by the arrow at the bottom of the figure. The
PMB-modified ß-chains (lanes 4, 5, 6, and 7)
are to the left of the figure and the PMB modified -chains
(lane 4) are toward the right.
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To determine the number of sulfhydryls in these purified fractions of
Hb A and Hb H, we performed spectrophotometric titrations with DTNB.
This reagent reacts with protein sulfhydryls accessible to the aqueous
environment. When it reacts, it gives rise to a chromophore with an
absorption maximum at 412 nm. Although Hb A (oxygenated conformation)
has one sulfhydryl on each -chain and two sulfhydryls on each
ß-chain, only one sulfhydryl on each ß-chain is "reactive" with
iodoacetamide (14). However, both sulfhydryls are reactive
on each of the four ß-chains of Hb H (14). Our data
obtained with DTNB confirm these findings (Table 1
).
Figure 2
shows the absorption spectra of purified oxygenated (solid
line) and deoxygenated (dashed line) Hb A. In comparison, Fig. 3
shows the spectra of oxygenated Hb H (solid line), highly
deoxygenated Hb H (PO2 = 32.9 mmHg,
dashed line), and Hb H fully deoxygenated by the use of dithionite
(dashed/dotted line). Although the spectra for oxyhemoglobin A and Hb H
are very similar, there are small shifts in the absorption maxima near
415 and 540 nm for Hb H. The spectra for fully deoxygenated Hb A and Hb
H are nearly identical. A major difference between the two is that Hb H
has a very high oxygen affinity and remains almost fully oxygenated at
the PO2 values present in the venous
circulation (Fig. 3
, dashed line). Hb H has been shown to have a
P50 (oxygen partial pressure resulting in 50%
saturation) of <1 mmHg (pH 7, 30 °C), whereas Hb A had a
P50 of 16.6 mmHg under the same conditions
(15). Therefore, to achieve full deoxygenation of Hb H,
dithionite must be used.
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Figure 2. Absorption spectra of Hb A.
Absorption spectrum of oxyhemoglobin A (110 µmol/L) in 0.1 mol/L
phosphate buffer, pH 7.4 (solid line). The sample was placed
in a closed 1-mm pathlength cuvette (Precision Cells, Hicksville, NY)
for spectral measurements. Absorption spectrum of deoxyhemoglobin A
(92.3 µmol/L) in 0.1 mol/L phosphate buffer, pH 7.4 (dashed
line). Hb A was deoxygenated (1.0 mL, 2.2 mmol/L) with nitrogen
for 15 min with an IL Model 237 tonometer (Instrumentation Laboratory,
Lexington, MA). The sample was then transferred anaerobically with a
gas-tight syringe and diluted ~20-fold into a 1-mm pathlength cuvette
containing phosphate buffer that had also been deoxygenated in the
tonometer.
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Figure 3. Absorption spectra of Hb H.
Absorption spectra of oxyhemoglobin H (110 µmol/L; solid
line), highly deoxygenated Hb H (110 µmol/L,
PO2 = 32.9 mmHg; dashed),
and fully deoxygenated Hb H (86.4 µmol/L with 0.01 mol/L sodium
dithionite; dashed/dotted line). Because of significant
absorption of dithionite, the spectrum of dithionite-treated Hb H is
not shown below 400 nm. All samples were dissolved in 0.1 mol/L
phosphate buffer, pH 7.4 and all spectral measurements were made in a
1-mm pathlength cuvette.
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When NO gas or sodium nitrite is allowed to react with Hb H in the
presence of oxygen, methemoglobin H is formed in a manner similar to
that described for Hb A (7). The spectrum obtained after
exposure of oxyhemoglobin A to NO gas is shown in Fig. 4
(dashed line). The reaction occurred in a closed vial as
described in Materials and Methods. Note the presence of the
characteristic peak for methemoglobin at 630 nm. Similar spectra are
obtained after exposure of Hb H to NO gas (data not shown). If NO is
generated in situ by adding solid sodium nitrite to buffered solutions
of oxyhemoglobin A or Hb H containing ascorbate, methemoglobin
formation is prevented. Under these conditions, NO is bound to heme
iron and the absorption spectrum shown in Fig. 4
(solid line) is
obtained. The reaction of NO with reactive sulfhydryls has no
significant effect on the absorption spectra above 500 nm
(7). Therefore, the spectral changes in Fig. 4
(solid
line) are related to the binding of NO to heme groups. Interestingly,
if Hb A is allowed to react with sodium nitrite in the absence of
ascorbate, methemoglobin is also formed (spectra not shown) and the
spectra show no significant change for at least 1 h. If, however,
Hb H is treated with sodium nitrite in the absence of ascorbate,
methemoglobin is formed initially (Fig. 5
, dashed line) but is rapidly converted to (ferrous)
nitrosylated Hb (Fig. 5
, solid line). Note that the spectrum of Hb H
obtained ~45 min after treatment of Hb H with sodium nitrite is
almost identical to that of nitrosylated Hb H (Fig. 5
, dashed/dotted
line) and nitrosylated Hb A (Fig. 4
, solid line) produced by sodium
nitrite/buffered ascorbate.
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Figure 4. Absorption spectra of Hb A treated with NO.
Absorption spectra of Hb A (131 µmol/L in 0.1 mol/L phosphate, pH
7.4) treated with NO gas (dashed line) and Hb A nitrosylated
in situ with sodium nitrite/buffered ascorbate (solid line).
See Materials and Methods for details. All spectral
measurements were made in a 1-mm pathlength cuvette.
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Figure 5. Absorption spectra of Hb H treated with sodium nitrite.
Absorption spectra of Hb H (69.8 µmol/L in 0.1 mol/L phosphate, pH
7.4) immediately after treatment with 10 g/L sodium nitrite
(dashed line) and after 45 min (solid line).
Absorption spectrum of Hb H (55.1 µmol/L in 0.1 mol/L phosphate, pH
7.4) nitrosylated in situ with sodium nitrite and buffered ascorbate
(dashed/dotted line). All spectral measurements were made in
a 1-mm pathlength cuvette.
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Jia et al. (7) have shown that NO also binds to the
reactive sulfhydryls of Hb A. Nitrosylation of these Hb sulfhydryls is
most readily achieved by transfer of the nitroso group from a
nitrosothiol to the protein sulfhydryls. To test whether the
sulfhydryls of Hb H could be nitrosylated in an analogous fashion, we
treated Hb H with SNAP, a nitrosothiol shown to be effective in
transnitrosylation reactions (12). The reaction mixture
was then passed through a G25 gel filtration column to remove excess
SNAP. The absorption spectrum of this modified Hb H containing
nitrosothiol groups on Cys residues was identical to Fig. 3
(solid
line) because heme groups were not modified by this procedure. The
nitrosothiolated Hbs were then analyzed for the number of nitrosothiols
with the Saville reaction (13). This method involved
mercuric ion-assisted hydrolysis of nitrosothiols to yield nitrous
acid. The nitrous acid produced was immediately reacted with
sulfanilamide and N-(1-naphthyl) ethylenediamine to form an
azo chromophore, which was monitored by its absorbance at 540 nm. The
number of nitrosothiols formed by reaction of Hb A and Hb H with SNAP
and detected by the Saville reaction is summarized in Table 2
. In contrast to Hb A, Hb H forms nitrosothiols with both of the
Cys residues on each of the four ß-chains. Hb H is therefore capable
of binding four times as many molecules of NO as Hb A.
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Discussion
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The ability of Hb solutions to affect blood pressure has been
known for some time (16)(17). For example,
Malcolm et al. (17) demonstrated that cross-linked Hb
solutions could increase mean arterial pressure as much as 25–30% in
a dose-dependent manner within 15 min of administration and that the
effect could last as long as 5 h. Although the effects are not
fully characterized, they are believed to be due largely to the
trapping of NO by Hb (18)(19). In vivo
experiments suggest that the endothelin-related pressor effect of
cross-linked Hb involves the stimulation of the conversion of
proendothelin to endothelin (19). NO is a smooth-muscle
relaxant that functions via activation of guanylate cyclase and the
production of cGMP (20) or by direct activation of
calcium-dependent potassium channels (21). Therefore, the
increased binding of NO, which could result from increased free Hb in
thalassemic patients or from the presence of an intraerythrocytic Hb
species that binds significantly more NO than Hb A, could result in
transient or sustained increases in blood pressure.
Jia et al. have demonstrated that NO is also bound to the reactive
sulfhydryls of Hb A and is transported to and from the tissues in a
manner analogous to the transport of oxygen by heme groups
(7). Our results indicate that Hb H also binds NO to heme
iron (Figs. 4
and 5
) as well as to reactive sulfhydryls (Table 2
).
Because the reaction of NO with reactive sulfhydryls of Hb to form
nitrosothiols does not give rise to a significant change in the
absorption spectrum in the region between 500 and 700 nm, direct
spectral measurements cannot be used to monitor these reactions.
However, hydrolysis of these nitrosothiols with mercuric ion results in
the release of eight molecules of NO per Hb H tetramer (Table 2
). Under
similar conditions, only two molecules of NO are released from Hb A
(Table 2
). Therefore, Hb H traps four times as many NO molecules (in
the form of nitrosothiols) as Hb A. Our data also suggest that Hb H is
less susceptible to oxidation in the presence of nitrite (and
presumably NO) since it has the ability to rapidly convert
methemoglobin H to ferrous nitrosylated Hb H. This may be related to a
more rapid rate of reduction of nitrosylated methemoglobin H because
reactions of ferric Hbs are more subject to subtle changes in protein
structure than ferrous Hbs (22)(23). For
example, the on and off rate constants for the binding of NO to ferric
ß-chains are greater than for binding to ferric -chains
(23).
On the basis of our observations and the data from Jia et al., we
propose the following model to explain the potential relation between
-thalassemia and hypertension (Fig. 6
). Fig. 6A
summarizes the transport of oxygen and NO by Hb A
(7). Note that NO is trapped by deoxyhemoglobin A (e.g.,
in tissues) but that the reaction of NO with oxyhemoglobin A in the
presence of oxygen (e.g., in the lungs) results in the formation of
methemoglobin A. This oxidized Hb cannot transport NO or oxygen at its
hemes and must be reduced in vivo by methemoglobin reductase. However,
oxyhemoglobin A can be nitrosylated at its sulfhydryls by
transnitrosylation with nitrosothiols in blood (e.g., nitrosocysteine,
nitrosoalbumin, etc.). The net result is that oxygen and NO (as
nitrosothiols) can be picked up by Hb A in the lungs and released at
tissue sites as the conformation of Hb changes from oxy to deoxy.
There is no significant change in protein conformation as Hb H (Fig. 6B
) passes through the lungs or through the tissues, since it has such
a high oxygen affinity. As a result, Hb H is unlikely to give up any NO
(whether bound to heme groups or sulfhydryls) as it passes through the
tissues. NO can be trapped by the heme groups of Hb H (Fig. 5
dotted
line) or it can be captured from nitrosothiols in plasma (Table 2
,
transnitrosylation of Hb H by SNAP). Therefore, in contrast to the
reversible transport of NO to and from tissues by Hb A, NO is
irreversibly trapped by Hb H. Although the amount of NO carried by Hb
is only ~1/10 000 that of oxygen (7)(24),
changes in this small fraction in vivo results in significant changes
in vascular contractility and blood pressure (7). Because
Hb H has four times the capacity to bind NO and once bound should not
give it up readily, small amounts of Hb H present in individuals with
-thalassemia could have a significant effect on mean blood pressure
unless compensatory mechanisms are activated.
The data presented here do not establish proof of the proposed
mechanism or describe all of the potential changes in -thalassemia
that could result in changes in hydrodynamic properties of blood or
vasoactive substances and lead to hypertension. However, further
studies of such changes could lead to a better understanding of the
pathophysiology of -thalassemia as well as its implications for
other clinical conditions.
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Acknowledgments
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We are very grateful for the excellent technical assistance
provided by Millicent Boykin and Darlene Morgan in assisting with some
of the data collection.
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Footnotes
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1 Nonstandard abbreviations: Hb, hemoglobin; PMB,
p-mercuribenzoate; DTNB, bis-dithionitrobenzoate;
and SNAP, S-nitroso-N-acetylpenicillamine. 
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Top
Abstract
Introduction
S