Concentration changes of nitric oxide (NO) were
monitored using an NO-sensitive electrode in phosphate-buffered saline
(PBS) with either free oxyhemoglobin (oxyHb) or red blood cells (RBCs). In aerated PBS, the half-life of 0.9 µM NO is
greater than 4 min. NO is undetectable (<50 nM) when added
to a solution of oxyHb because the reaction of NO with oxyHb is rapid.
The disappearance rate of NO in PBS containing RBCs is rapid, compared
with PBS, but it is much slower (by a factor of approximately 650) than with an equivalent solution of free oxyHb. The half-life of NO is
inversely proportional to the concentration of RBCs, independent of
oxyHb concentration inside RBCs, and the disappearance rate of NO is
first order in NO concentration and first order in the concentration of
RBCs. After all the oxyHb reacts with NO to form methemoglobin, the
disappearance rate of NO slows greatly. These data indicate that the
reaction of NO with oxyhemoglobin within RBCs is limited by the
diffusion of NO into the cell, which has also been shown previously for
the reaction of O2 with deoxyhemoglobin. Experimental data
show that the half-life of NO in the presence of 2.1 × 106 RBCs/ml is 4.2 s. From this value, we estimate
that the half-life of NO in whole blood (5 × 109
RBCs/ml) will be 1.8 ms. A simple analytical expression for the half-life of NO in PBS with RBCs was derived in this study based on a
spherical diffusion model. The calculated half-life of NO from the
expression is in good agreement with the experimental values.
 |
INTRODUCTION |
Nitric oxide (NO)1 is
one of the 10 smallest, stable molecules of the hundreds of millions in
nature (1). According to Stokes' Law, the diffusibility of a molecule
in the condensed phase is inversely proportional to its molecular
radius, which thus makes NO one of the most rapidly diffusible
molecules known. Its diffusion constant (D) is approximately
3300-3800 µm2/s, whether measured in aqueous solution
(2) or in intact tissue (e.g. brain (3)). Membranes and
other hydrophobic structures in tissue are no barrier to diffusion of
NO because of its solubility in hydrophobic phases (4).
The reaction of free NO with oxyhemoglobin is rapid (bimolecular rate
constant k = 3.4 × 107
M
1 s
1
(5)), and from this rate constant it can be calculated that the
half-life of NO in the presence of a concentration of hemoglobin equivalent to that in the bloodstream (15 g/dl) would be very short,
approximately 2 × 10
6 s. As we have
pointed out previously (6, 7), the extremely rapid diffusibility of NO
coupled with its rapid reaction with oxyhemoglobin apparently poses a
difficulty in the postulate that free NO is the endothelium-derived
relaxing factor.
Using an electrochemical method, we describe here the results of
measurements of the disappearance of NO upon reaction with either
oxyhemoglobin in solution or oxyhemoglobin when contained within intact
erythrocytes. We find that, as reported in 1927 for the reaction of
O2 with deoxyhemoglobin (8), the NO reaction with intact
RBCs is considerably slower than with an equivalent concentration of
free oxyhemoglobin. We present a mathematical analysis of this
phenomenon, which demonstrates that the rate of the reaction of NO with
intraerythrocytic hemoglobin is limited by the rate of diffusion of NO
into the cell. From our data, we estimate that in whole blood the
half-life of NO will be less than 2 ms, which, although quite rapid, is
considerably longer than in the presence of free hemoglobin.
 |
EXPERIMENTAL PROCEDURES |
Preparation of NO Solution--
6 ml of phosphate-buffered
saline (PBS: 15 mM phosphate (potassium) plus 0.09% NaCl
pH 7.4) in a plastic vial was used in preparing saturated NO solution.
The solution was bubbled with argon gas (Aldrich) for 30 min and then
changed to NO gas (Aldrich) for 20 min. The NO gas was passed first
through a gas-washing bottle containing 1 M deaerated KOH
solution.
RBC and Free Hemoglobin Preparation--
Blood was withdrawn
from rats and centrifuged at 2300 × g for 10 min. The
plasma and buffy coat were discarded, and the RBC pellet was washed 3 times with PBS (pH 7.4). The packed RBCs then were added to PBS and the
solution was stirred gently. Cells were counted with a hemocytometer
and were stored on ice for use. To prepare free oxyHb, 2 ml of counted
RBCs was centrifuged at 2300 g for 10 min (4 °C). The packed
RBCs were then added to 40 ml of 5 mM phosphate solution
(pH 8), stirred and allowed to incubate for 30 min for hemolysis.
Electrochemical Measurements--
All electrochemical
measurements were carried out at 25 ± 2 °C by a BAS 100B
electrochemical analyzer with a PA-1 preamplifier and C2 cell stand
from Bioanalytical Systems Inc. (West Lafayette, IN) (9). A platinum
disc microelectrode was used as the working electrode for detecting NO.
An Ag/AgCl wire and a platinum wire were used as the reference
electrode and the auxiliary electrode, respectively. The
electrochemical cell is a plastic vial (2 cm in diameter, 2.86 cm in
height), obtained from Fisher. The working electrode, reference
electrode, and auxiliary electrode were inserted in the vial containing
the test solution through three holes on the cap. The sample was added
through another hole (diameter less then 1 mm) using a syringe. A
stirring magnet (1 cm) was placed in the vial for stirring the solution
during electrochemical measurements.
Spectrophotometric Analysis--
Absorption measurements were
carried out using a Beckman DU-64 spectrophotometer. The absorbance was
recorded immediately after adding NO to oxyHb solution. The amount of
oxyHb present during the NO titrations was followed by monitoring
absorbance and using a molar extinction coefficient of 1.58 × 104 M
1
cm
1 at 576 nm (10).
 |
RESULTS |
Disappearance of Free NO by Reaction with Free OxyHb and
RBCs--
Measurements of NO (solid tracing) and oxyHb
concentrations (bar graph) after repeated additions of NO to
a solution of oxyHb in an enclosed well stirred reaction vessel
(conditions under which the rate of volatilization of NO is negligible,
data not shown) are presented in Fig. 1.
Initial additions of NO (9 µM each addition) resulted in
decreased oxyHb concentration at a 1:1 molar stoichiometry, consistent
with the rapid reaction of NO to produce methemoglobin (5). NO was not
detectable because of its rapid disappearance (within the response time
of the electrode). However, after complete reaction (after two
additions of 9 µM NO to the solution that contains 18.4 µM heme), the rate of NO disappearance is dramatically
slowed, and so NO becomes detectable.

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Fig. 1.
Repeated additions of 9 µM NO
into a well stirred 4.6 µM free oxyHb (18.4 µM heme) solution. Top tracing, NO
concentration. Bottom graph, oxyHb concentration. The
additions of NO are designated by the arrows. OxyHb
concentration was monitored by absorption measurement at 576 nm.
|
|
Fig. 2 shows the disappearance of NO in
the presence of oxyHb and of RBCs. Tracing A is a
recording of NO concentration after sequential additions of 0.9 µM NO to 0.62 µM oxyHb (2.48 µM heme). Tracings B and
C are recordings of NO concentration in the presence of
0.7 × 106 RBCs/ml (1.05 µM total heme
concentration) and 1.4 × 106 RBCs/ml (2.1 µM heme), respectively. As is also true for Fig. 1,
tracing A shows that the reaction of NO (added at
the down arrows) with oxyHb is more rapid than the response
time of the electrode. NO is detectable only after complete reaction
with oxyHb (up arrow). However, tracings
B and C show that the reaction of NO with oxyHb,
when it is contained within RBCs, is much slower, and the time course
of NO disappearance becomes measurable. Reduction of methemoglobin
within the RBCs will not be significant under our conditions because
this process is much slower than the changes we are observing
(requiring at least 1-2 h even in the presence of reductant (11)).
Comparison of B with C shows that the rate of NO
disappearance is faster with higher concentrations of RBCs (0.7 × 106 RBCs/ml for B and 1.4 × 106 RBCs/ml for C). This is shown quantitatively
in Fig. 3, where the NO decrease after
addition is plotted as for a first order reaction for each cell
suspension. The calculated NO half-life for the higher RBC suspension
(Figs. 2B and 3A) is 12.9 ± 0.3 s,
whereas it is 6.4 ± 0.1 s for the lower suspension (Figs.
2C and 3B). The fact that the half-life for the
higher concentration of RBCs is approximately 50% of the half-life for
the lower concentration means that the reaction is first order with RBC
concentration (i.e. the rate constant is twice as large). In
addition, the rates are identical for multiple additions of NO,
conditions under which there is incremental loss of oxyHb
(Fig. 1). This shows that the rate is independent of the internal
oxyHb concentration. At the time when complete hemoglobin oxidation
occurs, the disappearance of NO slows dramatically and becomes similar
to the rate with free oxyHb after complete oxidation (Fig.
2A). This result indicates that oxyHb is converted to
methemoglobin uniformly throughout the entire RBC population, as
opposed to a rapid and complete conversion in only a fraction of the
RBC population. This is because if the latter were true then we would
not see an abrupt "switch" in the rate upon complete oxidation of
all the hemoglobin but rather a gradual decrease, which would be
similar in effect to gradually decreasing the number of RBCs (see Table
I).

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Fig. 2.
Repeated additions (denoted by the down
arrows) of 0.9 µM NO into aerated PBS
containing: A, 0.62 µM of oxyHb (2.48 µM heme); B, 0.7 × 106/ml
RBCs (1.05 µM heme); C, 1.4 × 106/ml RBCs (2.1 µM heme). Up
arrows indicate the point where all oxyHb completely reacted with
NO.
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Fig. 3.
First order plot of data from Fig. 2.
A, ln ([NO]/[NO]o)
versus time after NO addition for the two NO additions in
Fig. 2B (0.7 × 106 RBCs/ml). Filled
circles, first NO addition; open circles, second NO
addition. B, ln ([NO]/[NO]o)
versus time after NO addition for the three NO additions in
Fig. 2C (1.4 × 106 RBCs/ml). Filled
circles, first NO addition; open triangles, second NO
addition; open circles, last NO addition.
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Table I
Half-life of NO calculated from experimental data for different
concentrations of RBCs and different concentrations of NO at 25 °C
|
|
Table I presents the results of a series of measurements of NO
disappearance with increasing numbers of RBCs and (for the highest RBC
concentration suspension) two NO concentrations. As shown, the
half-life of NO decreases as the number of RBCs increases, and this
decrease predicts a first order dependence on RBC concentration. If the
number of RBCs is fixed, repeatedly adding NO does not change the
half-life of NO until the point that all the oxyHb completely reacts
with NO (Figs. 2 and 3). After this point, the half-life of NO
dramatically increases to a much larger value. This result means that
the half-life of NO is independent of oxyHb concentration inside RBCs
because oxyHb concentration inside the RBCs decreases incrementally
when we repeatedly add NO to the solution (Figs. 1 and 2). Thus, the
reaction of NO is first order in NO and RBC concentration but
independent (zero order) of intracellular oxyHb concentration.
A Diffusion Model for Reaction of Free NO with RBCs--
Hartridge
and Roughton (8) reported in 1927 that reaction (binding) of
O2 with deoxyhemoglobin occurs much more slowly when the
deoxyhemoglobin is contained within erythrocytes than when free in
solution. Numerous studies, both experimental and theoretical, have
examined this phenomenon further, and it is now believed that its
origin lies in the existence of an unstirred layer surrounding the
erythrocyte; the reaction of O2 is determined by the rate
at which O2 can diffuse into the cell and become accessible to the deoxyhemoglobin (12-15). Once inside the cell, the free O2 disappears virtually instantaneously because of the
rapid reaction with the high internal concentrations of
deoxyhemoglobin, making the rate of diffusion through the unstirred
layer rate limiting for O2 disappearance. Because the
diffusional properties of NO are similar to O2 (NO and
O2 differ by one atomic number) and the rate of reaction of
NO with oxyHb is also rapid, it is perhaps not surprising that the same
phenomenon (diffusion-limited disappearance with intact erythrocytes)
should hold true.
As described for the O2 reaction with deoxyhemoglobin
within RBCs (14), the rate of NO disappearance can be described by the
following empirical relationship,
|
(Eq. 1)
|
where D is the diffusion constant for NO,
is an
empirical mass transfer coefficient, cb is the
NO concentration in the bulk solvent, and cs is
the NO concentration at the first cytoplasmic layer. We present here an
analytical model that predicts a value for
, which is close to our
experimentally determined value.
This diffusion-reaction problem is mathematically analogous to that
describing current flow in a microelectrode, where the rate of the
measuring event is limited by the diffusion of the detectable species
(diffusion to the interior of the RBC or diffusion to the surface of
the electrode). We can thus apply similar mathematical analyses.
We assume that 1) the reaction of NO with oxyHb is so fast that NO
concentration inside the cell is zero (until all oxyHb inside the cell
is completely reacted with NO); 2) the rate of NO diffusion through the
cell membrane is so rapid that NO concentration on the surface of the
cell is close to zero; 3a) a small number of RBCs exists in solution,
and the average distance, d, between two adjacent cells is
much larger than the diameter of the cell; 3b) the transient diffusion
time t* (the time required for the diffusion to reach steady
state) for NO is much shorter than the half-life of NO in the measured
solution; and 4) reactions of NO with other compounds such as
methemoglobin and O2 can be ignored because they are much
slower than the reaction of NO with oxyHb. Assumption 2 is based on
experimental observations on oxygen uptake by RBCs (16). The transient
diffusion time t* is about 10 ms for oxygen diffusion into
RBCs (14), and so we assume that it is also short for NO and assumption
3b holds.
With these assumptions, the diffusion problem can be described by the
following equation,
|
(Eq. 2)
|
where c is the concentration of NO at location x,
y, z and cb is NO concentration in the bulk
solution. In our experiments, cb decreases with
time, but its half-life is much longer than the transient diffusion
time t* according to assumption 4.
By analogy with the diffusion problem for an electrode (17), the
diffusion rate of NO into the RBC can be expressed by the following
equation,
|
(Eq. 3)
|
where v is the rate (in mol/s) that NO diffuses to the
surface of a RBC, DNO is the diffusion
constant for NO, S is the surface area of the cell,
g is a constant for a given cell shape (i.e. spherical, discoidal, etc.), and
(
c/
x)0 is the concentration gradient at the cell surface. For simplicity, we assume that the shape
of the RBC is spherical; we note that experimental data with
measurements of NO binding to deoxyhemoglobin within RBCs (which, as
described under "Discussion," is a phenomenon similar in principle
to what we measure here) show that changing the shape of the RBC from
discoidal to spherical has no measurable effect on the rate of the
reaction (18). The RBC radius (average radius for all cells) and
surface area are r and S = 4
r2, respectively. The factor g
for a spherical cell is (4
)1/2 (19). Substituting these
values into Equation 3 gives the following equation.
|
(Eq. 4)
|
From mass conservation, the rate v of NO diffusion to
the surface of the cell from the bulk solution, must be equal to the rate of NO diffusion through the cell membrane,
|
(Eq. 5)
|
where km is the rate constant of the mass
transfer for NO diffusion through the cell membrane. The findings from
Equation 5 are as follows.
|
(Eq. 6)
|
Substitution of Equation 6 into Equation 4 gives the following
expression.
|
(Eq. 7)
|
If km is large enough, or
rkm
DNO as we
assumed in the beginning of this section, Equation 7 can be converted
to the following.
|
(Eq. 8)
|
Assuming the number of RBCs is N per cm3
(in units of cm
3), the total disappearance
rate of NO in the measured solution is as follows.
|
(Eq. 9)
|
Equation 9 shows that the disappearance rate of NO in the bulk
solution is first order in NO concentration cb.
The following is the first order rate constant.
|
(Eq. 10)
|
Solving Equation 9, we find the follow expression.
|
(Eq. 11)
|
The half-life, t1/2, which can be obtained from
Equation 11.
|
(Eq. 12)
|
If N = 2.1 × 106
cm
3, DNO = 3.3 × 10
5 cm2/s (2), and
r = 2.44 × 10
4 cm (20),
we have the rate constant kNO = 0.212/s and
the half-life t1/2 = 3.3 s. The experimentally
determined values were kNO
0.167/s and
t1/2
4.2 s (Table I). Thus, the model accurately
predicts the experimental results.
Combining Equation 1 with Equation 4 we have,
|
(Eq. 13)
|
where r is the radius of the spherical cell. This is
similar in form to the equation presented previously (14) for
O2.
 |
DISCUSSION |
By using an NO-selective electrochemical sensor, we have found
that the reaction of NO with oxyHb to produce methemoglobin and nitrate
is slowed substantially when the hemoglobin is contained within RBCs.
From Table I, we can calculate that the average first order rate
constant for the reaction of NO with RBCs is 7.74 × 10
8 s
1 on a per
RBC/ml basis (i.e. the rate of NO disappearance in a volume
containing 1 RBC/ml). Using our data in Figs. 2 and 3 and assuming an
average rat RBC volume of 59.7 µm3 (21), the rate becomes
5.16 × 104 s
1 on a per
molar heme basis. Comparison of this number with that determined by
Eich et al. (5) reveals that the reaction with free oxyHb is
slowed by a factor of approximately 650 when the oxyHb is contained
within RBCs. That the reaction with RBCs is not occurring via a small
amount of hemoglobin released from a few lysed RBCs is ruled out
because the concentration of free oxyHb required to yield a half-life
of NO of 6-12 s (Fig. 2) would be 1.7-3.4 nM (based on
the value for k reported by Eich et al. (5)), far
too small an amount to react with 0.9 µM NO (Fig. 2). We
point out that although this reaction is greatly retarded relative to
free oxyHb, the value for the half-life of NO in normal blood (5 × 109 RBCs/ml (22)) would still be very short, less than 2 ms. This makes the blood vessel lumen a potent sink for free NO and
poses a difficulty in postulating that only free NO accounts for the actions of the endothelium-derived relaxing factor, as we have pointed
out previously (6, 7).
We also present a theoretical model for predicting the
diffusion-limited reaction of NO, based on the similarity of this
phenomenon to diffusion-limited reaction at an electrode (17). We
predict from this model that on a per RBC/ml basis the rate of NO
disappearance will be given by the following expression.
|
(Eq. 14)
|
Using the values DNO = 3.3 × 10
5 cm2/s (2) and
r = 2.44 × 10
4 cm (20),
we arrive at a value for kNO/N of 1.01 × 10
7 s
1, which
compares favorably to our experimental value (0.774 × 10
7 s
1).
Hakim et al. (23) reported that the half-life of NO in the
presence of oxyHb (1.55 µM) is 11.5 s. They used a
probe similar to the Clark-type sensor for oxygen. This probe is
impressively sensitive to NO (as low as 10 nM), but its
response time to concentration change of NO is slow, 2.2-3.5 s for
75% rise time at an NO concentration of 1 µM (24). The
working electrode does not directly contact the measured solution. NO
must pass through the membrane from the measured solution and then
diffuse across the inner solution before it reaches the surface of the
working electrode. NO is homogeneously distributed throughout the inner
solution of the electrode system because of diffusion. If we add oxyHb
to the measured solution containing NO, oxyHb will remove all NO in the measured solution immediately. However, oxyHb cannot directly react
with NO in the inner solution because oxyHb entry into the inner
solution is prevented by the membrane. NO concentration in the inner
solution will still decrease with time because NO will both diffuse out
of the membrane and also be oxidized at the electrode. The
disappearance rate of NO in the inner solution will thus depend on the
surface area of the electrode and the volume of inner solution. We feel
that the curves recorded by Hakim et al. (23) in the
presence of higher oxyHb concentration are not the disappearance rate
of NO concentration in the measured solution, which is too fast to be
recorded by their electrode, but rather the disappearance rate of NO in
the inner solution after NO in the measured solution was removed by
oxyHb. Our working electrode directly touches the measured solution, so
its response time to a concentration change is much faster (~0.3 s)
than the electrode Hakim et al. (23) used.
Our experimental data and theoretical analysis imply that if a solution
contains oxyHb and we add NO to the solution, the rate of NO
disappearance is dependent on the distribution of oxyHb in the
solution. If oxyHb concentration is uniform in solution, the rate of NO
disappearance will be greater than if the same total amount of oxyHb is
contained within discrete membrane-enclosed sites in the solution, for
example, located within cells. Our results explain the observation that
enclosure of hemoglobin within erythrocytes attenuates its hypertensive
actions (25, 26).
Scavenging of NO is believed to be the major explanation for the
hypertensive effect of acellular hemoglobin, which is under active
investigation as a potential artificial red blood substitute (27, 28).
Indeed, this is one reason why nitrosation of a cysteine residue in
hemoglobin has been proposed as an important phenomenon in
endothelium-derived relaxing factor-dependent vasodilation (29, 30); by this mechanism nitrosonium equivalents can be carried by
hemoglobin, thus apparently avoiding the potent scavenging activity of
oxyHb for free NO (7).
It is known that encapsulating Hb within liposomes greatly attenuates
its hypertensive effect (as well as increasing its lifetime in the
circulation) (31, 32). This effect is generally attributed to an
increased scavenging of NO by free Hb resulting from tissue extravasation (33). According to our results, even without tissue extravasation, free hemoglobin will have a more than 500-fold higher
scavenging ability for NO than the same amount of hemoglobin contained
within RBCs. Our data explain the observation that polymerization of
diaspirin cross-linked hemoglobin (34), undertaken to prevent movement
into the interstitial space, does not improve its hemodynamic properties; we postulate that hemoglobin must be enclosed within a
membrane or diffusion barrier (such as in the RBC) to prevent hypertension.
Rudolph et al. (35) recently showed that liposome
encapsulation of Hb decreases its vasoconstrictive activity by
30-60-fold in rabbit arterial segments. Data from stopped-flow
spectrophotometric measurements were interpreted as suggesting that
encapsulation did not retard NO reaction with oxyHb, contrary to our
conclusions here. However, under their conditions (0.96 mM
NO, concentration which is in excess over the Hb concentration) it can
be calculated that the half-life for this pseudo-first order reaction
will be 0.02 ms (k = 3.4 × 107
M
1 s
1).
With an instrumental dead time of 2 ms, it is highly doubtful that
either this reaction or even the slower reaction with encapsulated Hb
would have been observed, which is in fact what was reported (i.e. the rate in both cases was too fast for detection). A
slower subsequent spectrophotometric change and then gradual decrease (presumably because of the binding of NO by the Fe(III)-heme followed by nitrosative hydrolysis (36, 37)) was detectable and was similar in
rate for both acellular and free Hb. However, as we point out above,
the effects of encapsulation will only be manifested if the rate of
reaction within the cell or liposome is more rapid than diffusion into
the cell or liposome, which will be true for reaction with
oxyferrohemoglobin but not reaction with methemoglobin. Our results
here suggest that the antihypertensive effects of liposome
encapsulation of Hb are because of a diminution of NO scavenging as a
consequence of the diffusional limitation of NO reaction with oxyHb and
lend further support to the utility of encapsulation in ongoing
research to design effective hemoglobin-based blood substitutes
(38).
In relatively hypoxic areas, there will be appreciable amounts of
deoxyhemoglobin present within RBCs, and NO also reacts with deoxyHb,
forming a heme iron-nitrosyl complex (39, 40). The rate of this
reaction is very rapid, and in fact its magnitude (k = 2.2 × 107 M
1
s
1) is similar to the reaction of NO with
oxyHb (k = 3.4 × 107
M
1 s
1)
(5). In fact, earlier work by Carlsen and Comroe (18) showed that
reaction of NO with deoxyHb within intact RBCs is, like reaction with
oxyHb, so rapid that it is limited only by how rapidly the NO can
become accessible to the hemoglobin within the RBC. Their rate constant
for this reaction was 1.15 × 105
M
1 s
1,
which compares favorably to the rate we report here (0.52 × 105 M
1
s
1), indicating the diffusion into the cell
is limiting for both reactions. This means that formation of NOHb can
compete with NO reaction with oxyHb, which explains why the electron
paramagnetic resonance signal from NOHb has been observed in whole
blood from septic animals (41) and why its magnitude is greater in
venous compared with arterial blood (42).
In the body, hemoglobin is confined within RBCs and RBCs are found
within blood vessels. Thus, the half-life of NO in the spaces between
blood vessels will be dependent on the distribution of blood vessels
and especially on the distribution density of capillaries (7). This
means that the half-life of NO in vivo cannot be specified
by a single number but will be different at different locations,
depending on vascularity.