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Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5G 1L5, Canada
Submitted 3 March 2003 ; accepted in final form 22 April 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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47 Torr and Hb13 with PO2 
47 Torr. XLHb
increased Na-K-ATPase activity (86Rb uptake) in similar
PO2-dependent patterns. O2 consumption
(
O2) was measured in a
closed, well-stirred chamber. Ouabain- and oligomycin-inhibited
O2, reflecting
Na-K-ATPase activity and oxidative phosphorylation, respectively, mirrored the
PO2-dependent patterns of ATP and 86Rb
uptake. As PO2 fell below the midpoint of XLHb
desaturation, O2,
uncoupled from oxidative phosphorylation, transiently increased. The increase
was most pronounced with Hb34. Nitro-L-arginine methyl
ester had no effect on
O2. Inhibitors of NAD(P)H
oxidases and diamine oxidase partially prevented the
O2 surge with
Hb34. In conclusion, facilitated diffusion accounts for
PO2-dependent XLHb effects on ATP content and
Na-K-ATPase and for Hb13's effectiveness in hypoxic perfused
kidneys. NO scavenging was not a factor. O2-binding characteristics
influence XLHb effects on mitochondria and O2-sensitive enzymes
such as oxidases. ATP; Na-K-ATPase; Rb uptake; NAD(P)H oxidases; diphenyleneiodonium; aminoguanidine; nitro-L-arginine methyl ester; deferroxamine; oxidative phosphorylation
O2) was similar with the
two XLHb but venous PO2 was 40–60% lower when the
perfusate contained high-affinity XLHb
(4). High-affinity XLHB also
maintained tubular glucose and phosphate reabsorption more effectively. That
observation suggested that a high-affinity XLHb, which delivered O2
at very low PO2, maintained kidney function most
effectively. The XLHb with O2 affinity similar to that of
erythrocytes was less effective. Both XLHb facilitate O2 diffusion;
the only difference is the PO2 at which O2 is
released. Oxygenation of proximal tubules increases Na-K-ATPase activity and
converts the response to protein kinase C and protein kinase A from inhibition
to stimulation (19).
PO2 influences the activity of phospholipase A2, NAD(P)H
oxidases that produce reactive O2 species
(24), and oxygenases that
produce cytokines from arachidonic acid
(18). The products of these
various O2-dependent processes could modulate Na-K-ATPase and other
aspects of proximal tubule function. In the following experiments, we
investigated the biological mechanisms responsible for the beneficial effect
of high-affinity hemoglobin on proximal tubule function.
Another attribute of high-affinity XLHb may contribute to its superior
performance. It has a high affinity for nitric oxide (NO)
(29). NO inhibits oxidative
phosphorylation when PO2 is low
(1,
22). Under hypoxic conditions
that exist in the renal inner cortex and outer medulla, ATP production could
be inhibited by NO or limited by the availability of O2. Reduced
ATP production could limit Na-K-ATPase activity and Na cotransport of glucose
and phosphate (7). Therefore,
high-affinity hemoglobin could improve proximal tubular function by its
effects on O2 transfer or by scavenging NO. To examine these
possibilities, we measured
O2, ATP content, and
Na-K-ATPase activity (86Rb uptake) in proximal tubule fragments
incubated with highor low-affinity XLHb or bovine serum albumin (BSA).
Results obtained with tubule fragments exposed directly to XLHb in vitro may be extrapolated to tubules in vivo because of the close proximity between tubule cells and capillary lumens in the renal cortex. Two-thirds of epithelial cells' basal surface is only 0.15–0.4 µm distant from a capillary lumen. Capillary endothelium is thin and contains many fenestrae closed by 40–60 Å thick diaphragms (3). With this close approximation, XLHb circulating in capillary blood interacts almost directly with epithelial cells. Tetrameric XLHb, polymerized hemoglobin, and albumin all escape from capillaries and contact tubular epithelium before appearing in renal lymph (25). In vivo, some XLHb enters the tubule lumen and is reabsorbed in an O2-dependent manner (4).
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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1–82'); P50 34, Hill 2.5] or
Hb13 [trimesoyl-Hb (82–82'); P50
13, Hill 1.9] was prepared as previously described
(20,
21,
30). Hb34 contains
33% 
2(Val1)-Tm-(Lys82) and
67%
2(Val1,Lys82)-Tm-(Lys82).
Tm is the cross-linker trimesic acid. Hb13
(82-82'-Hb) contains 20%
2(Lys82,Lys144)-Tm-(Lys82) and
80%
2(Lys82)-Tm-(Lys82').O2
affinity was measured with a Hemox analyzer at 37°C; the parameters
measured in these lots of XLHb differ slightly from those described in our
previous publication in which P50 were 11 and 35. Rohlfs et al.
(29) report values of 15 and 2
for Hb13, which they called 82-Hb. Their values for
Hb34, which they called Tm-Hb, were 39 and 2.8. Tubules were prepared from outer cortical slices of Wistar rats as previously described (5). The kidneys were cleared of blood by infusing 60 ml of isotonic saline through the aorta. Slices were removed from the outer cortex with a Stadie-Riggs microtome placed in ice-cold saline and minced finely with a razor blade. The minceate was incubated at 37°C for 30 min in 6 ml of Krebs-Henseleit buffer containing 7.2 mg of collagenase (Sigma Chemical, St. Louis, MO) and 30 mg of BSA. The reaction was stopped with ice-cold buffer solution. The tissue was passed through a tea strainer, washed four times with buffer, and suspended in 30 ml of 45% Percoll in Krebs-Henseleit buffer. Tubules were separated from glomeruli by centrifugation in a 60TI rotor at 20,000 rpm for 20 min. Microscopic examination showed that the bottom layer containing 80–90% proximal tubule fragments with virtually no glomeruli. This layer was removed and washed four times with buffer and passed once through a 100-µm sieve. Tubules were kept on ice in a modified Krebs-Henseleit solution, which contained (in mM) 136 Na, 5 K, 111 Cl, 25 HCO3, 0.5 Mg, 1 Ca, 5 or 25 glucose, 2 lactate, 0.2 pyruvate, 2 glutamine, 1 arginine, 1 alanine, 1 heptanoic acid, and 10 g/l BSA. The pH was 7.4 at 37°C when equilibrated with 95% air-5% CO2.
For measurement of ATP concentration and 86Rb uptake, tubule fragments (0.8–1.6 mg protein) were incubated in 1.5–2 ml containing 1.1% BSA, 1% Hb13 + 0.1% BSA or 1% Hb34 + 0.1% BSA in a 50-ml Erlenmeyer flask at 37°C for 10 min with 95% air-5% CO2. The gas was then changed to 3–10% O2-5% CO2 for a further 10 min. ATP (ATP bioluminescent assay kit, Sigma Chemical) was measured with a Luminometer after addition of 30 µl of 70% perchloric acid.
Ouabain-sensitive 86Rb uptake by proximal tubule fragments was
used to measure Na-K-ATPase activity
(5) 86Rb
(Perkin-Elmer Life Sciences, Boston, MA) was added to produce an activity of
1 µCi/ml. Uptake was measured with and without 2.5 mM ouabain. Uptake
was terminated after 1 min by layering the tubule suspension onto 0.5 ml of a
2:1 mixture of dibutyl-dioctyl phthalate in a 1.5-ml centrifuge tube and
centrifuging for 10 s in an Eppendorf 5414 centrifuge. The medium above the
oil layer was removed, and the tubule was rinsed five times with distilled
water without disturbing the oil. The pellet of tubules was dissolved in 1 ml
of 0.1 N NaOH, and 200 µl of the solution were added to 10 ml of liquid
scintillation cocktail (Ready Safe, Beckman Coulter, Fullerton, CA) for
counting in a liquid scintillation counter. In preliminary experiments,
[3H]inulin was added with 86Rb. Less than 1% of the
3H passed through the oil with the tubules; therefore, in
subsequent experiments, we did not include [3H]inulin and did not
correct for trapped extracellular fluid.
Before measurement of
O2, tubules were
incubated at 37°C with 5% CO2-95% air for 30 min. In some
experiments, oligomycin (10 µmol/l), ouabain (2.5 mmol/l), aminoguanidine
(5 mM), or deferoxamine mesylate (1 mM) was added to the incubation solution
5–10 min before measurement of
O2.
Nitro-L-arginine methyl ester (L-NAME, 100 µmol/l)
was added 30 min before
O2 measurement.
Diphenyleneiodonium (DPI; 10 µM) was added directly to the measurement
chamber without preincubation. Chemicals were supplied by Sigma-Aldrich Canada
unless otherwise noted.
O2 measurement was
started by injecting tubules (0.8–1.6 mg of protein) into the 0.6-ml
analytic chamber (Diamond General Development). The chamber contained either
1% BSA or XLHb with sufficient albumin to maintain a constant 1% protein
concentration. Albumin and hemoglobin were dissolved in Krebs-Henseleit
solution and preequilibrated with 95% air-5% CO2 at 37°C.
PO2 was recorded polarographically with a YSI model 5300
biological oxygen monitor. Comparisons were done in duplicate or triplicate in
most experiments.
Many investigators have used the linear rate of change in O2
partial pressure (PO2) to measure
O2 by a variety of cells
and mitochondria. When tubules were incubated with 1% BSA alone, O2
content was calculated from PO2 and the solubility of
O2 in plasma at 37°C: O2 µmol =
PO2 (Torr) x 0.6 ml x 0.001257 µmol
· ml-1 · Torr-1
(12). A simple linear
relationship between PO2 and O2 content does
not exist when the solution contains hemoglobin. Instead, O2
content can be described by a function that includes
PO2, the concentration of hemoglobin, hemoglobin
O2 affinity (P50), and the Hill coefficient (Hill). For
solutions containing hemoglobin, the content was calculated as follows:
O2 µmol = PO2 (Torr) x 0.6 ml
x 0.001257 µmol · ml-1 · Torr-1 +
{Hb g/dl x 0.62 x 0.6 ml x (1-methemoglobin) x
[PO2Hill/(PO2Hill
+ P50Hill)]}.
Output from the YSI O2 monitor was recorded with a Biopac
Systems MP100 recorder at 2 samples/s. The data were transferred to an Excel
program, which calculated
O2 from an average of the
O2 content and
O2 over consecutive 15-s
intervals. The program also made corrections for atmospheric pressure and
changes in methemoglobin. For the latter calculations, it was assumed that
methemoglobin increased linearly with time during each run. Methemoglobin
content of the hemoglobin solution was measured before addition of tubules and
at the end of each run (13).
The accuracy of the equation was confirmed by equilibrating XLHb solutions
with various O2 concentrations and comparing calculated
O2 content with O2 content measured by a
LexO2Con analyzer (Lexington Instruments, Waltham, MA). Sensitivity
analysis of the predicted O2 content was done by varying
P50 and Hill coefficients used in the calculations.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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When tubules were incubated with BSA, ATP content increased in a sigmoidal fashion relative to PO2 (Fig. 1A). Incubation with XLHb at 47 and 72 Torr PO2 increased ATP content by 22–33%. Hb13 also sustained 10% higher ATP at 22 Torr PO2. Thus the effects of Hb13 on both ATP and Rb uptake were left-shifted relative to Hb34.
To analyze the relationship between ATP concentration ([ATP]) and Na-K-ATPase activity, [ATP] was calculated by assuming that tubule cells contained 2 µl water/mg protein (Fig. 2), which is within the range found by others (1.6–2.2 µl/mg protein) (10, 31, 32). During incubation with BSA, Na-K-ATPase activity was independent of [ATP] above 2 mM. We assumed that the relationship between [ATP] and 86Rb uptake is described by Michaelis-Menten kinetics. Km was 0.88 ± 0.37 mM, and Vmax was 147 ± 8 µmol · min-1 · mg-1 for the rectangular hyperbola fitted to the BSA data using Sigma Plot 4.01. This Km is in the range found by direct measurement of Na-K-ATPase in cell membranes (17, 31). The XLHb produced O2-dependent increases in Na-K-ATPase relative to [ATP], which suggests that factors in addition to [ATP] were responsible for increased activity.
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O2.
To examine the effects of XLHb on various components of
O2, we used several
inhibitors. Oligomycin blocks
O2 for ATP production by
oxidative phosphorylation, and ouabain blocks O2 and ATP
consumption by Na-K-ATPase. Other inhibitors were used to block O2
consumed by oxidases.
After addition of tubules to a BSA solution, PO2
decreased linearly down to 20 Torr or less
(8). O2 content of a
BSA solution is directly related to PO2; therefore,
O2 was calculated from
the rate of decrease in PO2. After addition of tubules
to a XLHb solution, PO2 decreased in a nonlinear fashion
(inset in Fig. 3).
O2 content was calculated by using the XLHb concentration and the
Hill coefficients and P50 values previously measured for
Hb34 with a Hemox analyzer at 37°C (central thick line). A
range of values for the Hill coefficient and P50 were used to show
sensitivity of the
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O2 calculation. The
P50 values and Hill coefficients reported by Rohlfs et al.
(29) yield slightly lower peak
O2 consumption but do not
produce major changes in the overall pattern. In preliminary experiments using
5 and 10 g/l of the XLHb, concentration had no significant effect on total
O2 nor on the shape of
the curves relating O2 to
PO2; therefore, we used 5 g/l for all subsequent
experiments. The initial methemoglobin content of hemoglobin solutions was 7.5
± 0.5% for Hb13 and 12.6 ± 0.1 for Hb34.
Methemoglobin increased by 0.28 ± 0.03%/min with Hb13 and
0.20 ± 0.03%/min with Hb34 reaching 10.6 ± 0.3 and
14.1 ± 0.2%, respectively, by the end of the measurement. In the
calculation of O2 content, we assumed that methemoglobin increased
at a constant rate.
Figures 4,
5,
6,
7 show
O2 curves sampled at
intervals of 5 Torr PO2. Duplicate or triplicate
measurements with a tubule preparation were averaged before carrying out
statistical analyses based on the number of tubule preparations. O2
content fell most rapidly when PO2 decreased below the
midpoint of the XLHb O2 dissociation curves. Consequently, as
PO2 fell from 65 to 25 Torr with Hb34,
O2 surged up briefly.
With Hb13, O2
increased below 15 Torr PO2, but to a much smaller
extent (Fig. 4). The surge in
O2 remained when
oxidative phosphorylation was inhibited by oligomycin
(Fig. 4 below).
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Both XLHb increased oligomycin-sensitive
O2 with Hb13
by up to 30% when PO2 was between 5 and 10 Torr
(P = 0.05–0.01). With Hb34, the increase was up to
20% when PO2 was between 20 and 40 Torr, but no single
point reached statistical significance (P = 0.15–0.2)
(Fig. 5A). Nystatin
(10 µM), which raises Na-K-ATPase activity by increasing Na entry into the
cell, stimulated oligomycin-sensitive
O2 with Hb34.
The effect was most notable between 20 and 40 Torr, which confirms the
PO2-dependent stimulation of Na-K-ATPase by
Hb34 (P = 0.02–0.005 for 4 paired measurements in 2
tubule preparations).
With XLHb, ouabain-sensitive
O2 also appeared to be
sensitive to PO2, but none of the points reached
statistical significance (Fig.
5B). The insensitivity of rat tubules to ouabain makes
oligomycin a more reliable index of mitochondrial ATP production than ouabain.
O2 affinity of the XLHb influenced the patterns of increased
ouabainand oligomycin-sensitive
O2
(Fig. 5). Likewise,
O2 affinity influenced the PO2-dependent
increases in 86Rb uptake (Fig.
1). Rb uptake was measured in a steady state with constant
O2 concentration, whereas
O2 measurements were made
under non-steady-state conditions as O2 concentration decreased
over 3–8 min.
Both methods indicated increased Na-K-ATPase activity with XLHb. XLHb did
not significantly alter the ratio of Rb uptake to ouabain-sensitive
O2. The ratio was
calculated for Rb uptake from 47 to 147 Torr and for
O2 from 30 to 110 Torr.
The averages were BSA 9.2, Hb34 9.8, and Hb13 10.9.
Under optimum conditions with oxidation of NADH-linked substrates,
mitochondria should synthesize 4–6 ATP for each O2 that is
consumed (16). Assuming that
Na-K-ATPase transports 2 Rb (or K) for each ATP consumed, then the ratio of Rb
uptake to ouabain-sensitive
O2 should be
8–12.
Several inhibitors of oxidases were tested. Hb34 increased
sensitivity to aminoguanidine, an inhibitor of diamine oxidase and nitric
oxide synthase activity (NOS) (Fig.
6B). L-NAME, which blocks NOS
(Fig. 6, inset),
tended to stimulate rather than inhibit
O2, but the change was
not significant; therefore, the response to aminoguanidine was not due to
inhibition of NOS. DPI, an NAD(P)H oxidase inhibitor, decreased the surge of
O2 caused by
Hb34. DPI-sensitive
O2 rose significantly as
PO2 fell to 20 Torr
(Fig. 6A). Oligomycin
added during DPI inhibition exerted its full effect; therefore, DPI and
oligomycin acted additively (Fig.
7). DPI only inhibited the burst of
O2. In this respect, it
differed from oligomycin, which reduced
O2 over the entire range
of PO2 (Fig.
4).
Deferoxamine (1 mM) tended to reduce
O2 with Hb34
or BSA, but the 4 nmol · min-1 · mg-1
decrease in peak O2 was
not statistically significant (paired t-test, P = 0.10,
n = 5). Deferoxamine also tended to decrease the production of
methemoglobin (95% confidence interval 0–6%).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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Hypoxic conditions in the inner renal cortex may limit oxidative
phosphorylation by reducing O2 delivery. Hypoxia also favors
competitive inhibition of oxidative phosphorylation by NO
(22). Hb13 and
Hb34 could support oxidative phosphorylation by facilitating
O2 diffusion to cells and by scavenging NO. Both XLHb are NO
scavengers, but Hb13 has a higher affinity for NO
(29), which might account for
its beneficial effects in the isolated perfused kidney. However, if NO
scavenging is a factor, then blocking NO synthesis with L-NAME
should have reduced the effects of Hb34 on
O2. This did not happen
(Fig. 6). Furthermore, in
previous experiments, L-NAME did not alter 86Rb uptake
by proximal tubules incubated with BSA
(6). Therefore, it is unlikely
that XLHb stimulated Na-K-ATPase or
O2 by removing
extracellular NO. XLHb is not likely to have entered tubule cells and altered
intracellular NO during the short duration of these experiments. Improved
O2 delivery through facilitated diffusion is a more likely
explanation for the beneficial effects of Hb13.
XLHb facilitate O2 diffusion by binding O2 at the high side of a PO2 gradient and releasing it at the low side. O2 transfer depends on the product of the diffusion constant and the amount of O2-hemoglobin in solution. Free O2 diffuses much more rapidly than O2 bound to hemoglobin, but the solubility of O2 is low, so the net transfer rate is low. XLHb diffuse more slowly but can hold much more O2 in solution. O2-binding equilibria determine the PO2 levels at which O2 is bound and released from the XLHb. To examine facilitated diffusion, McCarthy et al. (26) perfused an O2-permeable capillary surrounded by N2 gas with tetrameric Hb with low and high O2 affinity (P50 33 and 15 Torr, respectively). The high-affinity XLHb facilitated diffusion more effectively when PO2 was low. Hb13 and Hb34 are hemoglobin tetramers like those used by McCarthy et al.; therefore, we expected that Hb13 would deliver O2 most effectively at low PO2.
In our experiments, ATP content provides a biological index of facilitated
diffusion. ATP content decreased in a sigmoidal fashion as the equilibrating
O2 concentration was lowered
(Fig. 1). ATP production by
oxidative phosphorylation, indicated by oligomycin-sensitive
O2, and ATP consumption
by Na-K-ATPase, indicated by ouabain-sensitive
O2 and 86Rb
uptake, were insensitive to PO2 until it fell below 20
Torr (Figs. 1 and
5). Decreasing [ATP] with
relatively constant oxidative phosphorylation is consistent with a kinetic
model of oxidative phosphorylation developed by Korzeniewski
(23). His model predicts that,
when respiration is 70–80% of maximum, lowering mitochondrial
O2 concentration from 30 to 3 µM can decrease [ATP] by 80% with
only a slight decrease of
O2 and ATP production (B.
Korzeniewski, personal communication). The PO2-dependent
effects of Hb13 and Hb34 on [ATP] are consistent with
their effects on facilitated diffusion as a function of
PO2.
Na-K-ATPase activity was independent of [ATP] above 2.5 mM during incubation with BSA (Fig. 2). Yet Hb13 and Hb34 produced PO2-dependent patterns of increased Na-K-ATPase activity relative to [ATP], which suggests that increased O2 transfer stimulated Na-K-ATPase activity by factors in addition to [ATP]. There is precedent for this suggestion (14). Oxygenation with 100% O2 for 15 min increased total Na-K-ATPase hydrolytic activity in proximal tubule fragments by 25% when compared with tubules incubated with air. In those experiments, Na-K-ATPase activity was measured with exogenous saturating concentrations of ATP. Increased oxygenation also converted the Na-K-ATPase response to protein kinase C or protein kinase A from inhibition to stimulation (14, 19). Féraille et al. (14) attributed the Na-K-ATPase stimulation to reduced phospholipase A2 activity (28). Phospholipase A2 is activated by hypoxia and releases arachidonate. Lipoxygenases act on arachidonate to produce derivatives that inhibit Na-K-ATPase (14, 19). Oxygenation blocks this inhibitory pathway by preventing phospholipase A2 activation.
As PO2 fell and O2 began to escape from
XLHb, O2, unrelated to
oxidative phosphorylation, increased rapidly, reaching a peak at the midpoint
of Hb13 desaturation (P50 13 Torr) and 10–15 Torr
below the midpoint for Hb34 (P50 34 Torr)
(Fig. 4B). The burst
of O2 was most evident
with Hb34; therefore, we examined it in more detail. A nonspecific
inhibitor of NAD(P)H oxidases inhibited two-thirds of the brief
oligomycin-insensitive increase (Fig.
6A). NAD(P)H oxidase activity is found in plasma
membranes and is a source of reactive O2 species (ROS) in the
proximal tubule (24). NAD(P)H
oxidase in plasma membranes activates hypoxia-inducible factor-1 and may be a
factor in the cellular response to hypoxia
(27). Diamine oxidase,
inhibited by aminoguanidine, was also stimulated by Hb34. Absence
of a response to L-NAME indicates that NOS, which is also inhibited
by aminoguanidine, was not involved (Fig.
6). A PO2-dependent pattern of increased
then decreased O2 similar
to that shown in Fig. 4 could
be produced by O2-consuming processes with non-Michaelis-Menten
kinetics due to substrate (O2) inhibition. Juranek et al.
(18) show such a relationship
between O2 concentration and cyclooxygenase-1.
Uncoupling of oxidative phosphorylation triggered by increased ROS
production in the mitochondria or by redox reactions with hemoglobin
(2,
9) might also have contributed
to the O2 surge. However,
ROS production by oxidation of Hemoglobin to methemoglobin was low and similar
for Hb34 and Hb13 and is unlikely to have been a major
factor accounting for the difference in responses to the two XLHb.
Furthermore, blocking ROS with deferoxamine decreased peak
O2 with Hb34
by a statistically insignificant 4 nmol/min.
The effects of Hb13 and Hb34 on proximal tubular
function are summarized in Fig.
8. Hb13 facilitates O2 diffusion more
effectively than Hb34 under hypoxic conditions. This is manifested
by increased [ATP]. Stimulation of ATP production and Na-K-ATPase activity by
increase O2 delivery at low PO2 could account
for improved reabsorption of phosphate and glucose by proximal tubules in
perfused kidneys (4).
Additional factors such as reduced phospholipase activity may have contributed
to O2-dependent increase in Na-K-ATPase activity above that due
simply to increased [ATP]. NOS activity and ROS production do not appear to
have been major factors in the responses to XLHb. Within a narrow window of
O2 concentrations around its P50, Hb34
stimulated oxidase activity and other forms of
O2 unrelated to oxidative
phosphorylation. Increased XLHb-induced oxidase activity in vivo could produce
cytokines that influence the function of proximal tubules and surrounding
vascular tissue and contribute to the vasoconstriction that follows XLHb
transfusion (29).
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DISCLOSURES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESACKNOWLEDGMENTSREFERENCES |
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82)-(Lys82))-hemoglobin. J Org
Chem 59: 733-736,
1994.[Web of Science]
intensive work transition.
Biophys Chem 83:
19-34, 2000.[Web of Science][Medline]
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