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Blood, Vol. 91 No. 9 (May 1), 1998:
pp. 3467-3470
Comparative Oxidation of Hemoglobins A and S
By
Kuan Sheng,
Michelle Shariff, and
Robert P. Hebbel
From the Department of Medicine, University of Minnesota Medical
School, Minneapolis.
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ABSTRACT |
The mutant hemoglobin S (HbS) previously was reported to undergo
accelerated autooxidation during incubation in vitro. However, subsequent observations have raised the possibility that this might be
explained by adventitious association of molecular iron with HbS,
rather than reflecting an inherent property of HbS. Using purified HbA
and HbS obtained from genotypic HbAS donors, we found that the observed
oxidation rate of HbS, but not of HbA, is indeed exaggerated by
adventitious iron. This result suggests a preferential partitioning of
molecular iron to HbS over HbA, which was further supported by
experimentation. However, after elimination of this effect, there still
remains a significant increase in inherent autooxidation rate for HbS.
Physiologic oxidants (superoxide, peroxide, hydroxyl radical) and
various Fe(III) chelates all stimulate oxidation of oxyHb, but they do
so equivalently for HbA and HbS. Nevertheless, these mechanisms also
would contribute to excessive biologic oxidation of HbS because the
cytoplasm of sickle red blood cells, unlike that of normal cells, would
be exposed to abnormal amounts of oxidants and low-molecular-weight iron compounds.
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INTRODUCTION |
THE MUTANT HEMOGLOBIN (Hb) that defines
sickle cell anemia, HbS, is reported to exhibit an accelerated
autooxidation rate during incubation in vitro.1,2
Consistent with this, intact sickle red blood cells exhibit a
correspondingly exaggerated generation of activated oxygen species
during incubation in vitro.3 On the other hand, no
structural or biochemical explanation for this enhanced oxidation
tendency has been forthcoming. In fact, the two Hbs are isomorphic at
the level of x-ray crystallography, have multiple identical functions,
and show only minor differences at the N-terminal end of their
respective  -globin chains.4 HbS does manifest one
dramatic behavioral difference in the form of an abnormal surface
denaturation tendency4 that is derived from the mutant
Hb's hydrophobic surface substitution (6Glu Val).
While this probably underlies heme pocket disturbances during interfacial interactions between Hb and chemical
denaturants5 or phospholipids,6 it comprises an
uncertain proximate cause of abnormal Hb oxidation in solution.
Given this background, more recent observations require consideration
of the possibility that observation of augmented HbS oxidation actually
results from the presence of adventitious iron carried by the purified
Hb. In first place, the cytosolic aspect of the membranes of
sickle but not of normalred blood cells carries substantial amounts
of molecular iron.7 Despite its association with the
membrane with very high avidity,8 this Fe3+ is
redox-active. For example, it can form a redox couple that, even while
remaining membrane-bound, oxidizes soluble oxyHb to metHb.9
This presumably corresponds to the known ability of Fe3+
chelates to promote oxidation of oxyHb.10,11 Of particular relevance, binding studies demonstrated that Fe3+ binds to
hemichrome,12 denatured low-spin ferric Hb, should this be
present; HbS is more likely than HbA to form hemichrome from metHb as a
result of its surface denaturation behavior.4,13
Thus, the possibility exists that adventitious iron, whether
physiologic (eg, derived from cytoplasm) or an outright contaminant (eg, derived from experimental buffers), could explain the previous impression that HbS has an abnormal tendency to autooxidize. This is
important to resolve because a tendency for HbS to undergo enhanced
oxidization (either because of intrinsic properties of the Hb or
because of physiologic adventitious iron) has significant implications
for the cellular pathobiology of sickle cell disease, in which red
blood cells develop numerous membrane defects that are believed to be
oxidative in etiology.14
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MATERIALS AND METHODS |
Materials.
Chromatography supplies were obtained from BioRad (Hercules, CA), and
reagents from Sigma Chemical (St Louis, MO). With the exception of Hb
and iron itself, all reagents, solutions, and materials used in these
experiments were treated for at least 3 hours before use to remove any
contaminating iron using an iron-chelating resin, Chelex-100 (Sigma),
at 1 g per 100 mL solution.
Hb.
Fresh red blood cells were obtained from donors with sickle trait (ie,
having both HbA and HbS), so that both Hbs studied would be from the
same intracellular environment. The two Hbs were isolated, purified,
and stripped (of organic phosphates and counter ions) using four
sequential chromatographic steps (DEAE-cellulose, Sephadex G100,
AG11A8, and AG501×8), as recommended by Caughey and
Watkins.15 HbA and HbS were then dialyzed together against the same solution of experimental buffer: 140 mmol/L NaCl, 20 mmol/L
HEPES, pH 7.0.
Hb oxidation.
Hb at 1 mg/mL (15.5 µmol/L Hb tetramer, or 62 µmol/L in heme) and
the various reactants, each formulated in experimental buffer (with pH
adjusted as necessary to 7.0), were prewarmed separately and then
admixed in a temperature-controlled spectrophotometer (37 ± 0.1°C). Absorbance at 577 nm was monitored every 30 seconds for 30 minutes to directly measure loss of oxyHb. Simultaneous monitoring of an isosbestic point for oxyHb and metHb (587 nm) showed
no change in A587 over the course of these experiments, confirming that no species other than oxyHb and metHb appeared in the
system to introduce artifacts. Measurements verified that pH remained
stable at 7.0 ± 0.01 U during all of these experiments. At this pH,
some metHb would be in the form of ferrihemoglobinOH, but this would
comprise a small percentage of the total metHb (<10%) at this pH,
and its extinction coefficients indicate that its effect on results
measured in this fashion would be to blunt, not augment, apparent
oxidation rate.16
To calculate results, the measured absorbance changes were expressed on
a log scale and plotted against time, so that initial oxidation rates
could be calculated from the slope of the resulting linear plots. Hb
oxidation rates are expressed here as the disappearance of oxyHb in
units of micromolar heme per minute (µmol/L/min).
Variables.
Except as specifically noted later, all experiments were conducted with
Hb at 1 mg/mL. To identify the inherent oxidation rate and show the
presence of adventitious iron, the oxidation rate of the purified Hbs
was compared with and without inclusion of 10 µmol/L DTPA, a
hexadentate chelator that does not itself stimulate Hb oxidation (see
Results).
To identify the oxidation-stimulating effect of iron chelates, we
admixed Hb with iron chelates composed of 50 µmol/L Fe3+
and variable chelator concentrations chosen so that greater than 99%
binding of iron was assured in each case, based on reported chelator
affinities. Expressed as log of the cumulative stability constant,8 these affinities are as follows: deferiprone
(L1), 35.9; EDTA, 25.0; nitrilotriacetic (NTA), 24.3; citrate, 11.5; ATP, adenosine triphosphate, approximately 6; and adenosine
diphosphate, approximately 4. Affinities for 2,3-disphosphoglycerate
(23DPG) and glutathione (GSH) are not known. For these experiments,
results are reported as the absolute increment in Hb oxidation rate
measured in the presence of the iron chelate versus the presence of
chelator without iron and thus eliminate any potential effects of the
chelators themselves on absorbance measurements.
In other experiments, Hbs were exposed to enzymatically generated
physiologic oxidants. Superoxide (·O2 ) was
generated using 500 µmol/L xanthine, 1 × 104 U/mL
xanthine oxidase, plus 10 µmol/L DTPA; the addition of catalase did
not alter Hb oxidation rate in this system, so we did not routinely
include it. H2O2 was generated using 500 µmol/L glucose, 4 × 103 U/mL glucose oxidase, plus
10 µmol/L DTPA. Hydroxyl radical (·OH) was generated using glucose,
xanthine, and both enzymes as earlier, but with DTPA replaced by
EDTA/Fe3+ at 10 µmol/L each. For each of these
experiments, the negative control sample consisted of all relevant
reagents except the enzyme(s).
To examine for any effect of Hb concentration, we compared oxidation
rates for Hbs at several concentrations: 1 mg/mL (our standard
condition), 10 mg/mL, and 100 mg/mL. For these experiments, we
preserved the same Hb to DTPA ratio used for our standard conditions to
ensure that all adventitious iron would be inactivated. Although incubations were performed at the higher Hb concentrations, for analysis, the samples were carefully diluted to [Hb] = 1 mg/mL for
reading in the spectrophotometer. Therefore, the oxidation rate results
(expressed in µmol/L/min) can be directly compared with those
obtained in experiments at low Hb concentration. In similar fashion, we
also examined the effect of one chelate, iron/ADP, at Hb and iron and
ADP concentrations 10-fold higher than in our standard condition.
Iron binding to HbS.
To determine if added iron would bind preferentially to HbS over HbA,
purified preparations of both were treated with 10 µmol/L DTPA,
followed by extensive dialysis (against 1.2 × 105 vol
of experimental buffer) to remove DTPA and all adventitious iron as
iron/DTPA. Then, Hbs were exposed to iron by incubation with
Fe3+/ADP (50 µmol/L/1 mmol/L) as earlier, since the poor
solubility of iron in water precludes meaningful incubation with free
iron per se. After 30 minutes, the Hbs were dialyzed together against 1.2 × 105 vol of experimental buffer to remove ADP and
any Fe3+ not bound to Hb, and the oxidation rate was then
determined with and without 10 µmol/L DTPA.
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RESULTS AND DISCUSSION |
Inherent oxidation rate.
The comparative Hb oxidation rates we observed here under our standard
condition (Hb at 1 mg/mL) before removal of any adventitious iron
showed an average 52% rate augmentation for HbS (Table
1). The rates in the present report are
expressed in the fashion in which the raw data were collected, in units
of µmol/L/min. Conversion of these results to pseudo first-order rate
constants (units of h1) yields corresponding values for
HbA and HbS of 0.027 h1 and 0.041 h1,
respectively, which are consistent with those previously
reported.1,2
Inclusion of 10 µmol/L DTPA resulted in a significant decrement in
the observed oxidation rate for HbS (Table 1). However, even after any
adventitious iron was thus entirely inactivated by DTPA, a
significantly increased oxidation rate is still evident for HbS (Table
1), substantiating the existence of an inherent increase in an
autooxidation tendency for HbS. Interestingly, even though the two Hbs
were derived from the same intracellular environment, addition of DTPA
did not reduce the rate of HbA oxidation. These oxidation rates
recorded in presence of DTPA represent the minimal rate observable
under these conditions, as evidenced by the fact that higher DTPA
concentrations exerted no additional protective benefit (data not
shown). Also, oxidation rates dropped no further even after removal of
all DTPA and iron/DTPA by extensive dialysis against iron-free buffer,
confirming that the observed rate in the presence of DTPA really
represents a minimal value (Table 1).
Effect of Hb concentration.
Since the degree of dimerization increases as the Hb concentration
decreases, our standard experimental conditions would entail substantially greater dimerization than under physiologic Hb
concentrations (~320 mg/mL in heme). Therefore, we also determined
oxidation rate at a 100-fold higher Hb concentration that would
diminish the proportion of dimers by an order of
magnitude.* Results of this experiment
(Table 1) were identical to those observed under our standard
conditions, establishing that our data are indeed relevant to
physiologic Hb concentrations.
Iron partitioning to HbS.
Since the previous data suggest that adventitious iron associates
preferentially with HbS, we designed an experiment to test this
directly (Table 2). We obtained iron-free
preparations of Hbs (by addition of DTPA and subsequent removal of
iron/DTPA by dialysis) and determined their oxidation rates (Table 2).
Then, we reexposed them to Fe3+/ADP, after which the two
Hbs were extensively dialyzed together to remove ADP and any
Fe3+ not bound to Hb. Subsequent examination of the
oxidation rate showed reinstitution of a greater increase for HbS than
for HbA (Table 2). Since direct exposure of HbA and HbS to the same
concentration of iron/ADP exerts exactly the same effect on oxidation
rate (as shown later), this experiment suggests preferential binding of iron to HbS. Consistent with this, final addition of DTPA restored the
basal oxidation rate identified in the iron-free preparations in the
first place (Table 2).
Effect of iron chelates.
Any molecular iron in red blood cells that is not associated with Hb or
the membrane is likely to be in the form of low-molecular-weight chelates. Since these are known to promote Hb
oxidation,10,11 we tested whether HbS was abnormally
susceptible to this effect. All of the tested Fe3+ chelates
significantly stimulated Hb oxidation, but in each case this
incremental effect was equivalent for HbA and HbS (Table 3). This excludes an abnormal inherent
susceptibility for HbS to be oxidized by Fe3+ chelates.
However, the abnormal decompartmentalization of iron characteristic of
the sickle red blood cell nevertheless predicts that this is a
mechanism relevant to biology, because the sickle cell cytoplasm may
have low-molecular-weight Fe3+ chelates not present in
normal red blood cells.
The magnitude of oxidation effected by the different Fe3+
chelates varied enormously in these experiments, although discerning the reasons why is far beyond the scope of this investigation. Clearly,
our expectation, based on earlier and more limited data,10 that oxidation rates would be simply related inversely to chelator affinity for Fe3+ is not substantiated by our data. The
reason for the observed variation must be related to the multiplicity
of factors that are known to influence rate of chelate-induced
oxidation.10 Significantly, the incremental change in
oxidation rate seen in presence of Fe3+/ADP was the same
for Hbs examined at 1 mg/mL (Table 3) and at 10 mg/mL (.342 ± .020
for HbA and .332 ± .018 for HbS; n = 3).
Effect of oxy radicals.
All three physiologic oxidants (·O2,
H2O2, and ·OH) significantly accelerated Hb
oxidation, but no difference in susceptibility between HbA and HbS was
noted in this regard (Table 4). Again, however, this mechanism remains relevant to biologic HbS oxidation, since the sickle red blood cell generates excessive amounts of these
activated oxygen species compared with normal red blood cells.3
Discrepancy of GSH results.
Because our observation of minimal oxidation promotion by the
Fe3+ chelate with GSH (Table 5)differs from the large GSH effect reported earlier,17 we
examined this further. Our data demonstrate that GSH has a hemoglobin
oxidizing effect even in the absence of iron (Fig
1). However, this effect is dose-dependent,
and is only seen at GSH to heme ratios of 2:1 and greater, ratios that far exceed physiologic conditions (ratio of  1:4). In fact, these unusual conditions actually were used in that earlier
report,17 so this explains why our results differ.
Additional experiments performed at two different Hb concentrations
substantiate the importance of the GSH to Hb ratio (Table 5).
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| Fig 1.
GSH in the absence of iron accelerated Hb oxidation, but
only at very high GSH to Hb ratios. Experiments were performed in the
presence of DTPA to ensure that observed effects were iron-independent. The [Hb] was 62 µmol/L in heme, and oxidation was accelerated only
when the GSH to heme ratio was  2:1; the physiologic ratio is closer
to 1:4.
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Conclusion.
These results document that purified HbA and HbS preparations, even
from the same cellular source (ie, genotypic HbAS donors), exhibit
different autooxidation rates. The described data suggest that this is
accounted for by differential association of adventitious molecular
iron so that sufficient amounts to augment the Hb oxidation rate are
carried on HbS, but not on HbA (although our data set does not include
direct measurement of such adventitious iron). However, elimination of
this effect still leaves an exaggerated oxidation rate for HbS,
clarifying the earlier reports of this fact.1,2 In addition
to this inherent autooxidation tendency, oxidation of HbS would also be
influenced by exogenous stimuli, such as low-molecular-weight
Fe3+ chelates and physiologic oxidants (ie, oxygen
radicals), as shown here. Our data indicate that HbS has no exaggerated
inherent susceptibility to such stimuli. However, these mechanisms are
still relevant to sickle disease pathobiology, because sickle cells
would be subjected to abnormal levels of such stimulation derived from the abnormal iron decompartmentalization7 and excessive
oxidant generation3 characteristic of red blood cells in
this disease.
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FOOTNOTES |
Submitted July 2, 1997;
accepted December 17, 1997.
*
For example, for our standard experimental condition of Hb at 1 mg/mL (62 µmol/L heme) and 37.0°C, we calculate that the
proportion of heme in dimers is 12.5%, compared with 4.1% at
620 µmol/L heme, 1.3% at 6,200 µmol/L heme, and 0.7% at the
physiologic 20 mmol/L heme. Yet the corresponding absolute
dimer concentrations would be 2.9 × 106 mol/L, 12.8 × 106 mol/L, 41.0 × 106 mol/L, and
74.1 × 106 mol/L, respectively. This calculation
assumes that the tetramer-dimer equilibrium can be expressed by
D2/T = K, where T = H/4  D/2, H = [heme], D = [dimer], T = [tetramer], and K = 1.1 × 106 mol/L
at 37.0°C. This value for K is calculated from a value of 1.5 × 106 mol/L at 21.5°C assuming a  H of 3,900 cal (1 cal = 4.18 J).18
Supported by National Institutes of Health Grant No. HL37528.
Address correspondence to Robert P. Hebbel, MD, Box 480 UMHC,
Department of Medicine, Harvard St at E River Rd, Minneapolis, MN
55455.
The publication costs of this article were defrayed in part by page
charge payment. This 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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Abstract
Introduction
Abstract