Báo cáo khoa học: The oxidative effect of bacterial lipopolysaccharide on native and cross-linked human hemoglobin as a function of the structure of the lipopolysaccharide A comparison of the effects of smooth and rough lipopolysaccharide ppt

The oxidative effect of bacterial lipopolysaccharide on native and
cross-linked human hemoglobin as a function of the structure
of the lipopolysaccharide
A comparison of the effects of smooth and rough lipopolysaccharide
Douglas L. Currell and Jack Levin
Department of Laboratory Medicine, University of California School of Medicine and Veterans Administration Medical Center,
San Francisco, CA, USA
The binding of lipopolysaccharide (LPS, also known as
bacterial endotoxin) to human hemoglobin is known to
result in oxidation of hemoglobin to methemoglobin and
hemichrome. We have investigated the effects of the LPSs
from smooth and rough Escherichia coli and Salmonella
minnesota on the rate of oxidation of native oxyhemoglobin
A
0
and hemoglobin cross-linked between the a-99 lysines.
For cross-linked hemoglobin, both smooth LPSs produced a
rate of oxidation faster than the corresponding rough LPSs,
indicating the importance of the binding of LPS to the
hemoglobin. The effect of the LPS appeared to be largely on
the initial fast phase of the oxidation reaction, suggest-
ing modification of the heme pocket of the a chains. For
hemoglobin A
0,
the rates of oxidation produced by rough
and smooth LPSs were very similar, suggestingthe possibility
that the effect of the LPSs was to cause dissociation of
hemoglobin into dimers. The participation of cupric ion in
the oxidation process was demonstrated in most cases. In
contrast, the rate of oxidation of cross-linked hemoglobin by
the LPSs of both the rough and smooth E. coli was not

affected by the presence of chelators, suggesting that cupric
ion had previously bound to these LPSs. Overall, these data
suggest that the physiological effectiveness of hemoglobin
solutions now being developed for clinical use may be
decreased by the presence of lipopolysaccharide in the
circulation of recipients.
Keywords: bacterial endotoxin (lipopolysaccharide); human
hemoglobin; oxidation of hemoglobin; cross-linked hemo-
globin.
The interaction between bacterial lipopolysaccharide (LPS,
also known as bacterial endotoxin) and human hemoglobin
(Hb) has been shown in previous studies to affect the
properties of both the Hb molecule and the LPS [1–3]. The
binding of Hb to the smooth LPSs, Escherichia coli 026:B6
and Proteus mirabilis S 1959, was demonstrated and shown
to cause disaggregation and an increase of the biological
activity of the LPS [1]. In a related study, Hb similarly
enhanced activation of Limulus amebocyte lysate and
stimulation of endothelial cell tissue factor production by
smooth or rough P. mirabilis [2]. Rough LPS lacks the
polysaccharide side-chain that is present in the complete
(smooth) LPS molecule. In contrast, Limulus amebocyte
lysate activation either by lipid A (which consists of a
phosphorylated disaccharide backbone with several long-
chain fatty acids) or partially deacylated Salmonella minne-
sota 595 (Re) LPS was not enhanced in the presence of Hb.
The effect of Hb on the LPS and purified lipid A of rough
E. coli has been recently investigated, and significant
physical changes in the purified lipid A and in the lipid A
moiety of intact LPS were reported [4].

The binding of LPS to oxyHb results in the oxidation of
theHbtometHbandhemichrome[3].Incontrasttothe
lack of effect of Hb on the biological activity of partially
deacylated LPS from S. minnesota 595, this LPS was more
effective in producing oxidation of Hb than the LPS of
either rough S. minnesota 595 or smooth P. mirabilis [3]. To
further clarify these structure–function relationships, we
have extended these studies to compare the effects of
smooth and rough LPSs of E. coli and S. minnesota on the
oxidation of native and cross-linked Hb. Because the auto-
oxidationofHbhasbeenshowntodependonthepHand
the presence of heavy metal cations [5–8], we have also
investigated the effects of pH, EDTA and neocuproine on
the LPS-mediated oxidation of Hb.
MATERIALS AND METHODS
Bacterial lipopolysaccharides
Smooth E. coli lipopolysaccharide 026:B6 (Westphal
method [9]) was obtained from Difco Laboratories (Detroit,
MI, USA). Rough E. coli J5 (Rc) and smooth S. minnesota
(Galanos method [10]) were generously provided by
K. Meyers (RIBI Immunochem Research, Inc., Hamilton,
MT, USA). Deep rough S. minnesota 595 (Re) lipopoly-
saccharide (Westphal method [9]) was obtained from List
Biological Laboratories, Inc. (Campbell, CA, USA).
The lipopolysaccharides (5.0–5.9 mg) were suspended in
l.0 mL NaCl/P
i
(0.9% NaCl), pH 7.4, by treatment for
Correspondence to J. Levin, V. A. Medical Center (111-H2) 4150
Clement Street, San Francisco, CA 94121, USA.

Fax: + 1 415 831 2506, Tel.: + 1 415 750 6913,
E-mail:
Abbreviations: LPS, lipopolysaccharide; Hb, human hemoglobin.
(Received 19 April 2002, revised 11 July 2002, accepted 1 August 2002)
Eur. J. Biochem. 269, 4635–4640 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03163.x
5 min in an ultrasonic bath (Branson Ultrasonic Cleaner,
Shelton, CT, USA), after initial suspension with a vortex
mixer. The LPS suspensions were stored at 0–4 °Cand
immediately before use were retreated with the vortex mixer.
Reagents
LPS-free NaCl/P
i
was obtained from Irvine Scientific (Santa
Ana, CA, USA) and was diluted with deionized water to
produce a buffer, pH 7.4, 0.1
M
phosphate and 0.15
M
NaCl. All other phosphate buffers used were prepared from
monobasic NaH
2
PO
4
(Fisher Scientific Co., Fairlawn, NJ,
USA) and dibasic K
2
HPO
4
(J. T. Baker Chemical Co.,
Phillipsburg, NJ, USA) and used as 0.2

M
solutions. Tricine
was obtained from Sigma Chemical Co. (St Louis, MO,
USA)andusedasa0.15
M
solution. Neocuproine and
EDTA were obtained from Sigma Chemical Co. and used
as a 0.01
M
aqueous solution and a 0.1
M
solution in NaCl/
P
i
, respectively.
Hemoglobin
Hemoglobin A
0
,58mgÆmL
)1
, in Ringer’s lactate, pH 8.0,
which had been purified by ion-exchange HPLC as
described previously [11], was provided by the Blood
Research Detachment, Walter Reed Army Institute of
Research, Washington, D.C., USA and stored at )70 °C
until use. The initial metHb concentration of Hb A
0
was
always < 5%. Human Hb, cross-linked between the Lys99
residues of the a chains by treatment of deoxyHb with

bis(3,5-dibromosalicyl) fumarate, also was provided by the
Blood Research Detachment [12]. The stock solution was
71 mgÆmL
)1
in Ringer’s acetate, pH 7.4. It was sterile and
essentially LPS-free (< 100 pgÆmL
)1
as assessed by the
Limulus amebocyte lysate assay [13]) and stored at )70 °C
until use. The initial metHb concentration of the cross-
linked Hb was always < 7%.
Copper analysis
All reagents, buffers, Hb stock solutions and LPS suspen-
sions (containing 5.0–5.9 mgÆmL
)1
LPS) were analyzed for
cupric ion by M. Qian in the laboratory of J. W. Eaton,
James Graham Brown Cancer Center, University of
Louisville, Louisville, KY, USA, by the method of Makino
[14]. The results are presented in Table 1.
Oxidation experiments
To 360 lL of buffer was added 6.0 lL of a cross-linked Hb
solution, 71 mgÆmL
)1
,or7.0lLofaHbA
0
solution,
58 mgÆmL
)1
,andthen80lL of a suspension of LPS,

5.0–5.9 mgÆmL
)1
, to produce an LPS/Hb suspension of
approximately equal concentrations (mgÆmL
)1
): the final
Hb concentration was 0.8–1.0 mgÆmL
)1
.Insomeexperi-
ments, 4.4 lLEDTA,0.1
M
,or4.4 lL neocuproine, 0.01
M
,
was added. The absorption spectrum from 400 nm to
800 nm was measured at selected time intervals during a 2-h
period, using a Beckman DU-7400 spectrophotometer
(Beckman Instruments, Inc., Fullerton, CA, USA). All
experiments were carried out at 37 °C. The temperature was
maintained by a circulation water bath, Lauda K-2/RD9
(Brinkman Instruments, Westbury, NY, USA). The neces-
sary correction for light scattering in all suspensions that
contained lipopolysaccharide was performed with a pro-
gram in the spectrophotometer software. The relative
concentrations of oxyHb, metHb and hemichromes were
obtained by the method of Winterbourn [15] from simul-
taneous measurements of absorbances at 560, 577 and
630 nm. The major oxidation product was metHb. The
amount of hemichrome produced during a 2-h reaction was
typically less than 10% (data not shown). The decrease in

concentration of oxyHb with time was utilized as a measure
of the rate of oxidation of oxyHb.
RESULTS
The effect of pH on the auto-oxidation of cross-linked Hb
was studied. The rate of decrease of the concentration of
oxyHb increased as the pH was lowered over the range from
pH 9.0–5.8 (data not shown). As was observed previously
by others [5], the reaction is biphasic at pH 7.4 and below,
with an initial fast phase followed by a slower phase. To
determine the optimum pH at which to study the effect of
LPS on the oxidation rate, a comparison of the effects of the
LPSs of smooth E. coli and rough S. minnesota on the
oxidation rate over the pH range 5.8–9.0 was undertaken
(data not shown). Because at pH 7.0 both LPSs produced
marked but distinguishable effects, all further experiments
were carried out at this pH.
The contribution of the polysaccharide component of
LPS to its effect on the oxidation of cross-linked Hb was
then investigated by a comparison of the effects of rough
andsmoothLPSsofE. coli, both in the presence and
absence of EDTA (Fig. 1). The rate of oxidation was
increased in the presence of the LPSs of both the smooth
and rough E. coli, but the rate of oxidation in the presence
of the LPS of smooth E. coli was much faster than for the
LPS from rough E. coli. Although EDTA markedly
decreased the rate of auto-oxidation, its effect on the
oxidation rate of cross-linked Hb in the presence of LPS was
negligible for both the smooth and rough LPSs (Fig. 1).
The effect of the LPSs of smooth and rough S. minnesota
on the oxidation rate of cross-linked Hb also was compared

(Fig. 2). The rate of oxidation in the presence of the LPS of
smooth S. minnesota was much faster than in the presence
Table 1. Copper concentration in Hb stock solutions, buffers and LPS
suspensions.
Sample Cu concentration (l
M
)
Hemoglobin
a,a-Hb (71 mgÆmL
)1
) 1.0
Hb A
0
(58 mgÆmL
)1
) 2.5
LPS (5.0–5.9 mgÆmL
)1
)
a
S. minnesota (R) 1.8
S. minnesota (S) 3.4
E. coli (R) 5.0
E. coli (S) 6.4
Buffers
Phosphate buffers, 0.2
M
0.8
Phosphate, 0.1
M

buffered-saline, 0.15
M
0.3
Tricine, 0.15
M
0.0
a
Cu concentrations are the mean of two determinations.
4636 D. L. Currell and J. Levin (Eur. J. Biochem. 269) Ó FEBS 2002
of the LPS from rough S. minnesota, both in the presence
and absence of EDTA. In contrast to the results with the
LPSs of E. coli, EDTA decreased the rate of oxidation.
However, the rate of oxidation mediated by the smooth LPS
was less affected by the presence of EDTA. The rough
S. minnesota LPS increased the initial fast phase of the
reaction, but decreased the rate of the slow phase of
oxidation in the presence of EDTA.
A comparison of rough and smooth LPSs of E. coli and
S. minnesota in the presence of EDTA revealed that both in
the presence and absence of EDTA, the oxidation of cross-
linked Hb was faster in the presence of the smooth LPSs
(Figs 1 and 2). In addition, the rate of oxidation mediated
by the smooth E. coli LPS was faster than that produced by
the smooth S. minnesota LPS. The rate of oxidation in the
presence of the rough S. minnesota LPS was slower than
that produced by the other three LPSs studied.
A comparison of the auto-oxidation of cross-linked Hb
with that of Hb A
0
is shown in Fig. 3A. The effect of the

presence of EDTA, known to bind heavy metal cations [16],
on the oxidation of both Hbs is also presented in Fig. 3A.
The rate of auto-oxidation of cross-linked Hb was greater
than that of Hb A
0,
both in the presence and absence of
EDTA, as has been observed previously [17]. In addition, the
rates of auto-oxidation of both cross-linked Hb and Hb A
0
were markedly reduced by EDTA, suggesting catalysis of the
oxidation by heavy metal cations, as previously observed by
Rifkind [8,18]. To determine whether the heavy metal cation
was cupric ion as indicated by the results of Rifkind [8], the
effect of a chelator specific for cupric ion, neocuproine
[19,20], was studied. The results in Fig. 3B,C show that the
effects of neocuproine and EDTA on the oxidation rate were
identical, confirming that the cupric ion was the heavy metal
cation primarily responsible for the catalysis. The concen-
trations of cupric ion in the solutions used were determined
by chemical analysis (Table 1).
Fig. 1. Comparison of the effects of the LPSs of smooth E. coli 026:B6
and rough E. coli J5 (Rc), in the absence and presence of EDTA, on the
oxidation of a,a-cross-linked Hb (XL Hb). Hb concentration was
0.8 mgÆmL
)1
, in phosphate buffer, 0.2
M
,pH7.0.LPSconcentration
was 0.8–1.0 mgÆmL
)1

. The mean ± SD of three independent experi-
ments is shown. Each experiment was performed with aliquots of a
single sample of Hb. Therefore, apparent differences in the starting
oxyHb concentrations are the result of an immediate drop in the
oxyHb concentration upon addition of the LPS.
Fig. 2. Comparison of the effects of the LPSs of rough S. minnesota 595
(Re) and smooth S. minnesota, in the absence and presence of EDTA, on
the oxidation of a, a-cross-linked Hb. Themean±SDofthreeinde-
pendent experiments is shown. Other conditions as in Fig. 1.
Fig. 3. The effect of EDTA or neocuproine on the auto-oxidation of
a,a-cross-linked Hb (XL) and Hb A
0
(A
0
). Hb concentration was
0.8 mgÆmL
)1
, in phosphate buffer, 0.2
M
,pH7.0.Themean±SDof
three or four independent experiments is shown.
Ó FEBS 2002 Oxidative effects of LPS on human hemoglobin (Eur. J. Biochem. 269) 4637
Our studies of the effect of the structure of the LPS on the
oxidation reaction were extended to native human Hb A
0
to
determine whether the above effects were general or specific
to cross-linked Hb. A comparison of the effects of the LPSs
of rough and smooth E. coli on the oxidation of Hb A
0

,
both in the presence and absence of EDTA, is provided in
Fig. 4. The rate of oxidation was increased in the presence
of the LPSs of both smooth and rough E. coli,butin
contrast to the results obtained for cross-linked Hb, the
difference in the oxidation rates mediated by the two LPSs
was slight. In both cases EDTA reduced the oxidation rate,
in contrast to the results obtained with cross-linked Hb in
the presence of LPS (Fig. 1), upon which EDTA had no
effect. An interesting characteristic of the reaction mediated
by the LPS of rough E. coli was a lag phase of 10 minutes
both in the presence and absence of EDTA. The lag phase
was followed by a very rapid second phase only in the
absence of EDTA. Significantly, this lag phase was not
observed during the oxidation of cross-linked Hb produced
by the LPS of rough E. coli (Fig. 1).
The effect of the LPSs of smooth and rough S. minnesota
on the oxidation rate of Hb A
0
was compared (Fig. 5). In
contrast to the results observed with cross-linked Hb, the
rates of oxidation were identical for the LPSs of smooth and
rough S. minnesota, both in the presence and absence of
EDTA. For both the smooth and rough LPSs, the effect of
EDTA was to reduce the oxidation rate. A comparison with
the auto-oxidation rate (data from Fig. 3) revealed that in
the presence of EDTA, the increase in the rate of oxidation
of Hb A
0
produced by the LPSs of rough and smooth

S. minnesota was solely due to a sharp increase in the initial
rate (Fig. 5).
A comparison of the effects of the rough and smooth
LPSs of E. coli and S. minnesota on the oxidation of Hb A
0
in the presence of EDTA revealed that the oxidation of
Hb A
0
was somewhat faster in the presence of the smooth
LPSs (Figs 4 and 5). The rate of oxidation mediated by the
smooth E. coli LPS was slightly faster than that produced
by the smooth S. minnesota LPS in the presence of EDTA.
The rate of oxidation produced by the rough S. minnesota
LPS was slower than for the other three LPSs studied.
Indeed, the LPS of rough S. minnesota hadnoeffectonthe
oxidation of Hb A
0
. The previously reported increase in the
oxidation rate of Hb A
0
in the presence of rough
S. minnesota [3] was probably due to the presence of cupric
ion, as no EDTA was present.
DISCUSSION
The effect of pH on the auto-oxidation of Hb A
0
has been
the subject of several studies. Mansouri and Winterhalter [5]
reported that the oxidation of the a chains of Hb A
0

was 10
times faster than that of the beta chains and that the
oxidation of the beta chains was not influenced by pH. The
biphasic reaction was shown to consist of a rapid initial
reaction followed by a slower second phase over a wide pH
range from 5.3 to 8. Tsuruga and Shikama [21] confirmed
that the fast phase of oxidation was due to the a chains and
the slow phase was due to the bchains. Tsuruga et al. found
that the beta chain of the tetramer does not exhibit any
proton-catalyzed auto-oxidation [22]. These authors found
further that upon dissociation of tetrameric oxyHb A
0
into
dimers by dilution, the rate of the fast phase was increased
markedly while the rate of the slow phase remained
unchanged.
The observation that cross-linked Hb oxidizes faster than
Hb A
0
(Fig. 3A) is consistent with the results of others who
reported that the rate of auto-oxidation is inversely propor-
tional to the oxygen affinity of the Hb [17]. Therefore, the
demonstration of more marked auto-oxidation of the cross-
linked Hb than was observed for Hb A
0
can be attributed to
the lower oxygen affinity of the cross-linked derivative.
The data in Fig. 2 indicate that at pH 7.0, in the
presence of EDTA, the oxidation of cross-linked Hb
mediated by the LPS of rough S. minnesota was very

slow. In contrast, the effect on the oxidation reaction of
the LPS of smooth E. coli was marked and not altered in
the presence of EDTA (Fig. 1). The binding of the LPS
molecule of the smooth E. coli to Hb, shown previously in
this laboratory [1], apparently increases the oxidation rate
while shielding the Hb from the effect of heavy metal
cations, perhaps through binding of the cations by the
LPS. Support for this idea is provided by the report of the
binding of 1.5–2 mol of iron in either the ferrous or
ferric state to the LPS of smooth E. coli. Such binding
Fig. 4. Comparison of the effects of the LPSs of smooth E. coli 026:B6
and rough E. coli J5 (Rc), in the absence and presence of EDTA, on the
oxidation of Hb A
0
. The mean ± SD of three independent experi-
ments is shown. Other conditions as in Fig. 1.
Fig. 5. Comparison of the effects of the LPSs of rough S. minnesota 595
(Re) and smooth S. minnesota, in the absence and presence of EDTA, on
the oxidation of Hb A
0
. The mean ± SD of three independent
experiments is shown. Other conditions as in Fig. 1.
4638 D. L. Currell and J. Levin (Eur. J. Biochem. 269) Ó FEBS 2002
resulted in a slight decrease in the biological activity of the
LPS [23].
The results show that for both S. minnesota and E. coli,
the smooth (wild type) LPS was more effective in increasing
the oxidation rate of cross-linked Hb than the rough LPSs
lacking the O-specific polysaccharide moiety (Fig. 6). It was
previously found that the singly deacylated derivative of

rough S. minnesota 595 was more effective than the rough
LPS [3]. Partial deacylation probably disturbs the supra-
molecular structure of the rough LPS, exposing the fatty
acids of the lipid A component. Therefore, it was suggested
that the lipid A moiety (Fig. 6) is crucial in catalyzing the
oxidation of Hb [3]. However, as the lipid A constitutes a
much smaller proportion of the molecular mass of smooth
LPSs, their greater effect on the oxidation rate may be due
to more effective binding of Hb. The effects of the various
LPSs on the oxidation rate of cross-linked Hb can be
compared in Figs 1 and 2. The relative rates both in the
presence and absence of EDTA are: smooth E. coli >
smooth S. minnesota > rough E. coli >roughS. minne-
sota. In the presence of EDTA, the oxidative effect of the
rough E. coli LPS approaches that of the smooth S. min-
nesota LPS.
The results of experiments, in which the oxidation of
Hb A
0
by the four LPSs was studied, indicated that their
effects upon Hb A
0
and cross-linked Hb differ (Figs 4 and 5
vs. Figs 1 and 2). This difference in behavior of native
human Hb A
0
and cross-linked Hb, in the presence of the
LPSs utilized, must lie in the principal differences in the
properties of the two Hbs [24]. The reduced oxygen affinity
of the cross-linked Hb would be expected to maximize any

effects due to the resultant increase in oxidation rate. At
equilibrium, the concentration of deoxyHb is much greater
in the low affinity cross-linked Hb. Therefore, another
difference between cross-linked Hb and Hb A
0
may be in
the binding of the deoxyHb to the LPS. Another obvious
difference between the two Hbs is the possibility of
dissociation of the Hb A
0
into dimers, which is not possible
for the cross-linked Hb, as cross-linking the a chains
prevents dissociation into ab dimers. Dissociation into
dimers is known to increase the oxidation rate of Hb [25].
Thus, if the oxidative effect of LPS on native human Hb A
0
is primarily due to the enhancement of the dissociation of
the Hb into dimers, then the observed rates of oxidation
caused by each of the LPSs studied should be similar to each
other, i.e. simply that of the rate of oxidation of dimers.
The increase in rate of oxidation in the presence of rough
E. coli was striking for Hb A
0
(Fig. 4). This effect was
reduced in the presence of EDTA, suggesting that the
binding of heavy metal cations to smooth E. coli may be
greaterthantoroughE. coli, as binding of heavy metal
cations to the LPS would be expected to prevent catalysis of
oxidation by the cations. However, it is not clear why the
effect of EDTA was negligible in the oxidation of cross-

linked Hb mediated by the rough LPS of E. coli (Fig. 1).
The mechanism by which LPSs accelerate the oxidation
of cross-linked Hb is not clear. Because the achains are
covalently linked, dissociation into ab dimers is not possible.
It is conceivable that binding of the Hb tetramer to the LPS
molecule makes the heme cavity of the a chains more
accessible to a water molecule which can then accelerate the
displacement of the protonated superoxide anion, as was
suggested by Tsuruga and Shikama [21] to explain the
increase in oxidation rate of the a chains in the ab dimer. It
had been demonstrated earlier by Wallace et al. [26] that
nucleophiles such as water are important in the proton
assisted displacement of superoxide during the auto-oxida-
tion of Hb.
In general, the increase in the oxidation rate of cross-
linked Hb mediated by LPSs is due to an increase in the rate
of the initial fast phase, i.e. oxidation of the a chains. The
rates of oxidation are reduced in the presence of chelators of
heavy metal cations in most cases. An exception is the lack
of alteration of the increased oxidation rates of cross-linked
Hb in the presence of the LPSs of smooth or rough E. coli.
This lack of effect of the chelator suggests that the LPS itself
binds the heavy metal cations (probably at the phosphate
groups) and thus prevents catalysis of Hb oxidation by the
heavy metal cations. For cross-linked Hb, the smooth LPSs
were more effective than the rough LPSs, suggesting that
binding of the Hb by the LPS was more important than the
lipid content of the LPS. Overall, the E. coli LPSs were
more effective than the S. minnesota LPSs in increasing the
rate of oxidation, suggesting a difference in binding of the

two types of LPSs. The effect of the LPSs on the rate of
oxidation of Hb A
0
was much less than on cross-linked Hb
and furthermore, the differences in structures of the LPSs
were less important, suggesting that the effect of the LPS on
Hb A
0
was possibly due to enhancement of dissociation
into dimers.
An exception to the above general statements is the
behavior of the LPS of rough E. coli. This LPS exhibited
a lag phase of approximately 10 min, followed by a very
rapid phase of oxidation for Hb A
0
but not for cross-
linked Hb. Furthermore, the rate of the oxidation reaction
mediated by this LPS was decreased by EDTA for Hb A
0
but not for cross-linked Hb. It is possible that the binding
of Hb A
0
by the LPS of rough E. coli exposes the binding
site for heavy metal cations on the Hb which then leads to
catalysis of oxidation. The b-2 histidine has been sugges-
ted as the binding site for cupric ion in Hb [18], but its
distance from a heme makes it difficult to understand its
involvement in the oxidation process. Another potential
binding site, the b-93 sulfydryl, is close to the heme [27]
and thus more likely to be involved in the oxidation of

Hb. Indeed, interaction between cupric ion bound to the
b-93 sulfhydryl and the heme center has recently been
demonstrated [28].
Many types of preparations of Hb, including cross-linked
Hb, are now under development as red blood cell substitutes
[29]. It is likely that LPS will be present in the circulation of
many of the potential recipients of Hb solutions, as
endotoxemia may occur in patients who are hypotensive
Fig. 6. Schematic representation of smooth LPS, rough LPS and lipid
A.
Ó FEBS 2002 Oxidative effects of LPS on human hemoglobin (Eur. J. Biochem. 269) 4639
and/or have experienced trauma and hemorrhage. The
studies described in this investigation, in conjunction with
our previous reports of the effects of LPS on both native
and cross-linked Hb [1,3], suggest the possibility that the
presence of circulating LPS may significantly decrease the
ability of Hb solutions to satisfactorily function as oxygen
carriers.
ACKNOWLEDGEMENTS
Supported in part by the Veterans Administration and the REAC
Committee of the University of California School of Medicine, San
Francisco.
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4640 D. L. Currell and J. Levin (Eur. J. Biochem. 269) Ó FEBS 2002