We have investigated the effects of the LPSs from smooth and rough Escherichia coli and Salmonella minnesotaon the rate of oxidation of native oxyhemoglobin A0and hemoglobin cross-linked
Trang 1The 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
minnesotaon the rate of oxidation of native oxyhemoglobin
A0and 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 A0,the rates of oxidation produced by rough
and smooth LPSs were very similar, suggesting the 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-sota595 (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 the Hb to metHb and hemichrome [3] In contrast to the 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-oxidation of Hb has been shown to depend on the pH and 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
M A T E R I A L S A N D M E T H O D S 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 (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: levinj@medicine.ucsf.edu
Abbreviations: LPS, lipopolysaccharide; Hb, human hemoglobin.
(Received 19 April 2002, revised 11 July 2002, accepted 1 August 2002)
Trang 25 min in an ultrasonic bath (Branson Ultrasonic Cleaner,
Shelton, CT, USA), after initial suspension with a vortex
mixer The LPS suspensions were stored at 0–4C and
immediately before use were retreated with the vortex mixer
Reagents
LPS-free NaCl/Piwas obtained from Irvine Scientific (Santa
Ana, CA, USA) and was diluted with deionized water to
produce a buffer, pH 7.4, 0.1M phosphate and 0.15 M
NaCl All other phosphate buffers used were prepared from
monobasic NaH2PO4(Fisher Scientific Co., Fairlawn, NJ,
USA) and dibasic K2HPO4 (J T Baker Chemical Co.,
Phillipsburg, NJ, USA) and used as 0.2Msolutions Tricine
was obtained from Sigma Chemical Co (St Louis, MO,
USA) and used as a 0.15M solution Neocuproine and
EDTA were obtained from Sigma Chemical Co and used
as a 0.01Maqueous solution and a 0.1Msolution in NaCl/
Pi, respectively
Hemoglobin
Hemoglobin A0, 58 mgÆmL)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 A0 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)1in Ringer’s acetate, pH 7.4 It was sterile and
essentially LPS-free (< 100 pgÆmL)1 as assessed by the
Limulusamebocyte 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)1LPS) 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, or 7.0 lL of a Hb A0 solution,
58 mgÆmL)1, and then 80 lL 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 In some experi-ments, 4.4 lL EDTA, 0.1M, or 4.4 lL neocuproine, 0.01M, 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 37C 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
R E S U L T S 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 and smooth LPSs of E 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.
Hemoglobin
LPS (5.0–5.9 mgÆmL)1)a
Buffers
Phosphate buffers, 0.2 M 0.8
Phosphate, 0.1 M
buffered-saline, 0.15 M
0.3
a Cu concentrations are the mean of two determinations.
Trang 3of 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 minnesotain 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 A0is 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 A0,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 A0 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 ofthe effects ofthe LPSs ofsmooth E coli 026:B6
and rough E coli J5 (Rc), in the absence and presence ofEDTA, on the
oxidation of a,a-cross-linked Hb (XL Hb) Hb concentration was
0.8 mgÆmL)1, in phosphate buffer, 0.2 M , pH 7.0 LPS concentration
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 ofthe effects ofthe LPSs ofrough S minnesota 595
(Re) and smooth S minnesota, in the absence and presence ofEDTA, on
the oxidation of a, a-cross-linked Hb The mean ± SD of three
inde-pendent experiments is shown Other conditions as in Fig 1.
Fig 3 The effect ofEDTA 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 , pH 7.0 The mean ± SD of three or four independent experiments is shown.
Trang 4Our studies of the effect of the structure of the LPS on the
oxidation reaction were extended to native human Hb A0to
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 A0,
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, but in
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 A0was 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 A0 produced by the LPSs of rough and smooth
S minnesotawas 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 A0
in the presence of EDTA revealed that the oxidation of
Hb A0was 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 had no effect on the oxidation of Hb A0 The previously reported increase in the oxidation rate of Hb A0 in the presence of rough
S minnesota[3] was probably due to the presence of cupric ion, as no EDTA was present
D I S C U S S I O N The effect of pH on the auto-oxidation of Hb A0has been the subject of several studies Mansouri and Winterhalter [5] reported that the oxidation of the a chains of Hb A0was 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 b chains 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 A0into 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 A0(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 A0can 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 ofthe effects ofthe LPSs ofsmooth E coli 026:B6
and rough E coli J5 (Rc), in the absence and presence ofEDTA, on the
oxidation ofHb A 0 The mean ± SD of three independent
experi-ments is shown Other conditions as in Fig 1.
Fig 5 Comparison ofthe effects ofthe LPSs ofrough S minnesota 595
(Re) and smooth S minnesota, in the absence and presence ofEDTA, on
the oxidation ofHb A 0 The mean ± SD of three independent
experiments is shown Other conditions as in Fig 1.
Trang 5resulted 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 > rough S
minne-sota In the presence of EDTA, the oxidative effect of the
rough E coli LPS approaches that of the smooth S
min-nesotaLPS
The results of experiments, in which the oxidation of
Hb A0by the four LPSs was studied, indicated that their
effects upon Hb A0and cross-linked Hb differ (Figs 4 and 5
vs Figs 1 and 2) This difference in behavior of native
human Hb A0and 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 A0may 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 A0into 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 A0
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 A0 (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
greater than to rough E 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 a chains 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 A0was 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 A0 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 A0 but not for cross-linked Hb Furthermore, the rate of the oxidation reaction mediated by this LPS was decreased by EDTA for Hb A0 but not for cross-linked Hb It is possible that the binding
of Hb A0by 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 ofsmooth LPS, rough LPS and lipid
A.
Trang 6and/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
A C K N O W L E D G E M E N T S
Supported in part by the Veterans Administration and the REAC
Committee of the University of California School of Medicine, San
Francisco.
R E F E R E N C E S
1 Kaca, W., Roth, R.I & Levin, J (1994) Hemoglobin, a
newly recognized lipopolysaccharide (LPS)-binding protein that
enhances LPS biological activity J Biol Chem 40, 25078–
25084.
2 Kaca, W., Roth, R.I & Levin, J (1994) Human hemoglobin
increases the activity of bacterial lipopolysaccharides in activation
of Limulus amebocyte lysate and stimulation of tissue factor
production by endothelial cells in vitro J Endotoxin Res 1, 243–
252.
3 Kaca, W., Roth, R.I., Vandegriff, K.D., Chen, G.C., Kuypers,
F.A., Winslow, R.M & Lev in, J (1995) Effects of bacterial
endotoxin on human cross-linked and native hemoglobins.
Biochemistry 34, 11176–11185.
4 Ju¨rgens, G., Mu¨ller, M., Koch, M.H.J & Brandenburg, K (2001)
Interaction of hemoglobin with enterobacterial lipopolysaccharide
and lipid A Physicochemical characterization and biological
activity Eur J Biochem 268, 4233–4242.
5 Mansouri, A & Winterhalter, K.H (1973) Nonequivalence of
chains in hemoglobin oxidation Biochemistry 12, 4946–4949.
6 Brooks, J (1931) The oxidation of haemoglobin to
methaemo-globin by oxygen Proc Roy Soc., Series B 109, 35–50.
7 Brooks, J (1935) The oxidation of haemoglobin to
methaemo-globin by oxygen II The relation between the rate of oxidation
and the partial pressure of oxygen Proc Roy Soc., Series B 118,
560–577.
8 Rifkind, J.M (1974) Copper and autoxidation of hemoglobin.
Biochemistry 13, 2474–2481.
9 Westphal, O & Jann, K (1965) Bacterial lipopolysaccharides.
Extraction with phenol-water and further application of the
pro-cedure Methods Carbohydrate Res 5, 91–93.
10 Galanos, C., Luderitz, O & Westphal, O (1969) A new method
for the extraction of R-lipopolysaccharides Eur J Biochem 9,
245–249.
11 Winslow, R.M & Chapman, K.W (1994) Pilot-scale preparation
of hemoglobin solutions Methods Enzymol 231, 3–16.
12 Christensen, S.M., Medina, R., Winslow, R.M., Snell, S.M.,
Zegna, A & Marini, M.A (1988) Preparation of human
hemoglobin A 0 for possible use as a blood substitute J Biochem.
Biophys Methods 17, 145–154.
13 Levin, J & Bang, F.B (1968) Clottable protein in Limulus: its localization and kinetics of its coagulation by endotoxin Thromb Diath Haemorrh 19, 186–197.
14 Makino, T (1989) A sensitive direct colorimetric assay of serum copper using 5-Br-PSAA Clinica Chimica Acta 185, 7–16.
15 Winterbourn, C.C (1990) Oxidative reactions of hemoglobin Methods Enzymol 186, 256–274.
16 Sille´n, L.G & Martell, A.E (1964) Stability Constants of Metal-Ion Complexes, pp 625–626 The Chemical Society, London.
17 MacDonald, V (1994) Measuring relative rates of hemoglobin oxidation and denaturation Methods Enzymol 231, 480–490.
18 Rifkind, J.M., Lauer, L.D., Chiang, S.C & Li, N.C (1976) Copper and the oxidation of hemoglobin: a comparison of horse and human hemoglobins Biochemistry 15, 5337–5343.
19 Nebesar, B (1964) Spectrophotometric determination of copper in tellurium and related thermoelectric compounds of the bismuth telluride type with 2,9-dimethyl-1,10-phenanthroline Anal Chem.
36, 1961–1965.
20 Stephens, B.G., Felkel, H.L & Spinelli, W.M (1974) Spectro-photometric determination of copper and iron subsequent to the simultaneous extraction of bis (2,9-dimethyl-1,10-phenanthroline) copper (l) and bis[2,4,6-tripyridyl)-1,3,5-triazine]iron (ll) into propylene carbonate Anal Chem 46, 692–696.
21 Tsuruga, M & Shikama, J (1997) Biphasic nature in the auto-xidation reaction of human oxyhemoglobin Biochim Biophys Acta 1337, 96–104.
22 Tsuruga, M., Matsuoka, A., Hachimori, A., Sugawara, Y & Shikama, K (1998) The molecular mechanism of autoxidation for human oxyhemoglobin J Biol Chem 273, 8607–8615.
23 Roth, R.I., Panter, S.S., Zegna, A.I., Arellano, F.A & Levin, J (1997) Effects of iron on bacterial endotoxin J Endotoxin Res 4, 273–278.
24 Chatterjee, R., Welty, V., Walder, R.Y., Pruitt, S.L., Rogers, P.H., Arnone, A & Walder, J.A (1986) Isolation and characterization
of a new hemoglobin derivative cross-linked between the a chains (lysine 99a 1 fi lysine a 2 ) J Biol Chem 261, 9929–9937.
25 Zhang, L., Levy, A & Rifkind, J.M (1991) Autoxidation of hemoglobin enhanced by dissociation into dimers J Biol Chem.
266, 24698–24701.
26 Wallace, W.J., Maxwell, J.C & Caughey, W.S (1974) The mechanisms of hemoglobin autoxidation: evidence for proton-assisted nucleophilic displacement of superoxide by anions Biochem Biophys Res Comm 57, 1104–1110.
27 Muirhead, H., Cox, J.M., Mazzarella, L & Perutz, M.F (1967) Structure and function of haemoglobin III A three-dimensional Fourier synthesis of human deoxyhaemoglobin at 5.5 A˚ resolu-tion J Mol Biol 28, 117–156.
28 Bonaventura, C., Godette, G., Tesh, S., Holm, D.E., Bonaventura, J., Crumbliss, A.L., Pearce, L.L & Peterson, J (1999) Internal electron transfer between hemes and Cu (II) bound
at cysteine b93 promotes methemoglobin reduction by carbon monoxide J Biol Chem 274, 5499–5507.
29 Stowell, C.P., Levin, J., Spiess, B.D & Winslow, R.M (2001) Progress in the development of RBC substitutes Transfusion 41, 287–299.