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1Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287; 2Consiglio Nazionale della Ricerca, Institute of Molecular Biology and Pathology and Department Biochemical Sciences University La Sapienza, Rome, Italy; and 3Department of Chemistry, Temple University, Philadelphia, Pennsylvania 19122
Submitted 15 January 2003 ; accepted in final form 4 April 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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-human and 
-bovine chains. Polymerization of Hb
Minotaur through S-S intermolecular cross-linking was obtained by introducing
a Cys at position 9 and substituting the naturally occurring Cys. This
homogeneous polymer, Hb Polytaur, has a molecular mass of 
500 kDa and was
resistant toward reducing agents present in blood. In mice, the circulating
half-time (3 h) was fivefold greater than adult human Hb (HbA). The half-time
of autooxidation measured in blood (46 h) exceeded the circulating retention
time. Hypervolemic exchange transfusion resulted in increased arterial blood
pressure similar to that with albumin. The increase in pressure was less than
that obtained by transfusion of cross-linked tetrameric Hb known to undergo
renovascular extravasation. The nitric oxide reactivity of Hb Polytaur was
similar to HbA, suggesting that the diminished pressor response to Hb Polytaur
was probably related to diminished extravasation. Transfusion of 3% Hb
Polytaur during focal cerebral ischemia reduced infarct volume by 22%.
Therefore, site-specific Cys insertion on the Hb surface results in uniform
size polymers that do not produce the large pressor response seen with
tetrameric Hb. Polymerization maintains physiologically relevant oxygen and
heme affinity, stability toward denaturation and oxidation, and effective
oxygen delivery as indicated by reduced cerebral ischemic damage.
hemoglobin recombinant; hemoglobin synthetic; hemoglobin polymeric; blood substitutes; transfusional fluids; artificial oxygen carriers; hemoglobin retention time; exchange transfusion; extravasation; focal cerebral ischemia
Alternatively, Hb can be expressed in microorganisms
(19,
29,
34,
44). The continuous
development of recombinant techniques will allow the production of Hb in
microorganisms in a potentially limitless supply, free from mammalian
infectious agents. This approach also offers the possibility to design and
construct mutant Hbs with specific conformational and functional
characteristics that render them suitable for applications as therapeutic
agents in different clinical situations. In the search for recombinant Hb
molecules possessing properties suitable for therapeutic applications, we
previously described a polymer of HbA, Hb Prisca
(HHS9C+C93A+C112G),
containing six to eight tetrameric molecules intermolecularly cross-linked
through S-S bonds introduced at position 9 on the Hb surface
(16). Hb Prisca was modeled
after the naturally occurring mutant Hb Porto Alegre
(HHS9C)
(7). Hb Prisca has an oxygen
affinity similar to that of plasma-based HbA. It was obtained by refolding
recombinant -globin, initially expressed in Escherichia coli as
a fusion protein. Refolding occurred in the presence of heme and native
-subunits (19). The
recombinant hemoglobins obtained with this system had biophysical
characteristics nearly identical to that of natural Hb; however, the yield of
the protein was too low for the implementation of in vivo studies.
Alternatively, Hb can be expressed in E. coli in a soluble form,
thus allowing the possibility to produce recombinant Hbs in an amount
sufficient for animal experimentation
(13). For the study presented
in this article, we constructed Hb Minotaur, containing -human and
-bovine chains. This hybrid Hb was selected because of the high yield of
expression and protein recovery. Here we describe the construction and
characterization of a homogeneous and stable polymer of Hb Minotaur, with
functional characteristics suitable for an artificial oxygen carrier. We
determined the plasma retention time and investigated whether polymerization
would decrease the hypertensive response associated with peripheral
vasoconstriction typically observed after infusion of nondissociable
tetrameric cross-linked Hb solutions. The efficacy as an oxygen carrier was
evaluated by measuring infarct volume after transient focal cerebral
ischemia.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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-globin gene. The A
allele of HbBV has been constructed from 21 synthetic deoxy oligonucleotides
that were synthesized and gel purified by DNAgency (Berwyn, PA). The protein
sequence was that previously determined by Schroeder et al.
(43) and by Schimenti and
Duncan (42). The DNA codon
usage was optimized for E. coli expression, and restriction enzyme
recognition sites were incorporated into the sequence at potentially useful
locations. The gene was constructed in four parts, I, II, III, and IV, of
blunt-ended, double-stranded DNA. The fragments were then joined in pairs,
i.e., I-II and III-IV, by first digesting parts I and II with BstEII
and III and IV with PstI, followed by ligation. I-II was digested
with NcoI and KpnI and inserted into a vector obtained by
treating pNFMb with NcoI and KpnI. The resulting plasmid was
designated pNF-Bov/2. pNFMb is a plasmid constructed by replacing the human
-globin gene of pJKO5 with the sperm whale myoglobin gene from pMb413
(38). After treatment of gene
parts III and IV with PstI, they were ligated to give part
III–IV, which in turn was digested with KpnI and
EcoRI, and inserted into pNFBov/2, which had been digested with the
same two enzymes. The resulting plasmid pNFBovine was sequenced by the DNA
Sequencing Facility in the Department of Genetics, University of Pennsylvania,
verifying the sequence shown in the accompanying figure
(Fig. 1). For expression of the
fusion protein NS1-FX- bovine, E. coli strain AR120 was
transformed with the plasmid and induced with nalidixic acid as previously
described for pJKO5 (19).
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For expression of the hybrid hemoglobin
HBv designated Hb Minotaur, the Arg codon
of the Factor X recognition sequence in pNFBovine was changed from CGC to CAT,
creating a NdeI site at the Met1 codon. The pAlter
Mutagenesis system of Promega was utilized for this and other mutagenesis
reactions. The bovine gene was then extracted by digesting
with NdeI and HindIII and inserted into pDLIII-13e
(24), which had been
completely hydrolyzed with HindIII and partially hydrolyzed with
NdeI. The resulting plasmid has the human -globin gene of
pDLIII-13e replaced by the bovine -globin gene and is designated
pDLHBv. During the purification procedure,
an excess of Bv-chains was observed relative to
H-chains. In a effort to more nearly equalize expression of
the two globins, a second human gene was inserted into the
plasmid. This was accomplished by taking advantage of two XbaI sites,
one on each side of the human gene. Partial digestion of the
plasmid with XbaI provided the vector (5,639 bp) and complete
digestion provided the insert (508 bp). The sequence of the plasmid containing
two human genes is identical regardless of which
XbaI site receives the insert. The amount of hemoglobin isolated from
expression of this plasmid is 40% greater than from the plasmid
containing only one human gene.
Expression and purification of tetrameric Hb molecules in E. coli.
Soluble hemoglobin was expressed in pDLHBv
or in pDL2HBv. Cells were grown in a
5-liter fermentor in DM-4 medium
(30) supplemented by
continuous glucose feeding. With these conditions, we obtain 30.0 g of
cells per liter of medium. After equilibration with CO, harvesting, and two
cycles of freezing and thawing, the cells were suspended in 3 vol/g cells
paste of buffer containing (in mM) 40 Tris, 1 benzamidine, 0.3 dithiothreitol,
and 0.1 triethylenetetramine (TETA) at pH 7.4. The cells were lysed by
treatment with lysozyme (1 mg/ml) and benzonase (50 µl/l). Nucleotides were
precipitated in 0.5% polyethylenimine. Hb is purified by a two-step ammonium
sulfate precipitation (45% and 75%), followed by chromatography on a CM
SepharoseFF column connected to a ÄKTA Prime chromatography system
(Amersham Pharmacia Biotech). The protein and the column were equilibrated
with (in mM) 10 phosphate buffer, pH 6.5, 1 benzamidine, 0.3 dithiothreitol, 1
EDTA, and 0.1 TETA. A linear gradient uses 10 mM dibasic phosphate as a second
buffer. The Hb obtained is pure as determined by reversed-phase HPLC
(Fig. 2A) indicating
the presence of the heme peak (ET = 11.3 min) followed by only two
peaks. Edman degradation (10 cycles) confirmed the purity and identified the
peak with ET = 46.4 min as H-globin and the peak
with ET = 72.6 min as Bv-globin. In the absorption
spectrum of the purified protein shown in
Fig. 2B, the presence
of contaminating proteins or residual nucleotides was not detected in the far
UV region. The absorption spectrum of the oxy derivative, shown in the
inset of Fig.
2B, indicates the absence of the adduct of heme and
hydrogen sulfide (sulfHb).
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Protein polymerization. Polymerization spontaneously occurred
through the oxidation of thiol groups and formation of S-S bonds between Cys
residues introduced at position 9 of tetrameric Hb. Polymerization was
carried out under one atmosphere of O2 in 20 mM Tris, 1.0 mM
benzamidine, and 0.1 mM TETA, pH 8.4. The Hb in the oxygenated form was
concentrated to 40–60 mg/ml and filtered through a 0.45-µm
filter into a sterilized amber bottle. The extent of polymerization was
measured using a prepacked (1.6 x 60 cm) Fractogel EMD BioSec column (EM
Industries). The elution buffer was 20 mM phosphate and 300 mM NaCl at pH 7.2.
The column detects molecular mass differences between 5 and 900 kDa and was
calibrated using a set of known standard proteins. Peak areas were calculated
using Prime-View data collection software (Amersham Pharmacia Biotech).
Polymerization time varied with temperature from 2 days at 30°C to 4 wk at
4°C. Methemoglobin formed during the polymerization process was reduced
using the enzymatic system
(23).
Endotoxin removal. For samples to be used in animal experimentation, the protein was dialyzed against lactate-Ringer solution. After filtration through a 0.45-µm filter, endotoxins were removed by gentle mixing for 18 h with Detoxy-gel (Pierce) following the procedure described by the manufacturer that reduces the endotoxin content to less than 2 EU/ml. The endotoxin content of our samples was, however, not determined. The protein, was then stored in sterile glass vials at –80°C. Methemoglobin formation upon storage was minimal.
CD spectroscopy. The CD spectra were recorded at 15°C in 20 mM phosphate buffer at pH 7.0 on an AVIV model 202 spectropolarimeter (Aviv Associates, Lakewood, NJ). Each spectrum represents an average of eight scans. The Hb samples in the carbonmonoxy form were prepared by filtration through a Sephadex G25 column equilibrated with CO-saturated buffer. Immediately before use, 10 mg of dithionite dissolved in 100 µl of buffer were adsorbed on the top of column to completely reduce any methemoglobin present. This treatment did not affect the degree of polymerization. Soret spectra were measured between 470 and 380 nm; Hb concentration was 0.10 mg/ml.
Thermal denaturation. An AVIV model 202 spectropolarimeter with Peltier temperature controller was used to determine the median temperature (Tm) of denaturation. The Hb samples, in the carbonmonoxy form, were at a concentration of 0.1 mg/ml in 10 mM phosphate buffer at pH 7.0. The change in ellipticity was recorded at 222 nm under continuous stirring in a capped 1-cm cuvette. The temperature was increased at a rate of 10°C/min from 25°C to 100°C with a step size of 2.0°C. Equilibration at each temperature step was continued for 30 s before data collection, which was averaged over an additional 30 s.
Autooxidations. These measurements were performed either in buffer or in whole human blood. Measurements in buffer were carried out in a HP 8452A diode array spectrophotometer at 37°C with a protein concentration of 1.0 mg/ml. Buffer was 0.1 M phosphate at pH 7.0 with 1 mM EDTA and beef liver catalase at a molar ratio of 0.003 M heme–1 (28). Spectra were recorded every 30 min. Turbidity was corrected using as baseline the spectrum of a cuvette wrapped with parafilm (16). The absorption spectra were decomposed linearly into a series of prerecorded standard spectra. The fraction of oxyhemoglobin was plotted as function of time and then fit to a first- or second-order kinetic equation using Origin 6.1 (OriginLab, Northampton, MA).
For the measurements in whole blood, human blood (K. M. Bobofchak) collected in citrate was divided in 2-ml aliquots in capped 4-ml test tubes and equilibrated at 37°C. To each tube, 200 µl of a 3% solution of Hb in the polymerization buffer were added. The tubes were rotated slowly using a Labquake shaker (Labindustries) and 100-µl blood aliquots were collected at intervals and immediately centrifuged at 3,000 g, and the supernatant was collected and diluted with buffer. The absorption spectra, recorded between 480 and 380 nm in an AVIV spectrophotometer, were decomposed linearly into prerecorded spectra. The control for these measurements contained plasma from the same blood sample to which 100 µl of polymerization buffer were added, but no Hb.
Heme affinity. The affinity of Hb for heme was measured following
the method of Hargrove et al.
(22) using apomyoglobin
His64 
Tyr. Spectra were recorded using an HP 8452A
spectrophotometer with a water-controlled temperature cell holder. Hb
concentration was 3 µM in heme and apomyoglobin was 15 µM, temperature
25°C. The protein was buffered in 150 mM phosphate and 0.45 M sucrose, pH
7.0. Spectra were recorded every 5 min for 600 min. As heme is released from
Hb and bound to apomyoglobin, a decrease and red-shift of the Soret peak at
406 nm is observed. The absorbance at 406 nm was plotted and fitted with a
second-order kinetic equation using the program Origin version 6.1 (OriginLab)
to obtain the rate constants.
Oxygen affinity. A Hemox model B (TCS Medical Products) with computer interface was used for the measurements. Oxygen dissociation curves were obtained using the enzymatic deoxygenation method of Vandegriff et al. (51). Measurements were carried out in 0.1 M phosphate and 0.1 M NaCl at pH 7.4 at 37°C at a protein concentration of 1 mg/ml. The protein was equilibrated with 1 mM protochatechuic acid in a cuvette, which was then sealed with a custom-made adaptor containing a silicon membrane to eliminate air from the cell. Deoxygenation was initiated by injection of protochatecuate-3,4-dioxygenase through the membrane in a ratio of 0.06 U/mol heme. Raw data were fitted to the Adair function to determine the P50 and Hill coefficient, using the program Origin version 6.1 (OriginLabs). The percentage of ferric Hb was measured spectroscopically before and after each experiment; any runs with >10% methemoglobin were discarded.
NO reactivity. The rate constant for the combination of Hb with NO
was measured by stopped flow at 436 nm. The instrument SX-18-MV from Applied
Photophysics, (Leatherhead, UK) has a dead time of 2 ms and can measure
rate constants up to 250 s–1. Buffer was 0.1
M bis-Tris, pH 7.2, containing 1 mM EDTA.
A 2 mM stock solution of NO was prepared by allowing the gas (at 1 atm and 25°C) to dissolve in degassed water; this solution was then diluted with degassed buffer to obtain NO concentrations ranging from 5 to 20 µM. The effective concentration of the stock solution was checked by titration with HbO2 in an anaerobic, gas-free cell, because the gas converts stoichiometrically oxyhemoglobin into methemoglobin.
Hb solutions were prepared by diluting the protein to a final concentration
of 1.5 µM (heme) in deoxygenated buffer. Immediately before the
measurements, 0.1 mg/ml of sodium dithionite was added to the samples to
ensure the complete oxygen removal from the buffer. The extent of
polymerization was not modified by dithionite in the time frame of the
measurements (<10 min). Dithionite destroys NO at a much slower rate than
that for the combination of the gas with unliganded Hb (rate constant,
k = 1,400 M–1s–1)
(30). This entails an error
<10% on the measured rate constants.
Transfusion experiments. Transfusions were performed on C57Bl/6
mice (25 g body wt). All procedures were approved by the institutional
animal care and use committee. Mice were anesthetized with 1.0–1.5%
halothane administered via face mask, and a femoral artery was catheterized.
After the surgery was completed, the inspired halothane concentration was
reduced to 0.7%. Rectal temperature was maintained with a heating lamp. To
determine the plasma retention time of the hemoglobin, 200 µl of a 3% Hb
solution were infused over a 5-min period. Arterial blood samples (50 µl)
were drawn at 10, 20, 40, 60, 80, 120, and 180 min after the transfusion for
analysis of plasma Hb concentration. Plasma was diluted 60-fold with a
CO-saturated buffer containing 1 mg/ml dithionite to reduce any methemoglobin
present. Hb concentration was determined at 419 nm using the molar extinction
coefficient of carboxymonoxy-heme (
heme) = 190,000
M–1 cm–1.
In a second experiment, an exchange transfusion was performed with either a 5% albumin solution or a 3% polymerized Hb solution to determine whether there are differences in the arterial blood pressure response. As a positive control, an additional group of mice was transfused with a 6% sebacoyl cross-linked tetrameric Hb solution (8), which has previously been shown to increase arterial pressure in cats (48). The exchange transfusion through the femoral artery catheter was performed by infusing and withdrawing fluid in three-step increments over a 20-min period. The total amount of fluid infused was 650 µl, and the amount withdrawn was 350 µl. The exchange transfusion was hypervolumetric to allow subsequent blood sampling (50 µl) at 15, 40, 80, and 120 min after the transfusion without inducing hypovolemia. Differences in mean arterial pressure between groups transfused with albumin and the two hemoglobin solutions were compared by two-way analysis of variance over time. Comparisons among the three groups of the change in arterial pressure from baseline were made at each time point by one-way analysis of variance and the Newman-Keuls multiple range test at the 0.05 significance level. Data are presented as means ± SE.
In a third in vivo experiment, an exchange transfusion was performed with either a 5% albumin solution or a 3% polymerized hemoglobin solution during transient focal cerebral ischemia. A control group with no transfusion was also studied. Focal ischemia was produced by inserting a 6-O monofilament with a blunted tip 6 mm into the internal carotid artery so that in flow into the middle cerebral artery was occluded. At 10 min of ischemia a 700-µl exchange transfusion was performed over a 20-min period. After we obtained a blood sample at 45 min of ischemia, an additional 250 µl were infused. Catheters were removed, incisions were closed, and anesthesia was discontinued. At 2 h of ischemia, the mouse was briefly reanesthetized with halothane, and the monofilament was withdrawn from the internal carotid artery to allow reperfusion. At 24 h the mice were killed by an overdose of halothane anesthesia, and the brains were harvested and cut into five coronal slabs (2-mm thick). These slabs were incubated at 37°C in a 1% solution of 2,3,5-triphenyltetrazoleum chloride for 20 min followed by fixation in 10% buffered formalin. Infarct volume corrected for tissue swelling was measured by integrating regions that stained red with this vital dye in the ipsilateral hemisphere and subtracting this value from the volume of the contralateral hemisphere. Comparisons of infarct volume among the control, albumin-, and Hb-transfused groups were carried out by analysis of variance and the Newman-Keuls multiple range test.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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bovine gene was used for
expressing Hb Minotaur containing -human and -bovine subunits
(HBv). This expression system was selected
because of its high yield of protein expression and recovery introduced by the
Bv gene.
For the polymerization of Hb Minotaur, the mutant
(HV1M+C104SBvA9C+C93A)
was constructed and designated as Hb Polytaur. In the
H-chains 1Val was replaced by Met to closely
approximate the conformation of the natural -chains
(27), and 104Cys was
replaced by Ser, the residue present at this site in HbBv. In the
Bv-chain, 9Ala was replaced by Cys (this residue is
external and exposed to the solvent), and 93Cys was replaced by Ala
(52). The substitutions of the
naturally occurring Cys were introduced to facilitate the in vivo refolding
and to confine the intermolecular cross-linking to the Cys residues introduced
at the 9 position. In Fig.
3A the elution profile of size exclusion chromatography
of Hb Minotaur and of Hb Polytaur is shown. The dotted line corresponds to the
molecular mass of tetrameric Hb Minotaur in equilibrium with its dimeric form.
The solid line shows the elution profile of Hb Polytaur after 30 days of
polymerization at 4°C. The symmetric shape of the peak at 47 ml indicates
the presence of a major homogeneous fraction with a molecular mass of 500
kDa, corresponding to a polymer of seven to eight tetrameric Hb molecules. A
small peak at 60 ml (<10% total protein) indicates the presence of some
nonpolymerized tetrameric Hb. After removal of this fraction by gel
filtration, Hb Polytaur maintains an invariant elution profile.
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Because polymerization was obtained through the formation of intermolecular
S-S bonds, we determined the stability of the polymer toward the reducing
agents present in the blood. The S-S bonds are 2.5 Å long, and our
working hypothesis was that their accessibility would be hindered by the
numerous interactions resulting from the intermolecular cross-linking of the
subunits. To test the polymerization stability, the protein in 40 mM phosphate
buffer at pH 7.4 was mixed with human blood at a ratio of 1 ml 3% Hb solution
to 9 ml blood for 24 h at 37°C. In a control sample only buffer was added
to the blood. The samples were centrifuged at 800 g, and a plasma
aliquot was analyzed on a gel filtration column. Optical density changes were
recorded at 405 nm in the heme-sensitive Soret region of the spectrum.
Figure 3A shows the
elution profile of Hb Polytaur used for this experiment, and
Fig. 3B shows the
elution profile of the plasma sample containing Hb Polytaur corrected for the
absorption profile of the control plasma sample. Two new peaks are observed:
one with an elution volume (VE) equaling 38 ml, corresponding to a
molecular mass of 860 kDa, and a second with a VE equaling 52 ml,
corresponding to a molecular mass of 125 kDa. The amount of tetrameric Hb
(VE equaling 60 ml) present in the plasma was not increased.
Conformational characteristics and stability to denaturation. The
conformation of the heme pocket of Hb Minotaur and Polytaur was investigated
by observing the CD spectra in the Soret. In this region the spectra are
sensitive to the position of the heme in relation to the aromatic residues
lining the heme pocket. HbA and HbBv have similar CD spectra
(18), and HbA is taken as the
reference spectrum. A decreased ellipticity is observed in Hb Minotaur and Hb
Polytaur with respect to HbA (Fig.
4A). Spectral differences are also present between the
two recombinant hemoglobins. The stability toward denaturation of the
carbonmonoxy derivatives of these hemoglobins was investigated as a function
of temperature. Figure
4B shows the traces of the temperature-scanning
measurements. The three hemoglobins have a very similar resistance to heat
denaturation with a Tm 76°C.
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Autooxidation. Autooxidation rates were measured at 37°C
either in phosphate buffer or in whole blood. In phosphate buffer, HbA
autooxidation was described by a single exponential with a rate of 0.02
h–1. In Hb Minotaur and Hb Polytaur, the
autooxidation was described by two exponentials with 20% of the protein
having a fast autooxidation rate 50- to 150-fold increased with respect to
HbA. The rate of the remaining fraction (80%) was also increased, about
eightfold with respect to HbA. The half-time of autooxidation, T
ox, is 33 h for HbA and 3.2 h for Hb Minotaur and Hb Polytaur.
Representative first-order plots are shown in
Fig. 5A, and the
results of the data analysis are listed in
Table 1.
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For the measurements in whole blood, fewer points could be collected, and
the autooxidation rate was described by a linear decay as shown in
Fig. 5B. The
T ox was 160 h for HbA and 46 h for Hb Polytaur
(Table 1). These data were not
distorted by the release of Hb from the erythrocytes during the incubation
time of the measurements. As shown in the inset of
Fig. 5B, Hb is
released in minimal and similar amounts from all samples, indicating that,
under the conditions of this experiment, the addition of free Hb to the plasma
did not increase hemolysis of the erythrocytes.
Heme affinity. The rate of heme transfer was determined according
to the method of Hargrove et al.
(22), in which the transfer
occurs to an apomyoglobin in the solution. Representative time courses are
shown in Fig. 6. As listed in
Table 1, in HbA and Hb Minotaur
the data were described by a fast and a slow rate of the same fixed amplitude
(0.50) and one order of magnitude difference. The fast and a slow rate
constants are assigned to heme release from the - and -chains,
respectively (22). In Hb
Minotaur the rate of heme transfer was twofold increased with respect to HbA.
In contrast, in Hb Polytaur 40% of the total heme was not released by the
protein. In the remaining 60% the heme transfer rates were twofold decreased
with respect to HbA.
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Oxygen affinity. The oxygen affinities of solutions of HbA, Hb
Minotaur, and Hb Polytaur are similar under physiological conditions (37°C
at pH 7.4 in the presence of 0.1 M NaCl) with a P50 of
16–18 mmHg (Fig. 7).
The inset in Fig. 7 shows a decreased cooperativity of the recombinant hemoglobins with respect to
HbA. This is usually observed with recombinant hemoglobins and is particularly
evident in this hybrid system.
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NO reactivity. Figure 8 shows the time course of NO binding to HbA and Hb Polytaur at 7.5 µM NO (concentration before mixing). The traces, analyzed by a nonlinear least squares minimization routine, were found to be compatible with a single second-order process; most often even a pseudo first-order approximation was found to provide a satisfactory fit to the experimental data. The rate constant for the combination of NO with the deoxygenated forms of HbA and Hb Polytaur is similar, 19–25 µM–1s–1, respectively. This value is very close to that reported in the literature for HbA (k = 25 µM–1s–1) (32, 33).
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Plasma retention time. Figure 9 shows the pooled data of plasma Hb concentration normalized by the initial concentration after transfusion. Mice transfused with Hb Polytaur had a fivefold greater retention time than mice transfused with HbA. When half-time was analyzed individually on each mouse (n = 4 per group), the half-time was significantly greater after Hb Polytaur transfusion (176 ± 23 min) than after HbA transfusion (34 ± 4 min). Hemoglobinuria was evident after HbA but not after Hb Polytaur transfusion.
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Exchange transfusion. Exchange transfusion with a 3% solution of Hb Polytaur in mice (n = 5) resulted in a decrease in arterial hematocrit from 42 ± 1% to 35 ± 1% at 15 min after completion of the transfusion. Hematocrit decreased further (31 ± 1%) with subsequent blood sampling over a 2-h period. Similar decreases occurred after exchange transfusion of a 5% albumin solution (n = 5) and a 6% solution of sebacoyl-cross-linked tetrameric Hb (n = 6) (8). The plasma concentration of Hb Polytaur was 1% and 0.5% at 15 min and 24 h after transfusion, respectively. The plasma samples were analyzed spectrophotometrically; methemoglobin formation was not observed. The exchange transfusion was hypervolumetric (650 µl infused; 350 µl withdrawn) to allow for subsequent blood sampling in the mouse. Mean arterial blood pressure increased in all three groups (Fig. 10A). Two-way analysis of variance with repeated measures indicated a significant effect of time (P < 0.001) and a significant interaction among the three treatment groups over time (P < 0.001). Data were further analyzed by performing one-way analysis of variance with the Newman-Keuls multiple range test at each time point. At 15 min after the hypervolumetric transfusion, the change in arterial pressure with the sebacoyl tetrameric Hb (25 ± 3 mmHg) was greater than the increase with albumin (11 ± 2 mmHg), whereas the increase seen with Hb Polytaur (16 ± 5 mmHg) was not different from that seen with albumin. The increase in pressure was sustained during subsequent blood sampling over a 2-h period in both Hb groups but gradually subsided in the albumin group (Fig. 10B). Consequently, the change in arterial pressure became significantly different between the albumin and Hb Polytaur groups 2 h after transfusion. The change in arterial pressure was significantly greater in the sebacoyl Hb group than in the Hb Polytaur group from 40 min through 120 min after the transfusion. There was no change in rectal temperature after transfusion in any group and no difference among groups (Fig. 10C).
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Focal cerebral ischemia. Exchange transfusion with 5% albumin
after the onset of focal cerebral ischemia decreased hematocrit from 41
± 1% to 31 ± 1% and decreased arterial Hb concentration from
11.1 ± 0.5 to 7.0 ± 0.5 g/dl. Exchange transfusion with 3% Hb
Polytaur decreased hematocrit from 45 ± 1% to 35 ± 1% and
decreased arterial Hb concentration from 11.7 ± 0.5 to 9.4 ± 0.3
g/dl. As expected, the change in total blood hemoglobin concentration
(equivalent gram of tetramer) was less in the Hb-transfused group. Infarct
volume after 2 h of transient ischemia was similar in the control and
albumin-transfused groups but was reduced by 22% in the Hb Polytaur
transfused group (Fig.
11).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Polymer engineering. For the design of Hb Polytaur, we have used the same approach described for Hb Prisca (16), consisting of the substitution of the endogenous Cys residues and introduction of a novel Cys on the Hb surface to elicit specific intermolecular cross-linking. The substitution of the endogeneous Cys residues was necessary for obtaining the homogeneous polymerization shown in Fig. 3A. This peak has an estimated molecular mass of 500 kDa, corresponding to the polymerization of approximately seven tetrameric Hb molecules. A similar value was obtained by dynamic light scattering for polymerized Hb Prisca in which the tetramers assemble in a globular shape. Polymerization of Hb Prisca and Hb Polytaur follows the same approach; these systems most likely have the same polymerization assembly. Some residual tetrameric Hb (<10%) is observed, which can be eliminated by gel filtration. After removal of the tetrameric fraction by gel filtration, the elution profile of Hb Polytaur remains invariant, suggesting that the tetrameric residual is not the result of an equilibrium between the polymeric and the tetrameric forms but is due to a limiting rate of tetrameric Hb incorporation into the polymer. On mixing of Hb Polytaur with whole blood, the amount of tetrameric Hb present in the plasma remains very small (Fig. 3B). However, some opening of the S-S bonds by the reducing agents present in blood occurs as indicated by the appearance of a peak with a molecular mass of 250 kDa. A similar polymer with a molecular mass of 250 kDa was observed in hemolysates of erythrocytes containing HbA and Hb Porto Alegre in which the glutathione reductase activity was threefold increased (47). We also obtained a second new peak corresponding to a protein with an average molecular mass of 850 kDa. This peak is not observed in experiments in which blood was mixed with HbA. It may be due to fragments of Hb Polytaur captured by the plasma proteins or to the assembly of the species with molecular masses of 500 and 250 kDa. Whatever its origin, the important result is that the amount of tetrameric Hb capable of extravasation is not increased in the plasma. It is worth stressing that, for these experiments, we used a large excess of blood over free Hb, which should maximize S-S bond reduction.
Conformational characteristics and stability. The CD spectra in the Soret region spectrum of Hb Minotaur and Polytaur (Fig. 4A) indicate differences in the tertiary structure around the heme pocket in both proteins with respect to HbA. Differences in the Soret CD spectra are also present between Hb Minotaur and Polytaur. They may be due to conformational effects resulting from the amino acid substitutions introduced in Hb Polytaur. Alternatively, they may indicate that the intermolecular cross-linking elicits conformational changes that propagate to the heme pocket.
The high helical content and the quaternary assembly contribute in
conferring on the HbA molecule a high stability to temperature denaturation
(Fig. 4B). The
stability is well maintained in Hb Minoatur and Polytaur and is consistent
with the high expression level of these hybrid hemoglobins. This is an
important aspect for the long-term storage of these proteins. Notably, in Hb
Prisca containing - and -human chains the stability to
temperature denaturation was decreased 8°C with respect to HbA
(16).
Autooxidation. The autooxidation rate of Hb is concentration
dependent, with Hb dimers having about a 20-fold higher autooxidation rate
than the tetramers (53). At
the concentration used in our experiments (1.0 mg/ml), the amount of dimers
present in HbA is <10% and does not contribute significantly to the
autooxidation rate measured, which appears to be monoexponential.
Heterogeneity in the rate of autooxidation has been observed in recombinant
Hbs (17). In Hb Minotaur and
Hb Polytaur, the autooxidation rates are described by two exponentials of
different amplitude and a 10-fold difference in the autooxidation rate
(Table 1,
Fig. 5A). The
increased rate of autooxidation present in Hb Minotaur could be explained by
the presence of a very large dimeric population; however, Hb Polytaur, which
is not in equilibrium with its dimeric form, has an autooxidation rate similar
to that of Hb Minotaur and is 8- to 10-fold faster than HbA. The
similarity of these data suggest that the large increase in the autooxidation
rate observed in Hb Minotaur and Hb Polytaur is an intrinsic property of the
hybrid molecule.
Measurements made in vitro provide information about the intrinsic
properties of proteins, which may be affected by components present in an in
vivo environment. Therapeutic applications of Hb solutions involve injection
into the circulation. Reducing systems are present in the blood, which may
affect the autooxidation rate of proteins dissolved in the plasma. Our data
indicate that, in the presence of blood, the autooxidation rates of
stroma-free HbA and Hb Polytaur are 5- and 10-times decreased, respectively,
compared with the rates measured in buffer
(Table 1, Fig. 5B) confirming a
previous report on the protective role of blood components toward heme
oxidation (45). In blood the
half-time of autooxidation of Hb Polytaur is increased from 4 h in buffer
to 46 h. This value is within the retention time in circulation of other
polymerized hemoglobins, 20 h in humans
(9,
25,
26).
Heme affinity. Hb Polytaur has a remarkable increase in heme
affinity with 40% of the total heme not released from the protein. This
effect can be attributed to heme stabilization by intermolecular cross-linking
because it was not observed in Hb Minotaur
(Fig. 6). This observation is
consistent with a globular assembly of Hb Polytaur similar to that proposed
for Hb Prisca (16), which
would make the hemes less accessible to solvent. Heme release triggers
oxidative stress and cytotoxicity in the endothelial cells
(2,
4), and these negative effects
should be reduced in Hb Polytaur.
Oxygen affinity. The oxygen-binding curves of Hb Minotaur and
Polytaur show an oxygen affinity similar to that of HbA but a decreased
cooperativity (Fig. 7). In the
construction of hybrid Hbs containing HbBv -chains, we hypothesized that
they would have an oxygen affinity lower than that of HbA, regulated by the
Cl– in the solvent. However, this was not observed. In the
presence of 100 mM Cl–, HbA, Hb Minotaur, and Hb Polytaur
have the same oxygen affinities. The absence of a decreased oxygen affinity in
the hybrid hemoglobins is tentatively attributed to the nine amino acid
differences present at the 11 interface
between HbA and HbBv (17). We
are currently investigating this hypothesis with hybrid hemoglobins having
either human or bovine residues at the 11
interface.
The optimal oxygen affinity and the relevance of cooperativity in oxygen
transport and delivery are currently under scrutiny
(50). The assumption that a
plasma-based O2 carrier should have an O2 affinity
similar to the red blood cell-based hemoglobin may not necessarily hold under
all circumstances. Plasma may account for a considerable resistance to
O2 diffusion in small vessels, and hemoglobin in the plasma may
facilitate O2 unloading by increasing the effective O2
solubility, even at low Hb concentrations in the plasma
(36,
37). A low O2
affinity would promote precapillary O2 loss and raise perivascular
PO2, which could produce counterregulatory arteriolar
constriction and uneven capillary perfusion
(41,
50). Our observation that
infusion of a 3% solution of Hb Polytaur decreases infarct volume implies
effective O2 delivery under ischemic conditions at a low plasma
concentration (1 g/dl) and high O2 affinity (P50
17 mmHg).
Reactivity with NO. NO combines with reduced unliganded Hb without discriminating between the T and the R allosteric states, and its reaction is virtually diffusion limited, the energy barrier for bond formation being almost nil (35). Thus the experiments of rapid mixing described herein probe the rate of diffusion of this gas into the heme pocket. The observation that Hb Polytaur combines with NO at the same rate as that of HbA (Fig. 8) demonstrates that polymerization does not hinder the diffusion of diatomic ligands into the heme pocket (33). It should be pointed out that the overall affinity of NO for heme is largely determined by the slow dissociation rate constant, which was not determined in these experiments. The latter measure is more likely to correlate with O2 affinity and cooperativity.
Plasma retention time. Hb may be cleared from the plasma in the
urine, lymph, or reticuloendothelial system
(6). The fivefold increase in
the plasma retention time of Hb Polytaur compared with HbA is largely due to
the lack of clearance in the urine and possibly in lymph
(Fig. 9). It has been shown
that removing chemically cross-linked tetramers and polymers of <300 kDa
nominal molecular mass prevented extravasation and appearance in renal lymph
draining renal peritubular capillaries
(31). Because the Hb Polytaur
solution has a homogeneous molecular mass of 500 kDa with <10% Hb
remaining unpolymerized, the amount cleared by the lymphatics is probably
relatively small. The 3-h half-life of Hb Polytaur may appear to be too brief
for some clinical applications. However, it should be recognized that the
plasma retention of chemically modified Hb polymers varies logarithmically
with body mass and could be substantially greater in humans.
Exchange transfusion. The hypervolemic exchange transfusion resulted in similar increases in arterial blood pressure in the albumin and Hb Polytaur groups (Fig. 10). The lack of a substantially greater increase in arterial pressure with Hb Polytaur than with albumin transfusion implies that the polymer does not produce the large pressor response often seen with other Hb solutions (3, 9, 49). The pressor response to exchange transfusions of polymeric Hb solutions can be prevented by removing molecular mass species <300 kDa, which also reduces extravasation in renal lymph (31). Thus the lack of a large pressor response with the Hb Polytaur solution is probably related to the large homogeneous size of the polymer, which presumably does not extravasate in most vascular beds. As discussed above, polymerization does not hinder NO diffusion into the heme pocket and is unlikely to interfere with binding of NO. Extravasated Hb will scavenge NO in the interstitial spaces surrounding vascular smooth muscle more effectively than Hb in the plasma and thereby increase vascular tone. Therefore, eliminating low-molecular-mass Hb components is important for preventing unwanted vasoconstriction in tissues such as the kidney and intestines. In support of this hypothesis, tetrameric cross-linked Hb extravasates in renal lymph (31), increases arterial pressure in the cat (48), and decreases renal and intestinal blood flow (49) but does not inhibit NO-dependent dilation evoked by acetylcholine or ADP in brain arterioles with tight endothelial junctions (3). Moreover, sebacoyl-cross-linked tetrameric Hb transfusion in the mouse was presently found to increase arterial pressure to a greater extent than Hb Polytaur. These results are consistent with Hb Polytaur producing less peripheral vasoconstriction than the sebacoyl tetramer.
A limitation of this study is that cardiac filling pressure and cardiac output were not measured in these mice to determine whether systemic vascular resistance changed after transfusion. The oncotic pressure of the 6% sebacoyl Hb solution is presumably greater than that of the 3% Hb Polytaur solution. Therefore, it is possible that rapid fluid shifts resulted in a somewhat greater cardiac output contribution to the pressor response in the sebacoyl Hb group compared with the Hb Polytaur group. Moreover, a greater oncotic pressure of 5% albumin compared with 3% Hb Polytaur may have helped sustain arterial pressure at a level similar to that obtained during the first 80 min after Hb Polytaur transfusion. The difference in arterial pressure at 120 min between the albumin and Hb Polytaur groups might be attributed to a subsequent decline in plasma volume in the albumin group or possibly to time-dependent extravasation and NO scavenging of the small amount of unpolymerized tetrameric Hb (<10%) remaining in the infused solution. Therefore, differences in arterial pressure responses could be influenced by shifts in blood volume as well as by changes in peripheral vascular resistance.
Transfusion of Hb Polytaur during 2 h of transient focal cerebral ischemia resulted in a decrease in infarct size relative to albumin-transfused mice with a similar 20–25% lowering of hematocrit (Fig. 11). This decrease in infarct volume presumably is attributable to increased oxygen delivery into the ischemic border regions. Plasma-based Hb is thought to increase the effective O2 diffusivity in plasma and thereby facilitates O2 unloading (36, 37). Moreover, with low blood flow associated with ischemia, red blood cell flux in individual capillaries may become heterogeneous, and a plasma-based O2 carrier may improve the homogeneity of O2 distribution. However, we cannot exclude that differences in arterial blood pressure among groups may have occurred after discontinuing the anesthesia and that an improved O2 delivery was secondary to a higher blood pressure in the Hb Polytaur group during the remaining ischemia period.
We conclude that site-specific introduction of Cys on the Hb surface
permits polymerization to a uniform size while maintaining physiologically
relevant oxygen affinity, cooperativity, stability toward denaturation, heme
affinity, and stability toward oxidation. The solution of Hb Polytaur can be
used in vivo without producing hemoglobinuria. Replacing 20% of the red
blood cells with this plasma-based polymer resulted in stable arterial blood
pressure. NO diffusion into the Hb Polytaur heme pocket is not hindered by
polymerization, and the absence of a large pressor response may be attributed
to the absence of extravasation. Reduced ischemic damage in the brain suggests
that the polymer can act as an effective oxygen transporter despite a
P50 of 17 mmHg. Therefore, this unique polymer of recombinant
Hb represents a viable alternative oxygen carrier that potentially could be
developed into a transfusion fluid.
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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.
1 In Bv the histidine present at position 2 in HbA is
missing. We assign a number to this position in Hb Polytaur for
consistency. 
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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-chains of adult bovine hemoglobins C-Rhodesia and
D-Zambia. Arch Biochem Biophys
152: 222–232,
1972.[Web of Science][Medline]
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): cytochrome c (molecular weight 12,400), cross-linked HbA
(molecular weight 64,500), and thyroglobulin (molecular weight 669,000).
Linear fit of the standards was used to calculate the molecular weight of the
hemoglobins. B: size-exclusion chromatograph of Hb Polytaur after
being mixed with blood for 24 h at 37°C and corrected for plasma
absorbance. Conditions same as in A. Two new peaks are present, one
with elution volume (VE) = 52.2 ml for molecular mass of 250 kDa,
the other with VE = 36.6 ml for molecular mass of 860 kDa. The
fraction with VE = 60.1 ml represents <10% of the total protein
as in A.
) was
recorded at 222 nm from 25 to 100°C with a step size of 2.0°C. Figure
represents the first derivative of the temperature scanning, with the peaks
corresponding to the median temperature of denaturation (Tm).
), Hb Minotaur
(
), and Hb Polytaur (
) is shown. 
A436, change
in absorption at 436 nm. Data analysis was performed using a first-order rate
equation minimized using the FMINS routine (Matlab). Overall fit is good, with
minimal systematic deviations and high significance of the estimated
parameters; accordingly, semilogarithmic linearization yields the following
values of R2: HbA 0.996, Hb Polytaur 0.994. Experimental
conditions were 0.1 M bis-Tris buffer containing 1 mM EDTA. Hb concentration
was 0.7 µM (per heme after mixing); NO concentration was 5 µM (after
mixing), 23°C. Solution of Hb contains 0.01 mg/ml dithionite, whereas the
NO was dithionite free; we carefully degassed both solutions to remove
oxygen.
,
n = 5), 3% Hb Polytaur (