Glutaraldehyde Modification of Recombinant Human Hemoglobin
Alters Its Hemodynamic Properties*
Michael P.
Doyle,
Izydor
Apostol, and
Bruce A.
Kerwin
From Baxter Healthcare Corporation, Boulder, Colorado 80301
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ABSTRACT |
Many cell-free hemoglobin solutions designed as
oxygen-carrying therapeutics produce a hypertensive effect in animals.
The response is likely due to oxidation of nitric oxide by hemoglobin. Since the site of oxidation may lie outside the vascular compartment, we tested the hypothesis that polymerization of hemoglobin, rHb1.1, by
glutaraldehyde would attenuate the hypertensive response. Two products
of the cross-linking reaction were isolated, a
glutaraldehyde-derivatized monomer (mono-glxrHb) and a glutaraldehyde
cross-linked polymer (poly-glxrHb), and evaluated for their effects on
systemic hemodynamics in conscious rats. Administration of rHb1.1
caused a mean arterial pressure elevation of approximately 20 mm Hg and
an increase in total peripheral resistance of approximately 30%.
Administration of mono-glxrHb induced changes in mean arterial pressure
and vascular resistance that were significantly diminished relative to
those observed with rHb1.1. Poly-glxrHb elicited a mean arterial
pressure response that was further reduced compared with that obtained with mono-glxrHb and a change in vascular resistance that was the same
as the response to mono-glxrHb. These results suggest that rHb
peripheral vasoconstriction elicited by rHb1.1 is significantly attenuated by glutaraldehyde modification of the hemoglobin monomer and
that the effect of glutaraldehyde polymerization is likely due to
surface modification and/or intramolecular cross-linking, rather than
an increase in molecular size.
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INTRODUCTION |
As reviewed elsewhere (1-3), the search for a safe and
efficacious oxygen-delivering therapeutic has been ongoing for many years. Limitations to the use of hemoglobin solutions as therapeutics have included renal toxicities due to the presence of stromal elements
(4, 5), dissociation into 
dimers (6), high oxygen affinities
(7), and short intravascular retention times (1). Multiple approaches
have been taken to modify hemoglobins to address these limitations (1,
3), including chemical cross-linking of purified human or bovine
hemoglobin to prevent dissociation and chemical modification to
increase the P50 to a physiologically suitable
level (approximately 25-35 mm Hg, (7)). Recombinant technology was
employed to genetically cross-link the
-globins to form a
di-
-globin and thereby prevent dissociation. In addition, the
P50 was increased by substitution of Lys for Asn
at the
108 position. The resultant hemoglobin molecule,
rHb1.1,1 is a stabilized
pseudotetramer of 64 kDa with a P50 of 32 mm Hg
(8).
Solutions of rHb1.1 and other hemoglobins have been shown to have
vasoactive properties (9-12). Nitric oxide (NO), an important mediator
of vasodilation and other physiological processes (13), is known to
react rapidly with the oxyHb (14) forming metHb(Fe3+) and
NO3
or to bind to deoxyHb, essentially
scavenging the NO and causing vasoconstriction. Scavenging of NO by
hemoglobin is thought to contribute to the hypertensive response
observed with many cell-free hemoglobin solutions in intact animals
(15, 16), perfused organs (17, 18), and isolated vascular preparations
(19, 20). We have recently shown that genetic modification of the distal heme pocket to reduce the rate of NO scavenging, measured in vitro, can essentially eliminate the hemoglobin-induced
pressor response (21).
Although an interaction between cell-free hemoglobin and NO seems
clear, the site at which NO scavenging occurs (e.g.
intravascular or interstitial) is unknown. Several researchers have
suggested that hemoglobin must leave the vascular space to cause
constriction through an NO-mediated mechanism (22-24). Direct evidence
supporting this hypothesis is scant. However, larger polymerized
hemoglobins are known to have longer vascular retention times (25-27),
suggesting that they are less likely to leave the vascular space. Such
findings indicate that it might be possible to decrease the vasoactive properties of hemoglobin by making the hemoglobin molecule larger, thereby preventing extravasation into the interstitium and blocking NO-induced oxidation.
Glutaraldehyde polymerization of hemoglobin is well known and produces
a multitude of cross-linked species, e.g. dimers, trimers, tetramers, and larger species (28). Mixtures of these polymerized hemoglobins have been reported to exhibit minimal vascular responses (29), but none of the glutaraldehyde-treated monomeric species have
been purified to investigate their associated vasoactive properties. We
tested the hypothesis that the size of rHb1.1 can have a direct impact
on its hemodynamic properties. We report here results of a series of
experiments in which we compared the hemodynamic responses of
underivatized recombinant hemoglobin (rHb1.1) and
glutaraldehyde-polymerized rHb1.1 (poly-glxrHb). To account for
possible effects of intramolecular cross-linking and/or surface
modification by glutaraldehyde without a change in size, we also
examined the responses to glutaraldehyde-derivatized monomeric rHb1.1
(mono-glxrHb).
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EXPERIMENTAL PROCEDURES |
Hemoglobin Preparation
Preparation and Purification of Mono-glxrHb--
Recombinant
hemoglobin was produced at Somatogen as described by Looker et
al. (8). The hemoglobin was polymerized with glutaraldehyde
(Sigma, grade I) according to the following methods. rHb1.1 (150 mg/ml
in 5 mM sodium phosphate, pH 7.4, and 150 mM sodium chloride) was deoxygenated in a 1-liter round-bottom flask by
passing humidified nitrogen gas over the hemoglobin solution while
mixing in a Rotovap (Brinkman Instruments) for 6 h at 10 °C.
Following deoxygenation, the hemoglobin was capped with a white rubber
septum, and a 10% solution of deoxygenated glutaraldehyde in deionized
water was added to a final molar ratio of 8 mol of glutaraldehyde per
mol of hemoglobin. After incubation overnight at 4 °C, deoxygenated
sodium cyanoborohydride (126 mg/ml) in 0.1 M sodium
hydroxide was added to a final molar ratio of 10 mol of sodium
cyanoborohydride per mol of glutaraldehyde and incubated at room
temperature for 4 h. The solution was diluted to 2 liters with
buffer (5 mM sodium phosphate, pH 7.4 and 150 mM sodium chloride) and diafiltered with a Pellicon
diafiltration system (Millipore) against 10 volumes of the same buffer.
The solution was stored at
80 °C until purified by size exclusion chromatography.
Polymerized hemoglobin was fractionated by size exclusion
chromatography through a 7.5-liter Sephacryl S200 column followed by a
7.5-liter Sephacryl S300 column each equilibrated with 150 mM sodium chloride, 5 mM sodium phosphate, pH
7.4. The isolated monomeric rHb fractions from multiple chromatography
runs were pooled and concentrated to a final volume of 200 ml using the Pellicon diafiltration apparatus. The oxidized hemoglobin was reduced
by addition of sodium dithionite to deoxygenated mono-glxrHb until the
methemoglobin concentration was <2%. Reaction by-products were
removed by rechromatographing the solutions on the 7.5-liter Sephacryl
S200 column equilibrated in 20 mM Tris, pH 8.9, at 4 °C.
Endotoxin was removed by rechromatographing the hemoglobin on a 500-ml
Superose-Q ion-exchange column (Amersham Pharmacia Biotech)
equilibrated in 20 mM Tris, pH 8.9, at 4 °C. After
loading, the column was washed with 2 volumes of loading buffer, and
the hemoglobin was eluted with 20 mM Tris, pH 6.5, at
4 °C. The purified mono-glxrHb and poly-glxrHb were each diafiltered
against 10 volumes of 150 mM sodium chloride, 5 mM sodium phosphate, pH 7.4, and concentrated to 50 mg/ml.
Aliquots were filtered through pyrogen free 0.2-µm filters (Gelman
Sciences) and stored at
80 °C in 15-ml polypropylene tubes. The
purified hemoglobins were analyzed for methemoglobin and endotoxin as
described below. Methemoglobin was less than 10% and endotoxin was
less than 1 e.u./ml.
Preparation and Purification of Poly1-glxrHb--
Deoxygenated
rHb1.1 (145 mg/ml in 150 mM sodium chloride, 5 mM sodium phosphate, pH 7.4 at 23 °C) was reacted with
glutaraldehyde (preparation described previously) at a 7:1 molar ratio
of glutaraldehyde:hemoglobin for 5 min at room temperature. The
reaction was quenched by addition of sodium borohydride (152 mg/ml) in
0.1 M sodium hydroxide, at a 4:1 molar ratio
borohydride:glutaraldehyde. The solution was incubated an additional 15 min at room temperature and then diafiltered against 15 volumes of
deoxygenated 20 mM Tris, pH 9.0, at 8 °C. The
glutaraldehyde-treated hemoglobin was applied to a Superose-Q ion-exchange column (Amersham Pharmacia Biotech) at a ratio of 50 g of hemoglobin per 1 liter of resin. The column was washed with 2 volumes of 20 mM Tris, pH 9, at 8 °C followed by 12 volumes of 20 mM Tris, pH 7.6, at 8 °C. The poly1-glxrHb
was eluted with 20 mM Tris, pH 7.4, at 8 °C, diafiltered
against 150 mM sodium chloride, 5 mM sodium
phosphate, pH 7.4, and concentrated to 94 mg/ml. Aliquots were filtered
through pyrogen-free 0.2-µm filters (Gelman Sciences) and stored at
80 °C in 15-ml polypropylene tubes. The purified hemoglobins were
analyzed for methemoglobin and endotoxin as described below.
Methemoglobin was less than 10% and endotoxin was less than 1 e.u./ml.
Preparation and Purification of
Poly2-glxrHb--
Glutaraldehyde-polymerized hemoglobin with a
molecular mass range between 200 and 1000 kDa was isolated from the
reaction mixture used for preparing mono-glxrHb. The desired molecular mass range polymerized hemoglobin was isolated by size exclusion chromatography, reduced with dithionite, and endotoxin removed as
described previously for the mono-glxrHb.
Hemoglobin Characterization
Methemoglobin Analysis--
Methemoglobin content in hemoglobin
samples was determined using the method described by Evelyn and Malloy
(30).
Endotoxin Analysis--
Bacterial endotoxin content in
hemoglobin samples was determined using the Limulus Amebocyte Lysate
assay (Pyrotell Inc.).
Oxygen Equilibrium Binding
Measurements--
P50 and
nmax were determined using a hemox analyzer as
described by Hoffman et al. (31) at 37 °C and pH 7.40 in
50 mM HEPES (free acid) and 150 mM NaCl.
Autooxidation Rates--
Autooxidation rates of the hemoglobins
were determined at 37 °C by the methods described by Brantley
et al. (32) with the following modifications. The buffer
consisted of 50 mM Tris·hydrochloride (pH 8.3 at
37 °C), 10 µM diethylenetriaminepentaacetic acid, 150 nM superoxide dismutase, 150 nM catalase, and
equilibrated with 1 atm of oxygen. The hemoglobin concentration was 50 µM in heme.
NO-induced Oxidation--
The rates of NO-induced oxidation were
determined at 20 °C using stopped-flow rapid mixing techniques as
described by Eich et al. (33). Briefly, hemoglobin (0.2-1
µm) in 0.1 M sodium phosphate, pH 7.4, was mixed with a
solution containing dissolved NO and the formation of metHb monitored
at 402 nm.
Tryptic Mapping--
Tryptic mapping was performed as described
by Lippincott et al. (34). The percent change from rHb1.1
referred to in Table II was derived from the following equation.
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(Eq. 1)
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Sodium Dodecyl Sulfate-Polyacrylamide Gel
Electrophoresis--
SDS-PAGE was performed based on the method of
Laemmli (35). Aliquots (5 µg) were diluted with 2 volumes of SDS
sample buffer (Novex Corp.) containing 0.1 M dithiothreitol
and heated at 65 °C for 5 min. Samples were electrophoresed on an
8-16% polyacrylamide gradient Tris·glycine gel for 2.5 h at
120 V. The gel was stained in Coomassie Blue and then destained with a
solution of 40% methanol, 10% glacial acetic acid, and 50% water.
Destained gels were digitized using an IS-1000 digital imaging system
(Alpha Innotech Corp.).
High Performance Size Exclusion Chromatography--
The
molecular mass distribution of the glutaraldehyde cross-linked
hemoglobin was monitored using high performance size exclusion chromatography on a Superose 12 column (1 × 30 cm, Amersham
Pharmacia Biotech) connected in tandem with a Superose 6 column (1 × 30 cm, Amersham Pharmacia Biotech) mounted on a HP1090 HPLC system (Hewlett-Packard) and equilibrated with 150 mM NaCl, 5 mM sodium phosphate, pH 7.8. The absorbance was monitored
at 280 nm. SEC molecular weight standards were obtained from Sigma.
Reverse Phase HPLC--
Samples were prepared by precipitation
with ice-cold acid/acetone as described by Witkowska et al.
(36) and Lippincott et al. (34). Pellets were solubilized in
0.1% trifluoroacetic acid, 20% acetonitrile at a final concentration
of 1 mg/ml. Reverse phase HPLC analyses were performed using a Zorbax
C3 analytical column (0.46 × 25 cm) mounted on an HP1090 HPLC
system (Hewlett-Packard). The oven temperature was maintained at
40 °C. Solvent A is 0.1% trifluoroacetic acid in water and solvent
B is 0.1% trifluoroacetic acid in acetonitrile. The flow rate was 1 ml/min. The column was equilibrated in 65% solvent A, 35% solvent B. Following sample injection the column was maintained at the starting
conditions for 5 min and then ramped to 51% solvent A, 49% solvent B
over 45 min.
LC-MS--
Mass spectrometry was performed as described by
Lippincott et al. (34) using a Finnigan Mat LCQ with an
HP1090 HPLC on the front end to run reverse phase separations prior to analysis.
Dynamic Light Scattering--
Dynamic light scattering was
performed using a Nicomp 370 Submicron Particle Sizer equipped with a
40-milliwatt HeNe laser. Data were collected and averaged over 10 min
and analyzed using the C370 version 12 software program provided by
Particle Sizing Systems (Santa Barbara, CA). Samples were passed
through 0.2-µm filters prior to analysis.
Oncotic Pressure of Hemoglobins--
The oncotic pressures of
hemoglobins used in this study were measured in vitro
directly in a colloid osmometer (model 4420, Wescor, Logan, UT). The
oncotic pressures were measured at the final hemoglobin concentrations
used for injection into the animals.
Evaluation of Hemodynamic Responses
All surgical and experimental procedures were approved by the
Somatogen Animal Care and Use Committee. Male Sprague-Dawley rats
(Charles River, 250-350 g) were used for all experiments. The animals
were chronically instrumented with pulsed Doppler flow probes on the
ascending aorta for cardiac output measurement and with indwelling
arterial and venous catheters as described previously (37) for blood
pressure measurement and hemoglobin infusion.
For all experiments, the animals were studied in a conscious, resting
state. On the day of the experiment, each rat was placed in a Plexiglas
experimental chamber that was of sufficient size (25 × 15 × 12.5 cm) to allow free movement. The chamber was flushed continuously
with fresh air, and fresh bedding covered the chamber floor. The
catheters and Doppler flow probe leads were fed through the top of the
chamber, and both catheters were opened and flushed with sterile,
heparinized saline. The arterial catheter was connected to a pressure
transducer for arterial pressure measurement, and the venous catheter
was connected to a syringe containing hemoglobin or human serum albumin
(HSA, Baxter Healthcare Corp.). HSA was used as a negative control with
the assumption that it does not consume NO. Since the molecular mass
and concentration of HSA (66.5 kDa, 50 mg/ml) were virtually the same
as those of rHb1.1 and mono-glxrHb, it served as a comparator for the
mass and volume of protein solution administered. The flow probe leads
were connected to a modified high velocity module (HVPD, Crystal
Biotech, Northborough, MA) that was used in the autotracking mode at a
pulse repetition frequency of 125 kHz to avoid detection of spurious,
aliasing signals (38). Arterial pressure, heart rate, and cardiac
output were continuously recorded at sampling frequency of 50 Hz using a Windaq data acquisition system (Dataq Instruments, Columbus, OH) and
a 160-MHz Pentium computer (Compaq).
After sufficient time for acclimatization to the experimental
surroundings and recording of base-line data (generally 30-60 min),
hemoglobin or HSA was infused at a rate of 0.5 ml/min until a dose of
350 mg/kg was administered (less than 3 min). Hemodynamic data were
collected continuously for 90 min following completion of the infusion.
At the end of the 90-min data collection period, phenylephrine (3 µg/ml) was infused at a rate of 6 µg/kg/min for 2 min to verify
proper catheter placement and provide a qualitative indication of the
vascular responsiveness of each animal. Data were collected for an
additional 15 min. Animals that did not exhibit a brisk response to
phenylephrine were not included in subsequent analysis (<5%
occurrence). Each animal received only a single dose of hemoglobin or HSA.
In order to determine the hemoglobin concentrations in plasma following
administration and during the period of hemodynamic measurement,
another set of rats was instrumented with venous catheters using the
methods described above. After 2-3 days of recovery, the rHb1.1,
mono-glxrHb, and poly-glxrHb were again administered to conscious rats
by intravenous infusion (350 mg/kg, 0.5 ml/min). At time points of 0, 30, 60, 120 min post-administration, the animals were physically
immobilized, and blood samples (0.3-0.4 ml) were obtained by tail vein
transection. The blood was collected in heparinized tubes, and
centrifuged at 5,000 × g for 5 min to obtain plasma
samples. Hemoglobin concentration in the plasma was determined using
the analytical technique of Tentori and Salvati (39).
Data and Statistical Analyses
Custom-designed software was used to process the raw hemodynamic
data. Mean arterial pressure, heart rate, and cardiac output values
were determined by averaging data over 30-s intervals every 5 min prior
to and for 30 min following hemoglobin administration. Thereafter, 30-s
averages were obtained every 10 min until the end of the experiment.
All data shown are mean ± S.E.
Both mean arterial pressure and heart rate are expressed as the change
from base line. Base-line values were calculated as the average of the
data collected for 30 min prior to hemoglobin or HSA administration.
Cardiac output is expressed as percent change from base line. Total
peripheral resistance was calculated from mean arterial pressure and
cardiac output and is also expressed as percent change from base line.
To facilitate statistical comparisons of the hemodynamic responses to
several molecules, the data from 10- to 90-min post-administration were
averaged for each animal to obtain an "overall" response. The
infusion generally required up to 3 min to complete, and the animals
were frequently active for another 2-3 min. By 10 min post-infusion,
the animals had returned to their control, resting state. Therefore,
data from the 5-min time point were excluded from the overall response
calculation to avoid potential inclusion of hemodynamic changes due to
activity. The overall response data were analyzed by one-way analysis
of variance and Newman-Keuls post hoc tests.
 |
RESULTS |
Hemoglobin Preparation
A monomeric and two polymeric glutaraldehyde-modified recombinant
hemoglobins were prepared for testing in rat hemodynamic studies. The
polymerization reactions were quenched with either cyanoborohydride or
borohydride. Cyanoborohydride is a Schiff base-specific reducing agent
(40), and borohydride can reduce both Schiff bases and aldehydes (41).
Both reducing agents were equally effective in quenching the reactions
as analyzed by SEC (data not shown). The mono-glxrHb was purified from
the polymer mixture by preparative size exclusion chromatography (SEC)
to 91% purity with the remainder being dimeric hemoglobin as assessed by analytical SEC (Fig. 1B).
The molecular mass of the mono-glxrHb fraction (Table
I) based on comparison with globular
protein standards corresponded to an apparent molecular mass of 51 kDa and a diameter of 8.6 nm as determined by dynamic light scattering. Purified rHb1.1 (molecular mass = 64.4 kDa) also showed an
apparent mass of 51 kDa (Fig. 1A) although its diameter was
7.3 nm, less than that observed for mono-glxrHb.

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Fig. 1.
Size exclusion chromatographic analysis of
the purified glutaraldehyde cross-linked rHb fractions mono-glxrHb,
poly1-glxrHb, and poly2-glxrHb. Recombinant hemoglobin was reacted
with glutaraldehyde and size-fractionated as described under
"Experimental Procedures." A, purified rHb1.1;
B, purified mono-glxrHb; C, purified
poly1-glxrHb; D, purified poly2-glxrHb. The molecular weight
reference guide was generated from a linear regression of retention
time versus log molecular weight for a series of protein
standards.
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Table I
Summary of size and functional characterization for the purified
fractions of glutaraldehyde-modified rHb1.1
The data shown here were from single determinations of each assay.
Analysis of purified hemoglobins from other batches demonstrated
similar results (data not shown).
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Large scale purification of polymeric hemoglobin was accomplished using
ion-exchange chromatography to fractionate the hemoglobin polymers. The
majority of cross-linked hemoglobins greater than 1000 kDa flowed
through the column at pH 9.0 and were discarded. Lowering the buffer pH
to 7.6 caused elution of the monomeric hemoglobin along with some
dimeric and trimeric hemoglobins which eluted over a number of column
volumes (data not shown). An additional decrease in the buffer pH to
7.4 caused elution of the polymeric hemoglobin fraction of interest.
The SEC profile of poly1-glxrHb is shown in Fig. 1C. This
fraction contained less than 1% as monomeric rHb, ~7% dimeric rHb,
and ~15% trimeric rHb with the majority ranging from 200 to 1000 kDa
(Table I). Approximately 14% of the polymers were greater than 1000 kDa. The apparent diameter was 15.8 nm as measured by dynamic light
scattering (Table I). Due to the presence of lower molecular weight
hemoglobins in the poly1-glxrHb fraction, a more highly purified
subfraction was prepared to discern if these species had any effect on
hemodynamic responses. This hemoglobin polymer (poly2-glxrHb) was
purified by multiple SEC fractionations to exhaustively remove the
monomeric, dimeric, and trimeric hemoglobins along with large polymers
>1000 kDa. The poly2-glxrHb had a range of 200-1000 kDa with a peak molecular mass of 439 kDa (see Fig. 1D and Table I) and an
apparent diameter of 16.2 nm. The material contained approximately 2%
as dimers and trimers and approximately 14% >1000 kDa.
Hemoglobin Characterization
Functional Characterization--
The mono- and poly-glxrHb
fractions were examined for changes in the oxygen affinity
(P50), cooperativity
(nmax), autooxidation rates, and NO oxidation
rates of oxyHb (Table I). The P50 for unmodified
rHb1.1 was 32 mm Hg with a maximum Hill coefficient of 2.2. Glutaraldehyde modification of the hemoglobin resulted in a slightly
decreased P50 for mono- and poly1-glxrHb (30mm
Hg) and a slightly increased P50 for
poly2-glxrHb (36 mm Hg). The Hill coefficients of the modified
hemoglobins were all similar and demonstrated significantly decreased
cooperativity when compared with unmodified rHb1.1
(nmax = 1.4 for mono-glxrHb, 1.5 for
poly1-glxrHb, and 1.3 for poly2-glxrHb). The autooxidation rates were
0.7 and 1.0 h
1 for the mono- and poly-glxrHb,
respectively. Unmodified rHb1.1 has an intrinsic autooxidation rate of
0.7 h
1 under identical conditions. Rates of NO-induced
oxidation of oxyhemoglobin for the mono- and poly2-glxrHb fractions
were all essentially identical to rHb1.1 which has a rate of 57 µmol
1·s
1.
Characterization of Isolated Fractions--
Mono-glxrHb and both
poly-glxrHb preparations were analyzed by SDS-PAGE (Fig.
2). Native rHb1.1 (Fig. 2, lane
1) showed a characteristic banding pattern with the expected bands
at approximately 15 and 32 kDa corresponding to the
-globin and
di-
-globin, respectively. Mono-glxrHb (Fig. 2, lane 2)
contained the expected
- and di-
-globin bands as well as three
additional bands with molecular masses corresponding to 44, 46, and 62 kDa. The bands at 44 and 46 kDa may correspond to the cross-link of the
-globin to the first or second half of the di-
-globin resulting
in differentiation by SDS-PAGE. The band at 62 kDa is consistent with
two molecules of the
-globin chain cross-linked to the one
di-
-globin chain. Similar banding patterns, albeit to a reduced
degree, were observed for the poly1-glxrHb and poly2-glxrHb fractions.
However, the band at 62 kDa was not visible in either of the poly1- or
poly2-glxrHb. Additionally, diffuse banding patterns of cross-linked
polypeptides with molecular masses greater than 64 kDa were observed in
the poly1- and poly2-glxrHb.

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Fig. 2.
Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis of the purified glutaraldehyde cross-linked rHb
fractions. Aliquots of the indicated samples were analyzed on an
8-16% SDS-PAGE as described under "Experimental Procedures."
Positions of molecular mass markers are indicated to the
right. Lane 1, rHb1.1 control; lane 2, mono-glxrHb; lane 3, poly1-glxrHb; lane 4, poly2-glxrHb.
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Glutaraldehyde modification of the purified hemoglobins was further
examined using reverse phase HPLC and in-line mass spectrometry (Fig.
3). Unmodified
- and di-
-globin
elute at 31.0 and 42.8 min, respectively, with molecular masses of
15,913 and 30,342 atomic mass units (Fig. 3A), and they are
in good agreement with the expected values (42). LC-MS of the
mono-glxrHb (Fig. 3B) demonstrated the presence of
unmodified
-globin eluting at 31.5 min along with a number of other
peaks near the unmodified
-globin eluting at 31.8, 32.7, and 33.9 min with masses of 15,981, 15,979, and 15,979 atomic mass units. The
majority of the globins eluted between 40 and 43 min. It was not
possible to define the masses of the polypeptides due to the
heterogeneity of the sample giving rise to a noisy m/z
spectrum. SDS-PAGE analysis of the polypeptides in the peak eluting
between 40 and 43 min demonstrated that it was composed of the 32- (di-
), 44-, 46-, and 62-kDa species. Similar chromatographic
profiles to that observed for the mono-glxrHb were observed for the
poly1- and poly2-glxrHb samples, although the modified
-globins
eluting between 31.5 and 33.9 min were present to a lesser degree than
that observed for mono-glxrHb. The elution time for the majority of the
globins shifted to between 41 and 46 min. Again, it was not possible to
deconvolute the spectrum due to the heterogeneity of the derivatized
globins.

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Fig. 3.
Reverse phase HPLC and LC-MS analysis of the
purified mono-glxrHb, poly1-glxrHb, and poly2-glxrHb. Hemoglobins
were analyzed by reverse phase-HPLC and LC-MS as described under
"Experimental Procedures." A, rHb1.1; B,
mono-glxrHb; C, poly1-glxrHb; D, poly2-glxrHb.
The masses for the peaks are expressed in atomic mass units.
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The sites of glutaraldehyde modification/cross-linking were examined
using tryptic mapping. The notation of Lippincott et al.
(34) is used here to denote tryptic fragments. The tryptic profiles of
the mono- and poly-glxrHb fractions were similar but demonstrated
significant differences when compared with unmodified rHb1.1 (Fig.
4). Peak height ratios to the
4
peptide were used to compare maps. The
4 peptide of the
-globin
is flanked by Arg residues that are not modified by glutaraldehyde.
Therefore, the degree of trypsin digestion of the
4 peptide was used
as an internal standard, and the ratio of the peak height of each peptide to the
4 peptide was used to elucidate differences between the control and glutaraldehyde-modified samples (Table
II). The majority of peptides that
decreased by >35% on the
-globin were
1,
8/9-1,
8/9-2,
9,
10/11,
11,
12,
13- and
14, indicating modification
or involvement of cross-linking by the following residues:
N
terminus, Lys66, Lys82, Lys95,
Lys108, Lys120, and Lys132. The
modified peptides of the di-
-globin included
1,
8,9-1,
8,9-2,
10/11,
11,
12, and
13 corresponding to the
modification of the following residues: di-
N terminus,
Lys61, Lys99, Lys127, and
Lys139 from the first half of the di-
-globin chain and
the corresponding residues in the second half Lys203,
Lys241, Lys269, and Lys281. Two
difference peaks at 44.8 (1) and 45.2 (2) min were detected in the
mono-glxrHb and poly-glxrHb tryptic maps. LC-MS analysis of peak 1 indicated a mass of 1052 atomic mass units which is consistent with a
mass of the N-terminal
peptide (
1, 984 atomic mass units) plus
an additional 68 atomic mass units. Peak 2 had a mass of 828 atomic
mass units which is consistent with the mass of the di-
N-terminal
peptide (761 atomic mass units) plus an additional 68 atomic mass
units. We were unable to detect any other difference peptides with
masses corresponding to either glutaraldehyde decoration or
cross-linking.

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Fig. 4.
Trypsin maps of purified
glutaraldehyde cross-linked rHb fractions. Aliquots were
analyzed by trypsin mapping as described under "Experimental
Procedures." A, rHb1.1; B, mono-glxrHb;
C, poly1-glxrHb; D, poly2-glxrHb. The peptides
denotations in A are the same as those used by Lippincott
et al. (34). The peptides with asterisks
demonstrated decreases in peak heights of at least 35% from rHb1.1
occurring in the mono-, poly1-, and poly2-glxrHb fractions. The same
peptides all decreased in C and D. Difference
peptides are denoted 1 and 2 in B and
were observed in C and D.
|
|
Evaluation of Hemodynamic Responses
Administration of rHb1.1 caused a rise in mean arterial pressure
to levels approaching 20 mm Hg above control (Fig.
5A) and a concomitant fall in
heart rate and cardiac output (Fig. 5, B and C).
Consequently, total peripheral resistance rose significantly and
reached a plateau of approximately 30% above base-line values. In
contrast to the responses elicited by rHb1.1, administration of the
same volume of a 5% protein solution (HSA) caused little change in
mean arterial pressure or vascular resistance (Fig. 5, A and
D). Heart rate and cardiac output rose slightly following HSA administration (Fig. 5, B and C).
Surprisingly, the changes in mean arterial pressure and total
peripheral resistance elicited by mono-glxrHb were depressed compared
with those induced by rHb1.1 (Fig. 5, A and D),
despite the similarities in molecular weight. The mean arterial
pressure response induced by poly1-glxrHb was significantly reduced
compared with those obtained with rHb1.1 and mono-glxrHb (Fig.
5A). However, the vascular resistance response was identical
to that observed with mono-glxrHb (Fig. 5D). Changes in
heart rate and cardiac output were not different for the three hemoglobin preparations (Fig. 5, B and C).
Base-line mean arterial pressure and heart rate values were not
different in any of the experimental groups (Table
III).

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|
Fig. 5.
Overall mean arterial pressure
(A), heart rate (B), cardiac output
(C), and total peripheral resistance (D)
responses determined as the average of the data obtained from 10- to
90-min post-administration. Bars represent mean ± S.E. *, p < 0.05 versus rHb1.1; ,
p < 0.05 versus mono-glxrHb; #,
p < 0.05 versus poly1-glxrHb.
|
|
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[in this window]
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|
Table III
Base-line hemodynamic values obtained prior to hemoglobin or HSA
administration
Data are mean ± S.E. obtained from at least 30 min of base-line
recording. n, number of animals; MAP, mean arterial
pressure; HR, heart rate; bpm, beats per minute.
|
|
From these results, it appears that glutaraldehyde decoration
(i.e. mono-glxrHb) significantly attenuates the hemodynamic response induced by rHb1.1 and that an increase in molecular size by
glutaraldehyde cross-linking (i.e. poly-glxrHb) further
decreases the mean arterial pressure response. Since poly1-glxrHb
contained small amounts of dimeric and trimeric rHb species (Fig. 1),
we tested the contributions of those lower molecular weight fractions by examining the responses to a more highly purified fraction (poly2-glxrHb) in a second set of experiments. These animals were instrumented with arterial and venous catheters as described under "Experimental Procedures," but pulsed Doppler flow probes were not
implanted. Responses to rHb1.1, mono-glxrHb, and HSA were also obtained
in animals prepared in the same manner. As with poly1-glxrHb, the
change in mean arterial pressure in response to poly2-glxrHb was
significantly lower than those obtained with rHb1.1 and mono-glxrHb
(Fig. 6A). The heart rate
responses to rHb1.1, mono-glxrHb, and poly2-glxrHb were all greater
than the response to HSA but were not different from each other (Fig.
6B).

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|
Fig. 6.
Overall mean arterial pressure
(A) and heart rate (B) responses
determined as the average of the data obtained from 10- to 90-min
post-administration. Bars represent mean ± S.E.
*, p < 0.05 versus rHb1.1; ,
p < 0.05 versus mono-glxrHb; #,
p < 0.05 versus poly2-glxrHb.
|
|
To determine if the observed differences in hemodynamic responses could
be accounted for by differences in plasma hemoglobin concentration,
plasma samples were obtained at several time points during the first
120 min following administration. As shown in Table
IV, the concentrations of rHb1.1 and
mono-glxrHb were identical and were both significantly less than the
concentration of poly2-glxrHb throughout the time course of hemodynamic
measurement. Therefore, the reduced pressor responses elicited by mono-
and poly-glxrHb cannot be attributed to lower plasma concentrations or
more rapid clearance from the vascular space.
View this table:
[in this window]
[in a new window]
|
Table IV
Plasma hemoglobin concentrations (mg/ml) obtained at several time
points corresponding to the period of hemodynamic measurement
0 min refers to samples taken immediately after hemoglobin
administration, and the other columns refer to samples taken at the
specified times post-administration. Data are shown as mean ± S.E.
|
|
 |
DISCUSSION |
The prevailing hypothesis for the mechanism of the
hemoglobin-induced rise in arterial pressure postulates that hemoglobin extravasates into the interstitium and causes vasoconstriction due to
scavenging of NO. Sharma and colleagues (16) reported that co-infusion
of L-arginine diminishes the hemodynamic responses to
diaspirin cross-linked hemoglobin, a monomeric hemoglobin. Similar
results with L-arginine were reported by Katsuyama et al. (43) who also showed that inhibition of NO synthesis by N
-nitro-L-arginine attenuates the
hypertensive response to diaspirin cross-linked hemoglobin. Studies
performed in vitro have similarly indicated a dependence on
an interaction between hemoglobin and NO (20). We have recently
reported that administration of recombinant hemoglobin in which the
distal heme pocket was genetically modified to reduce NO scavenging
rates by 20-fold demonstrated negligible hemodynamic responses in rats
(21). Mono- and poly-glxrHb had NO oxidation rates that were identical
to that of rHb1.1, indicating that differences in NO scavenging rates
cannot account for the observed reductions in the hypertensive
response. One possible explanation for our data is that glutaraldehyde
treatment reduced the reactive heme concentration at the site of NO
scavenging. Whereas the specific site of NO scavenging important for
vasoconstriction is unknown, several investigators have suggested that
it lies outside of the vascular compartment (22-24). Although there is little or no direct evidence available to support the hypothesis that
extravasation is required for the hemoglobin-induced hypertensive response, our results and those of others (22, 27, 44) with polymerized
hemoglobin are consistent with an obligatory role for hemoglobin
extravasation. If polymerized hemoglobin such as poly-glxrHb is less
able to leave the vasculature, the interstitial concentrations will be
lower than those for smaller hemoglobin molecules (e.g.
rHb1.1), and the amount of interstitial NO scavenging will be
correspondingly diminished. In this context, the reduced hypertensive
response elicited by mono-glxrHb is puzzling. If extravasation is
required for the hypertensive effect, our results would suggest that
molecular size is not the sole determinant of the rate of extravasation
and that glutaraldehyde modification of the mono-glxrHb prevents its
extravasation to the sites of NO scavenging.
The routes whereby proteins cross- the endothelium are still
controversial but likely include a paracellular pathway and one utilizing plasmalemmal vesicles (45, 46). Since plasmalemmal vesicles
apparently provide the primary mode of transport for molecules greater
than 4 nm in diameter (46), endocytotic transport of rHb1.1 and
mono-glxrHb (diameters of 7.3 and 8.5 nm, respectively) is the likely
route whereby these hemoglobins extravasate. In support of this
possibility, Milici et al. (47) have reported that albumin
(64 kDa) movement across the myocardial endothelium occurs via
plasmalemmal vesicles. Velky et al. (48) have provided additional evidence for endocytotic transport of hemoglobin in their
observation that inhibition of endocytosis by
m-dansylcadaverine decreased leakage of stroma-free
hemoglobin into the peritoneal cavity. Initiation of the albumin
transport process appears to require binding of albumin to either
SPARC, gp60, gp30, or gp18 receptors (49). The additional finding that
modification of the albumin with either colloidal gold particles or
maleic anhydride, which nonspecifically modify primary amines, inhibits
its binding to both SPARC and gp60 (49) suggests that similar surface
modification of hemoglobin could interfere with its transendothelial movement.
We have ample evidence that the surface of the hemoglobin is altered by
glutaraldehyde treatment. Glutaraldehyde is known to modify primary
amines (50) which on proteins are available as N-terminal amines and
-amines of the Lys residues. In addition, the x-ray crystallographic
structure of the hemoglobin (51) shows that the N termini of the
-
and di-
-globins and the majority of the Lys residues are on the
surface of the hemoglobin and readily available to the solvent. The
Lys
108 and Lysdi-
99 are buried within the
diphosphoglycerate-binding pocket of the hemoglobin. Indeed, our
tryptic mapping data suggest that the
Lys residues 66, 82, 95, 108, 120, and 132 and di-
Lys residues 61, 99, 127, 139, 203, 241, 269, and 281 were modified on average to greater than 35% by
glutaraldehyde. Other Lys residues were also modified, albeit to
varying degrees. The similarities between the tryptic maps of mono-,
poly1-, and poly2-glxrHb (Fig. 4 and Table II) suggest that these
hemoglobins are all modified comparably and may account for the similar
hemodynamic responses observed for the glutaraldehyde-modified
monomeric and polymeric hemoglobins. The nature of the modifications on
the Lys residues is not clear. We observed only two identifiable
difference peaks in the tryptic map of the mono-glxrHb (Fig.
4B), whereas a number of peptides were clearly modified.
Both difference peaks, based on MS/MS fragmentation (data not shown),
were assigned as 68-Da modifications of the N-terminal peptide from the
- and di-
-globins. The mass increase expected for a single
molecule of glutaraldehdye forming Schiff bases between two amines
followed by reduction with borohydride to imines is 68 Da. Reaction of
the N terminus with one of the aldehyde groups followed by reduction
with borohydride should result in a mass increase of 88 Da. Therefore,
the exact nature of the modification is unclear and under further investigation.
Cross-linking of the
- and di-
subunits may also be important in
masking potential binding sites buried within the rHb1.1 molecule or in
further stabilization by internal cross-linking. For example,
haptoglobin binds to hemoglobin following its dissociation into 
dimers exposing a previously buried binding site (52, 53). The SDS-PAGE
(Fig. 2) and LC-MS analysis (Fig. 3) both demonstrated the presence of
- to di-
-globin cross-linked species in both the mono- and
poly-glxrHbs. A single
cross-linked to di-
-globin was detected
by the SDS-PAGE as two distinct bands with either 44- or 46-kDa
molecular masses. A
/di-
cross-linked globin would be expected to
have an approximate molecular mass of 46.3 kDa eluting near the 46-kDa
band on the gel. The lower band may be due to additional intraglobin
cross-linking changing its electrophoretic mobility or due to the
presence of glutaraldehyde on the globin. Several
modifications/decorations to the
-globin were observed during LC-MS
analysis (Fig. 3). The differences in mobility observed by SDS-PAGE
were not seen following cross-linking of a second
-globin to produce
the
2/di-
cross-linked species. Disappearance of that
band in the poly1- and poly2-glxrHb fractions may indicate further
polymerization of the hemoglobin.
We found that glutaraldehyde polymerization decreased the mean arterial
pressure response to a greater extent than did glutaraldehyde decoration (Fig. 5A). However, the vascular resistance
responses were the same for both treatments (Fig. 5D). The
reason for this apparent discrepancy in unknown but could stem from
differences in oncotic or colloid osmotic pressures. The oncotic
pressure of rHb1.1 measured in vitro at the concentration
used (100 mg/ml) is 42 mm Hg, whereas the oncotic pressure of
poly-glxrHb at the same concentration is 11 mm Hg. The oncotic pressure
of HSA measured under the same conditions is 58 mm Hg. Although the
oncotic pressure of mono-glxrHb was not determined, it would not be
expected to differ significantly from the value obtained for rHb1.1.
Administration of a highly hypo-oncotic solution such as poly-glxrHb
may cause fluid to leave the vascular space, leading to contraction of
intravascular volume and reductions in cardiac output and mean arterial
pressure. This mechanism, in addition to the observed attenuation of
peripheral vasoconstriction, could thus account for the additional
attenuation in the mean arterial pressure response compared with that
seen for the mono-glxrHb. Although blood volume was not determined in
these studies, the observation that the concentration of poly-glxrHb was higher than that of either rHb1.1 or mono-glxrHb supports the
hypothesis that fluid moved out of the vascular space after administration of poly-glxrHb.
Vandegriff and Winslow (54) have suggested that the vasoconstriction
elicited by cell-free hemoglobin is due to an autoregulatory response
to accelerated O2 delivery to arterioles. According to this
hypothesis, the O2 affinity of many hemoglobin solutions is
sufficiently low that O2 is released in the arterioles, and because of the O2 sensitivity of these vessels,
vasoconstriction occurs. The P50 of hemoglobin
in whole blood is 26 mm Hg (55). The P50 values
of the hemoglobins used here were all similar with P50 values ranging from 30 to 36 mm Hg.
Furthermore, the P50 value of rHb1.1 (32 mm Hg)
was between those of mono- and poly-glxrHb (30 and 36 mm Hg,
respectively), making it highly unlikely that the observed attenuation
of the hypertensive response is due to differences in oxygen affinity.
To our knowledge, this study provides the first evidence that
modification of hemoglobin with glutaraldehyde and without
polymerization can directly impact the peripheral vasoconstriction
induced by hemoglobin. The results presented here suggest that the
effects of glutaraldehyde treatment on the hemodynamic responses to
hemoglobin are by and large due to surface modification and/or internal
stabilization. The mechanism(s) by which the modifications alter the
hypertensive response remain to be elucidated.
 |
ACKNOWLEDGEMENTS |
We gratefully acknowledge the excellent
technical expertise of Jon Vincelette, Anne Armstrong, and Michael
Suniga in carrying out the hemodynamic experiments and Dominic Madril,
Maria Pagratis, Tim Fattor, and Rita Vali for help with purification of
the glutaraldehyde-treated hemoglobins. We also thank Dr. Douglas Lemon
for measuring the NO-induced oxidation rates and Tim Fattor for
measuring autooxidation rates. This work would not have been possible
without the support of the Somatogen technical staff in manufacturing,
pilot operations, and assay services. Finally, we thank Drs. Gillian
Olins, David Foster, and Richard Gorczynski for critical review of the manuscript.
 |
FOOTNOTES |
*
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.
To whom correspondence should be addressed: Amgen, Inc., One Amgen
Center Drive, MS 8-1-C, Thousand Oaks, CA 91320. Tel.: 805-447-0712;
Fax: 805-498-8674; E-mail: bKerwin{at}amgen.com.
The abbreviations used are:
rHb1.1, recombinant
human hemoglobin; oxyHb, oxyhemoglobin; deoxyHb, deoxyhemoglobin; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid
chromatography; LC-MS, liquid chromatographic mass spectrometry; HSA, human serum albumin; SEC, size exclusion chromatography; mono-glxrHb, glutaraldehyde-derivatized monomer; poly-glxrHb, glutaraldehyde
cross-linked polymer.
 |
REFERENCES |
-
Keipert, P. E.
(1992)
in
Oxygen Transport to Tissue XIV (Erdmann, W., and Bruley, D. F., eds), pp. 43-464, Plenum Publishing Corp., New York
-
Tsuchida, E.
(1995)
in
Artificial Red Cells: Materials, Performances, and Clinical Study as Blood Substitutes (Tsuchida, E., ed), pp. 1-20, John Wiley & Sons Ltd., Chichester, UK
-
Winslow, R. M.
(1989)
in
The Red Cell, Seventh Ann Arbor Conference (Brewer, G. J., ed), pp. 305-323, Alan R. Liss, Inc., New York
-
Hamilton, P. B.,
Hiller, A.,
and Van Slyke, D. D.
(1947)
J. Exp. Med.
86,
477-487
-
Rabiner, S. F.,
Helbert, J. R.,
Lopas, H.,
and Friedman, L. H.
(1967)
J. Exp. Med.
126,
1127-1142[Medline]
[Order article via Infotrieve]
-
Bunn, H. F.,
Esham, W. T.,
and Bull, R. W.
(1969)
J. Exp. Med.
129,
909-924[Medline]
[Order article via Infotrieve]
-
Winslow, R. M.
(1992)
Hemoglobin-based Red Cell Substitutes, The Johns Hopkins University Press, Baltimore
-
Looker, D.,
Abbott-Brown, D.,
Cozart, P.,
Durfee, S.,
Hoffman, S.,
Mathews, A. J.,
Miller-Roehrich, J.,
Shoemaker, S.,
Trimble, S.,
Fermi, G.,
Komiyama, N. H.,
Nagai, K.,
and Stetler, G. L.
(1992)
Nature
356,
258-260[CrossRef][Medline]
[Order article via Infotrieve]
-
Gulati, A.,
Sharma, A. C.,
and Burhop, K. E.
(1994)
Life Sci.
55,
827-837[CrossRef][Medline]
[Order article via Infotrieve]
-
Keipert, P. E.,
Gonzales, A.,
Gomez, C. L.,
MacDonald, V. W.,
Hess, J. R.,
and Winslow, R. M.
(1993)
Transfusion
33,
701-708[Medline]
[Order article via Infotrieve]
-
Kida, Y.,
Yamakawa, T.,
Iwasaki, S.,
Furusho, N.,
Kadowaki, Y.,
Iwata, S.,
Iwashita, Y.,
and Nishi, K.
(1995)
Artif. Organs
19,
511-518[Medline]
[Order article via Infotrieve]
-
Sharma, A. C.,
and Gulati, A.
(1994)
J. Lab. Clin. Med.
123,
299-308[Medline]
[Order article via Infotrieve]
-
Moncada, S.,
Palmer, R. M. J.,
and Higgs, E. A.
(1991)
Pharmacol. Rev.
43,
109-142[Medline]
[Order article via Infotrieve]
-
Doyle, M. P.,
and Hoekstra, J. W.
(1981)
J. Inorg. Biochem.
14,
351-358[CrossRef][Medline]
[Order article via Infotrieve]
-
Barve, A.,
Sen, A. P.,
Saxena, P. R.,
and Gulati, A.
(1997)
Art. Cells Blood Substit. Immobil. Biotechnol.
25,
75-84[Medline]
[Order article via Infotrieve]
-
Sharma, A. C.,
Singh, G.,
and Gulati, A.
(1995)
Am. J. Physiol.
269,
H1379-H1388[Abstract/Free Full Text]
-
MacDonald, V. W.,
and Motterlini, R.
(1994)
Art. Cells Blood Substit. Immobil. Biotechnol.
22,
565-575[Medline]
[Order article via Infotrieve]
-
Motterlini, R.,
and MacDonald, V. W.
(1993)
J. Appl. Physiol.
75,
2224-2233[Abstract]
-
Freas, W.,
Llave, R.,
Jing, M.,
Hart, J.,
McQuillan, P.,
and Muldoon, S.
(1995)
J. Lab. Clin. Med.
125,
762-767[Medline]
[Order article via Infotrieve]
-
Rioux, F.,
Petitclerc, E.,
Audet, R.,
Drapeau, G.,
Fielding, R. M.,
and Marceau, F.
(1994)
J. Cardiovasc. Pharmacol.
24,
229-237[Medline]
[Order article via Infotrieve]
-
Doherty, D. H.,
Doyle, M. P.,
Curry, S. R.,
Vali, R. J.,
Fattor, T. J.,
Olson, J. S.,
and Lemon, D. D.
(1998)
Nat. Biotechnol.
16,
672-676[Medline]
[Order article via Infotrieve]
-
Abassi, Z.,
Kotob, S.,
Pieruzzi, F.,
Abouassali, M.,
Keiser, H. R.,
Fratantoni, J. C.,
and Alayash, A. I.
(1997)
J. Lab. Clin. Med.
129,
603-610[Medline]
[Order article via Infotrieve]
-
Gould, S. A.,
and Moss, G. S.
(1996)
World J. Surg.
20,
1200-1207[CrossRef][Medline]
[Order article via Infotrieve]
-
Snyder, S. R.,
and Walder, J. A.
(1991)
in
Bio/Technology of Blood (Goldstein, J., ed), pp. 101-116, Butterworth-Heinemann, Boston
-
DeVenuto, F.,
and Zegna, A.
(1983)
J. Surg. Res.
34,
205-212[Medline]
[Order article via Infotrieve]
-
Keipert, P.,
Minkowitz, J.,
and Chang, T. M. S.
(1982)
Int. J. Artif. Organs
5,
383-385[Medline]
[Order article via Infotrieve]
-
Sehgal, L. R.,
Gould, S. A.,
Rosen, A. L.,
Sehgal, H. L.,
and Moss, G. S.
(1984)
Surgery
95,
433-438[Medline]
[Order article via Infotrieve]
-
MacDonald, S. L.,
and Pepper, D. S.
(1994)
Methods Enzymol.
231,
287-308[Medline]
[Order article via Infotrieve]
-
Gould, S. A.,
Sehgal, L. R.,
Rosen, A. L.,
Sehgal, H. L.,
and Moss, G. S.
(1990)
Ann. Surg.
211,
394-398[Medline]
[Order article via Infotrieve]
-
Evelyn, K. A.,
and Malloy, H. T.
(1938)
J. Biol. Chem.
126,
655
-
Hoffman, S. J.,
Looker, D. J.,
Roehrich, J. M.,
Cozart, P. E.,
Durfee, S. L.,
Tedesco, J. L.,
and Stetler, G. L.
(1990)
Proc. Natl. Acad. Sci. U. S. A.
87,
8521-8525[Abstract]
-
Brantley, R. E., Jr.,
Smerdon, S. J.,
Wilkinson, A. J.,
Singleton, E. W.,
and Olson, J. S.
(1993)
J. Biol. Chem.
268,
6995-7010[Abstract/Free Full Text]
-
Eich, R. F.,
Li, T.,
Lemon, D. D.,
Doherty, D. H.,
Curry, S. R.,
Aitken, J. F.,
Mathews, A. J.,
Johnson, K. A.,
Smith, R. D.,
Phillips, G. N., Jr.,
and Olson, J. S.
(1996)
Biochemistry
35,
6976-6983[CrossRef][Medline]
[Order article via Infotrieve]
-
Lippincott, J.,
Hess, E.,
and Apostol, I.
(1997)
Anal. Biochem.
252,
314-325[CrossRef][Medline]
[Order article via Infotrieve]
-
Laemmli, U. K.
(1970)
Nature
227,
680-685[Medline]
[Order article via Infotrieve]
-
Witkowska, H. E.,
Bitsch, F.,
and Shackleton, C. H. L.
(1993)
Hemoglobin
17,
227-242[Medline]
[Order article via Infotrieve]
-
Doyle, M. P.,
and Walker, B. R.
(1991)
Am. J. Physiol.
260,
R1114-R1122[Abstract/Free Full Text]
-
Gardiner, S. M.,
Compton, A. M.,
Bennett, T.,
and Hartley, C. J.
(1990)
Am. J. Physiol.
259,
H448-H456[Abstract/Free Full Text]
-
Tentori, L.,
and Salvati, A. M.
(1981)
Methods Enzymol.
76,
707-715[Medline]
[Order article via Infotrieve]
-
Geoghegan, K. F.,
Cabacungan, J. C.,
Dixon, H. B. F.,
and Feeney, R. E.
(1981)
Int. J. Pept. Protein Res.
17,
345-352[Medline]
[Order article via Infotrieve]
-
March, J.
(1977)
Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, McGraw-Hill Inc., New York
-
Apostol, I.,
Levine, J.,
Lippincott, J.,
Leach, J.,
Hess, E.,
Glascock, C. B.,
Weickert, M. J.,
and Blackmore, R.
(1997)
J. Biol. Chem.
272,
28980-28988[Abstract/Free Full Text]
-
Katsuyama, S. S.,
Cole, D. J.,
Drummond, J. C.,
and Bradley, K.
(1994)
Artif. Cells Blood Substit. Immobil. Biotechnol.
22,
1-7[Medline]
[Order article via Infotrieve]
-
Migita, R.,
Gonzales, A.,
Gonzales, M. L.,
Vandegriff, K. D.,
and Winslow, R. M.
(1997)
J. Appl. Physiol.
82,
1995-2001[Abstract/Free Full Text]
-
Predescu, S. A.,
Predescu, D. N.,
and Palade, G. E.
(1997)
Am. J. Physiol.
272,
H937-H949[Abstract/Free Full Text]
-
Tedgui, A.
(1996)
Prostaglandins Leukotrienes Essent. Fatty Acids
54,
27-29[Medline]
[Order article via Infotrieve]
-
Milici, A. J.,
Watrous, N. E.,
Stukenbrok, H.,
and Palade, G. E.
(1987)
J. Cell Biol.
105,
2603-2612[Abstract]
-
Velky, T. S., Jr.,
Lee, E. S.,
Maffuid, P. W.,
Robinson, G. T.,
Yang, J. C.,
and Greenburg, A. G.
(1987)
Arch. Surg.
122,
355-357[Abstract]
-
Schnitzer, J. E.,
and Oh, P.
(1994)
J. Biol. Chem.
269,
6072-6082[Abstract/Free Full Text]
-
Habeeb, A. F. S. A.,
and Hiramoto, R.
(1968)
Arch. Biochem. Biophys.
126,
16-26[Medline]
[Order article via Infotrieve]
-
Kroeger, K. S.,
and Kundrot, C. E.
(1997)
Structure
5,
227-237[Medline]
[Order article via Infotrieve]
-
Andersen, M. E.,
Moffat, J. K.,
and Gibson, Q. H.
(1971)
J. Biol. Chem.
246,
2796-2807[Abstract/Free Full Text]
-
Nagel, R. L.,
and Gibson, Q. H.
(1971)
J. Biol. Chem.
246,
69-73[Abstract/Free Full Text]
-
Vandegriff, K. D.,
and Winslow, R. M.
(1995)
in
Blood Substitutes: Physiological Basis of Efficacy (Winslow, R. M., Vandegriff, K. D., and Intaglietta, M., eds), pp. 143-154, Birkhauser Boston, Inc., Cambridge, MA
-
Bunn, H. F.,
and Forget, B. G.
(1986)
Hemoglobin: Molecular, Genetic, and Clinical Aspects, W. B. Saunders Co., Philadelphia
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.