Biophysical Properties and Oxygenation Potential
of High-Molecular-Weight Glutaraldehyde-Polymerized
Human Hemoglobins Maintained in the Tense
and Relaxed Quaternary States
Ning Zhang, M.S.,1Yiping Jia, Ph.D.,2Guo Chen, Ph.D.,1Pedro Cabrales, Ph.D.,3and Andre F. Palmer, Ph.D.1
Recent clinical evaluation of commercial glutaraldehyde-polymerized hemoglobins (PolyHbs) as transfusion
solutions has demonstrated several adverse side effects. Chief among these is the hypertensive effect. For-
tunately, previous studies have shown that the hypertensive effect can be attenuated by removing free hemo-
globin (Hb) and low-molecular-weight (low-MW) PolyHbs from the PolyHb mixture. In this work, polymerized
human Hb (PolyhHb) solutions were synthesized in two distinct quaternary states with high MW and subjected
to extensive diafiltration to remove free Hb and low-MW PolyhHb components (<500kDa). The resultant
PolyhHb solutions possessed high MW, distinct quaternary state, distinct reactivities with O2and CO, similar
NO deoxygenating rate constants, distinct autoxidation rate constants, high viscosity, and low colloid osmotic
pressure. To preliminarily assess the ability of PolyhHb solutions to oxygenate surrounding tissues fed by a
blood vessel, we evaluated the ability of PolyhHbs to transport O2to cultured hepatocytes in a mathematical
model of a hollow fiber bioreactor. The structure of individual hollow fibers in the bioreactor is similar to that of
a blood vessel and provides an easy way to assess the oxygenation potential of PolyhHbs without the need for
expensive and time-consuming animal studies. It was observed that PolyhHbs with low O2affinities were more
effective in oxygenating cultured hepatocytes inside the bioreactor than high O2affinity PolyhHbs. Taken
together, our results show that it is possible to synthesize high-MW PolyhHbs with no free Hb and low-MW
PolyhHb components that are capable of transporting O2to cultured cells/tissues.
vasoconstriction and hypertension still remain significant
side effects.1–4Although the exact mechanism of vasocon-
striction upon HBOC transfusion is not known, there are two
major hypotheses in the literature, namely, nitric oxide (NO)
scavenging (by far the most popular one of the two) and
oxygen oversupply.5–7To limit/prevent vasoconstriction by
either mechanism, the size (i.e., molecular weight, MW) of
the HBOC should be increased and free hemoglobin (Hb)
and low-MW components should be removed from solution
to reduce the interaction of the HBOC with the endotheli-
um.6,8,9Therefore, strategies targeted toward increasing the
size of HBOCs should be able to reduce these unwanted side
effects. Polymerization of Hb represents such an approach.
emoglobin-based oxygen carriers (HBOCs) are be-
ing developed as red blood cell substitutes. However,
Two commercial polymerized Hb (PolyHb)-based HBOCs
were developed for clinical use, Hemopure?(OPK Biotech,
Evanston, IL). Hemopure?had an average MW of 250kDa10,11
and was composed of <5% of free Hb,12whereas PolyHeme?
had an average MW of 150kDa13and was composed of <1%
of free Hb.14Unfortunately, both PolyHbs elicited substantial
hypertension in vivo upon transfusion.4,11,15
Fortunately, a path exists to improve the safety of
PolyHbs. Sakai et al. was the first to demonstrate that the
extent of vasoconstriction and hypertension was inversely
proportional to the size of the HBOC.16Unfortunately, this
study was performed on a wide variety of HBOCs synthe-
sized with different chemistries ranging from acellular Hbs
to cellular Hbs. Hence, it was not clear if this principle would
be applicable to HBOCs synthesized with the same chemis-
try. However recently, Cabrales et al. demonstrated the same
1William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, Ohio.
2Laboratory of Biochemistry and Vascular Biology, Division of Hematology, Center for Biologics Evaluation and Research (CBER), Food
and Drug Administration (FDA), Bethesda, Maryland.
3Department of Bioengineering, University of California, San Diego, La Jolla, California.
TISSUE ENGINEERING: Part A
Volume 17, Numbers 7 and 8, 2011
ª Mary Ann Liebert, Inc.
heuristic on a small library of tense-state PolyHbs (>500kDa)
of varying size synthesized with the same polymerization
chemistry, thereby verifying Sakai’s observations.9The re-
sults of Cabrales et al.’s study identified two tense-state
PolyHbs (50:1 and 40:1) with MWs of 1.36–23.71 MDa,9
whereas another study identified a zero-link cross-linked
bovine Hb (ZL-HbBv) with an average MW of 42 MDa17;
both were reported to elicit no vasoconstriction in vivo.
Therefore, polymerization of high-MW Hb seems like an
effective and simple strategy to limit/prevent vasoconstric-
tion in vivo.
This current work will expand on the study by Cabrales
et al.,9which focused on bovine Hb, and focus solely on the
synthesis and in vitro biophysical characterization of poly-
merized human Hb (PolyhHb) solutions in two distinct
quaternary states, with both high MW and no free Hb or
low-MW PolyhHbs in solution. Glutaraldehyde will be used
to nonsite specifically cross-link/polymerize hHb to yield
high-MW PolyhHb solutions that are frozen in either the
tense (T) or relaxed (R) quaternary states. The PolyhHb
solutions will be subjected to extensive diafiltration on
a 500kDa filter to remove free Hb and low-MW PolyhHbs.
The biophysical properties of the PolyhHbs will then be ex-
tensively characterized and used in a mathematical model
that describes O2transport in a hollow fiber (HF) bioreactor.
The HF bioreactor is composed of individual HFs that
mimics the structure of a blood vessel. Therefore, the math-
ematical model will be used to preliminarily assess the
ability of PolyhHbs’ to oxygenate cultured cells in the device
and by extension tissues fed by a blood vessel. The results of
these simulations will inform the proper selection of
PolyhHbs for applications ranging from tissue engineering
to transfusion medicine.
Glutaraldehyde (70%), synapinic acid, NaCl (sodium
chloride), NaOH (sodium hydroxide), Na2S2O4(sodium di-
thionite), NaCNBH3 (sodium cyanoborohydride), NaBH4
(sodium borohydride), KCl (potassium chloride), CaCl2
(NALC), Na2HPO4(sodium phosphate dibasic), and NaH2-
PO4(sodium phosphate monobasic) were purchased from
Sigma-Aldrich (St. Louis, MO). KCN (potassium cyanide),
KFe(CN)6(potassium ferricyanide), and all other chemicals
were purchased from Fisher Scientific (Pittsburgh, PA). HF
cartridges were obtained from Spectrum Labs (Rancho
hHb was purified by tangential flow filtration as described
previously in the literature.18–20
T-state PolyhHb was synthesized as previously described
in the literature.9,21–23hHb was diluted with 20mM phos-
phate buffer (PB; pH 8.0) to yield 1.5 L of 0.3mM hHb that
was stored in a 2 L glass bottle submerged in an ice bath. The
contents of the glass bottle were then subjected to vacuum
for 1min, followed by argon purging for 1min. This gas
exchange step was repeated a total of 10 times. The partially
deoxygenated hHb solution was then purged with argon for
40min. After another two short gas exchange steps and long
argon purging step, Na2S2O4was used to remove any re-
sidual O2in the hHb solution. A stock solution of Na2S2O4
was prepared by dissolving 300mg Na2S2O4in 200mL of
cold PB, which was kept on ice in the dark. The Na2S2O4
stock solution was injected into the hHb solution under
constant stirring, while the pO2 of the hHb solution was
monitored after each 30mL infusion of Na2S2O4 using a
RapidLab 248 Blood Gas Analyzer (Siemens USA, Malvern,
PA). Once the pO2was out of range (<0mm Hg), the infu-
sion of Na2S2O4was terminated. Next, deoxygenated glu-
taraldehyde (70%) was added to the deoxygenated hHb
solution to polymerize hHb at the following glutaraldehyde
to hHb tetramer molar ratios: 50:1 and 40:1. The polymeri-
zation reaction was continued for 2h in the dark at 378C, and
then quenched with 22.5mL of 2M NaBH4in 20mM PB to
yield T-state PolyhHb.
R-state PolyhHb was synthesized as previously described
in the literature.21–23About 1.5 L of 0.3mM hHb in 20mM PB
was purged with pure O2for 2h in an ice bath, and its pO2
was continuously monitored with a RapidLab 248 Blood Gas
Analyzer. After the pO2was out of range (>749mm Hg),
glutaraldehyde was added at the following glutaraldehyde
to hHb molar ratios—30:1 and 20:1. At the end of the 2h
polymerization period in the dark at 378C, 3.5mL of 8M
NaCNBH3in 20mM PB was added to the reaction mixture to
reduce the resultant Schiff bases and methemoglobin
(metHb; i.e., oxidized form of Hb). The reaction mixture was
then stirred on ice for another 30min, and then 13.5mL of
2M NaBH4was added to quench the reaction to yield R-state
Diafiltration of PolyhHb
Both T-state PolyhHb and R-state PolyhHb were first
passed through a column packed with glass wool to remove
any large particles from solution. The clarified PolyhHb
solution was then subjected to 4 cycles of diafiltration on
a 500kDa MW cutoff HF cartridge (Spectrum Labs) with
a modified lactated Ringer’s buffer (NaCl 115mM, KCl
4mM, CaCl21.4mM, NaOH 13mM, sodium lactate 27mM,
and NALC 2g/L). The concentrated PolyhHb solution was
then stored at ?808C.
Protein concentration of PolyhHb
The total protein concentration of hHb/PolyhHb was
measured by the Bradford method24using the Coomassie
Plus protein assay kit (Pierce Biotechnology, Rockford, IL).
MetHb level of PolyhHb
The metHb level of hHb/PolyhHb was measured by the
Sodium dodecyl sulfate-polyacrylamide
gel electrophoresis of PolyhHb
Twenty-five micrograms of hHb or PolyhHb was mixed
with an equal volume of denaturation sample buffer (Bio-
Rad, Hercules, CA), and boiled for 5min before being
loaded on the sodium dodecyl sulfate-polyacrylamide gel
928ZHANG ET AL.
electrophoresis (SDS-PAGE) gel. Electrophoresis was con-
ducted in a Mini Format 1-D Electrophoresis System (Bio-
Rad) using a 12% SDS-PAGE gel. After electrophoresis, the
gel was fixed and stained in Staining Buffer (Bio-Rad), and
destained before image analysis. Raw photos were processed
by Quantity One (Bio-Rad) software.
Native-PAGE of PolyhHb
Twenty micrograms of hHb or PolyhHb was mixed with a
1/4 volume of native-PAGE Sample Buffer (Invitrogen,
Carlsbad, CA), and loaded on a Novex?4%–12% Tris-Glycine
gel (Invitrogen). After electrophoresis, the gel was fixed and
stained in Staining Buffer (Bio-Rad), and destained before
image analysis. Photos were taken and processed by Quan-
tity One (Bio-Rad) software.
Absolute MW distribution of PolyhHb
The absolute MW distribution of hHb and PolyhHb so-
lutions was characterized by size exclusion chromatography
coupled with multi-angle static light scattering as described
previously in the literature.9,26
Matrix-assisted laser desorption/ionization of PolyhHb
Protein samples were diluted with deionized water to a
concentration of 0.2mg/mL, and mixed in 1:1:1 (v/v/v) ratio
with 0.1M HCl and saturated sinapinic acid in 50% (v/v)
acetonitrile solution on a matrix-assisted laser desorption/
ionization (MALDI) plate (Bruker Inc., Billerica, MA).
Results were obtained on a Bruker Microflex machine (Bruker
O2affinity and cooperativity of PolyhHb
The equilibrium O2binding properties of hHb/PolyhHb
were measured using a Hemox-Analyzer (TCS Scientific
Corporation, New Hope, PA) at 378C. Samples were pre-
pared by thoroughly mixing 50–100mL of hHb/PolyhHb
with 5mL of Hemox buffer (pH 7.4), 20mL of Additive-A,
10mL of Additive-B, and 10mL of anti-foaming agent (all
purchased from TCS Scientific Corporation). The samples
were allowed to saturate to a pO2(i.e., partial pressure of O2)
of 145?2mm Hg using compressed air. After giving the
sample enough time to equilibrate, the gas stream was
switched to pure N2to deoxygenate the sample. The absor-
bance of oxygenated and deoxygenated Hb in solution was
recorded as a function of pO2via dual wavelength spec-
troscopy. O2-hHb/PolyhHb equilibrium curves were fit to a
four-parameter (A0, A?, P50, and n) Hill model (Equation 1).
Where Y is the fractional saturation of hHb/PolyhHb (Y has
no units and ranges from 0 to 1, a value of 0 corresponds to
fully deoxygenated hHb/PolyhHb, while a value of 1 cor-
responds to fully deoxygenated hHb/PolyhHb), while A0
and A?represent the absorbance at 0mm Hg and full sat-
uration, respectively. P50represents the pO2at which the
heme binding sites in the Hb sample are 50% saturated with
O2, while n is the Hill coefficient, a measurement of co-
operativity in O2binding.
The goodness of fit was verified by comparing the ex-
with the curve
Circular dichroism spectroscopy of PolyhHb
The circular dichroism (CD) spectra of hHb/PolyhHb was
measured on an AVIV CD spectrometer (Lakewood, NJ).
hHb/PolyhHb was first diluted to 1.7mg/mL in 20mM PB
(pH 8.0), and scanned from 500 to 250nm in a 0.1cm path
length quartz cell. Samples were also diluted to 0.085mg/
mL in 20mM PB (pH 8.0), and scanned from 250 to 200nm in
a 0.1cm path length quartz cell. Data were collected and
analyzed by AVIV data collection software. Each curve was
an average of 3 scans.
Fast kinetic analysis of PolyhHb and ligand reactions
Fast kinetic measurements of gaseous ligand reactions
with hHb/PolyhHb were carried out in an Applied Photo-
physics SF-17 micro-volume stopped-flow instrument as
previously described.27O2dissociation kinetics were mea-
sured by rapidly mixing 30mM of O2-hHb/PolyhHb (heme
concentration) with 1.5mg/mL of sodium dithionite (British
Drug House, Poole, England), and the deoxygenation pro-
cess was monitored by absorbance changes at 437.5nm in
0.05M Tris buffer, pH 7.4, at 258C. The averaged kinetic
traces were fit to exponential equations using Marquardt-
Levenberg fitting routines in the Applied Photophysics
program. The kinetics of CO association with deoxygenated
hHb/PolyhHb were measured in the stopped-flow instru-
ment following absorbance changes at 437.5nm in 0.05M
Tris buffer, pH 7.4, at 258C in the presence of 1.5mg/mL of
Na2S2O4. The CO saturated stock solution (about 1mM) was
prepared by flowing prewashed CO gas through degassed
The fast kinetics of NO oxidation with O2-hHb/PolyhHb
solutions were measured in the stopped-flow instrument as
previously described.28NO gas prewashed in deoxygenated
1M NaOH and buffer solutions was bubbled through de-
oxygenated 0.05M Tris buffer, pH 7.4, to make an NO-
saturated stock solution (*2mM) in a gas-tight serum bottle.
Specific amounts of the NO stock solution were then trans-
ferred with a Hamilton syringe into a gastight syringe con-
taining deoxygenated buffer solution to make appropriate
NO concentrations for the reaction with O2-hHb/PolyhHb
solutions. O2-hHb/PolyhHb solutions (1mM heme) were
mixed with NO solutions (0–50mM), and the absorbance
changes of the reaction were followed at 420nm. Multiple
traces were taken and averaged for each reaction. The av-
eraged trace was fit to exponential equations to obtain the
reaction rate constant.
Spontaneous oxidation of PolyhHb solutions
All hHb/PolyhHb solutions were reduced to O2-hHb/
PolyhHb immediately before autoxidation experiments. Ex-
periments were carried out with 20–25mM hHb/PolyhHb
(heme concentration) in sealed cuvettes with room air-
equilibrated 50mM Chelex-treated PB at 378C. Absorbance
changes in the range of 450–700nm due to spontaneous
POLYMERIZED HUMAN HEMOGLOBIN929
oxidation of O2-hHb/PolyhHb were recorded up to 24h in a
temperature-controlled photodiode array spectrophotometer
(Hewlet Packard 8453, Palo Alto, CA). Similar oxidation as-
says were also performed in the presence of superoxide
dismutase (4.6U/mL) and catalase (414U/mL). A multi-
component analysis was performed to calculate the oxy and
ferric species based on their individual extinction coeffi-
cients.29Autoxidation rates were obtained from plots of the
loss of O2-hHb/PolyhHb versus time using nonlinear least
squares curve fitting with a double exponential equation in
Sigma-Plot (SPSS, Chicago, IL).
Viscosity and colloid osmotic pressure of PolyhHb
hHb/PolyhHb viscosity was measured in a cone and plate
viscometer DV-II plus with a cone spindle CPE-40 (Brook-
field Engineering Laboratories, Middleboro, MA), at a shear
rate of 160/second. The colloid osmotic pressure (COP) of
hHb/PolyhHb was measured using a Wescor 4420 Colloid
Osmometer (Wescor, Logan, UT).
All data were analyzed by t-test, and a p-value of 0.05 or
less was considered significant.
Simulation of PolyhHb facilitated O2transport
in a HF bioreactor
The main function of these materials is to store and
transport O2. Therefore, the purpose of the mathematical
model is to preliminarily determine the degree to which
these polymerized human Hb (PolyhHb) molecules are ca-
pable of transporting O2to surrounding cells cultured in the
HF bioreactor and by extension tissues fed by a blood vessel
without performing expensive and time-consuming in vitro
(bioreactor) or in vivo (animal) studies. In reality, the HF
bioreactor is used to facilitate cell culture; however, it can
also be used to simulate the structure of a blood vessel sur-
rounded by tissue. The main advantage of the O2transport
model is that the model parameters have been experimen-
tally measured for all PolyhHbs.
The PolyhHb selection criteria for transfusion versus bio-
reactor applications will be different and will mainly depend
on a variety of factors such as inlet pO2, flow rate of solution,
viscosity of solution, concentration of PolyhHb, size of
PolyhHb, O2affinity of PolyhHb, O2dissociation rate con-
stant of PolyhHb, and cell/tissue O2 consumption rate.
A convenient feature of this model is that all of these pa-
rameters can be varied in the simulation and its effect on ox-
ygenation ofthesurrounding cells ortissuescan be examined,
to select PolyhHbs for either of these two applications.
Therefore, the aim of these simulations is to explore the
oxygenation potential of PolyhHbs in oxygenating a tissue-
engineered construct (i.e., a bioartificial liver assist device)
and by extension tissue fed by a cylindrical blood vessel. A
mathematical model previously developed by Chen and
Palmer was used to assess the ability of PolyhHbs in oxy-
genating mammalian cells (C3A hepatocytes) cultured in a
HF bioreactor.30These results should yield some preliminary
insight into how PolyhHbs would oxygenate tissues when
transfused in vivo.
The O2transport model is based on the geometry of a
single HF in a HF bioreactor, which mimics the in vivo cap-
illary/sinusoid structure (Fig. 1). In this geometry, cell cul-
ture medium supplemented with HBOC enters the HF
bioreactor at the entrance of the lumen to provide nutrients
to cultured cells, which reside in the extra capillary space
(ECS), and leave the HF bioreactor through the exit port of
the lumen carrying away waste products. The cell culture
medium recirculates throughout the entire HF bioreactor
system. The HF membrane has a MW cut-off of 35kDa
and therefore confines HBOCs (MW>64kDa) within the
system. (A) Schematic of the entire HF
bioreactor system. (B) Closer view of the
HF bioreactor. (C) Diagram of a single
HF used in O2transport simulations. HF,
hollow fiber. Color images available on-
line at www.liebertonline.com/tea.
Geometry of HF bioreactor
930ZHANG ET AL.
lumenal space of the HF without contacting the cells residing
in the ECS.
The velocity profile in each of the three subdomains
(lumen, membrane, and ECS) can be calculated from a set
of momentum transport partial differential equations, shown
in Equation 2.
Navier-Stokes equation (lumen):
(v¢ ? r)v¢¼ ?
Brinkman’s Equation (membrane and ECS):
vector and P0is the dimensionless pressure. l0, v0, r, and m,
represent the reference length, reference velocity, fluid den-
sity, and viscosity, respectively.
Equation 3 shows the mass conservation equations, which
describe the transport of dissolved O2, total HBOC, and O2-
HBOC in dimensionless form. C0can either represent the
dimensionless O2partial pressure (pO2), total HBOC con-
centration, or O2-HBOC concentration. C0can either repre-
concentration, or O2-HBOC concentration. D can either rep-
resent the diffusivity of O2or HBOC. R represents the rate of
formation of O2/O2-HBOC. The HBOC diffusivity is esti-
mated from the PolyhHb MW using Equation 4.
lv0=l0:v¢ is the dimensionless velocity
v¢ ? rC¢
The reaction between O2and HBOC is described by Equa-
tion 5, where m is the number of O2binding sites (i.e., heme
groups) on a single HBOC molecule. The number of O2binding
sites on PolyhHb can be calculated by dividing the weight
average MW of PolyhHb by the MW of tetrameric Hb multi-
plied by a factor of 4, which represents the number of heme
groups per Hb tetramer. Given the thermodynamic relation-
ship describing the equilibrium between HBOC and O2
(Equation 6, where a1–a4are the Adair constants), the rate of
formation of O2(RO2[mm Hg/s]) or O2-HBOC (RoxyHBOC
[mol/m3/s]) in the lumen is shown in Equation 7. S is de-
fined as the HBOC saturation; that is, the molar fraction of
HBOC that is saturated with O2and Seqis the HBOC satu-
ration at equilibrium. [O2] is the concentration of dissolved
O2, [HBOC] is the concentration of total HBOC and a is the
solubility of O2in the aqueous medium. The O2consumption
rate in the ECS is described by Michaelis-Menten (M-M)
a1? pO2þ2a2? pO22þ3a3? pO23þ4a4? pO24
4(1þa1? pO2þa2? pO22þa3? pO23þa4? pO24)
m? RO2¼k?? [HBOC]
Further details about the mathematical model and pa-
rameters/constants used in the simulations can be found in
the literature.30The experimentally measured biophysical
properties of the hHb/PolyhHb solutions were used in these
simulations (i.e., MW, P50, n, and k?). The coupled set of
partial differential equations was solved by the finite element
method in Comsol Multiphysics (COMSOL, Inc., Burlington,
MA) yielding numerical solutions.
Polymerization of hHb
The major chemical reaction in the polymerization of hHb
with glutaraldehyde involves Michael addition between a,b-
unsaturated oligomeric aldehydes and primary amine
groups on lysine residues that are present on the surface of
hHb (Fig. 2A). Some Schiff bases form during the polymer-
ization step, but they are all reduced via the addition of
NaBH4or NaCNBH3at the end of the polymerization reac-
tion. Therefore, polymerization of hHb is based on stable
C-N bonds that will not hydrolyze in solution.
Figure 2B shows the SDS-PAGE of hHb and all PolyhHb
solutions under denaturing conditions. After sample dena-
turation, hHb yields two bands in the gel. The lower band
corresponds to individual a/b subunits, whereas the upper
band corresponds to a-a/b-a/a-b dimers that do not disso-
ciate into individual subunits. In contrast, all PolyhHb so-
lutions under denaturing conditions display strong bands
>250kDa and weak bands close to 15, 30, 45, and 60kDa in
MW corresponding to individual a/b subunits, a-a/b-a/a-b
dimers, a/b trimers, and a/b tetramers. In fact, PolyhHb’s
with higher cross-link density locked in the same quaternary
tetramers compared to lower cross-link density PolyhHb.
Figure 2C shows the absolute MW distribution of hHb and
PolyhHb solutions under nondenaturing conditions. Tetra-
meric hHb displays a weight averaged MW of *62kDa,
whereas all PolyhHbs display weight averaged MWs
>62kDa. The weight averaged MW of hHb and all PolyhHbs
is displayed in Table 1. Light scattering results clearly show
that both T- and R-state PolyhHbs possess no individual a/b
subunits, dimers, trimers, and tetrameric hHb in solution.
Figure 2D shows the native PAGE analysis of hHb and
PolyhHb solutions under nondenaturing conditions. The
position of the hHb tetramer in the gel is labeled by an
arrow. None of the PolyhHb solutions display bands at
or lower than the position of the hHb tetramer. This dem-
onstrates that PolyhHb is free of Hb tetramers, trimers, di-
mers, or individual a/b subunits in solution monomers.
Figure 2E shows the MALDI mass spectral analysis of hHb
and PolyhHb solutions. It is evident that hHb is composed of
2 peaks corresponding to individual a and b subunits. There
is no peak corresponding to dimers as indicated in the SDS-
PAGE analysis of hHb (Fig. 2B). Also, there are no peaks
corresponding to a/b subunits, dimers, trimers, or tetramers
for all PolyhHb solutions, in stark contrast to the SDS-PAGE
results (Fig. 2B).
POLYMERIZED HUMAN HEMOGLOBIN931
(A) Reaction scheme showing the chemical reaction between primary amine groups on hHb and reactive moieties on
glutaraldehyde. (B) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of hHb and PolyhHb solutions under de-
naturing conditions. (C) Absolute MW distribution of hHb and PolyhHb solutions under nondenaturing conditions. (D)
Native gel of hHb and PolyhHb solutions under nondenaturing conditions. (E) Matrix-assisted laser desorption/ionization
mass spectra of hHb and PolyhHb. hHb, human hemoglobin; PolyhHb, polymerized human hemoglobin; MW, molecular
weight. Color images available online at www.liebertonline.com/tea.
Schematic of reaction between hHb and glutaraldehyde, and methods used to assess the extent of polymerization.
Table 1. Biophysical Properties of Native Human Hemoglobin and Polymerized Human Hemoglobin Solutions
SolutionWeight averaged molecular weight P50(mm Hg)Hill coefficient Methemoglobin level (%)
40:1 T-state PolyhHb
50:1 T-state PolyhHb
20:1 R-state PolyhHb
30:1 R-state PolyhHb
hHb, human hemoglobin; PolyhHb, polymerized human hemoglobin.
932ZHANG ET AL.
To estimate the stability of PolyhHb during long-term
storage, the MW distribution of 40:1 T-state PolyhHb was
analyzed immediately after synthesis, after 8 months of
storage at ?808C, and after 8 months of storage at ?808C
coupled with incubation at 378C for 5 days (Supplementary
Figure S1; Supplementary Data are available online at
www.liebertonline.com/ten). Eight months of storage at
?808C had no influence on the MW distribution of the 40:1
T-state PolyhHb, whereas incubation of the 40:1 T-state
PolyhHb at 378C for 5 days significantly increased the MW of
the PolyhHb. Analysis of the MW of the 40:1 T-state
PolyhHb under all conditions yielded no free Hb tetramers,
trimers, dimers, or a/b subunits in solution. For this study
the PolyhHb was in the oxygenated form and not the de-
oxygenated form. Therefore, it is more subjective to oxidative
reactions for prolonged exposures to high temperatures un-
like the deoxygenated version of the same compound.
O2binding affinity and cooperativity of PolyhHb
Figure 3A shows the O2-hHb/PolyhHb equilibrium
curves. T-state PolyhHb solutions are right-shifted compared
to the control of hHb, whereas R-state PolyhHb solutions are
left-shifted compared to the control of hHb. Figure 3B shows
the regressed O2affinity (P50) of T- and R-state PolyhHbs.
Compared to the control of hHb (13.27?0.55mm Hg),
T-state PolyhHbs display much higher P50s. In contrast, R-
state PolyhHbs has extremely low P50s compared to both
hHb and T-state PolyhHbs. Both T- and R-state PolyhHbs
display cooperativity coefficients <1, whereas unmodified
hHb has a cooperativity coefficient of 2.59?0.12 (Fig. 3C).
The O2affinity and cooperativity coefficient of hHb and all
PolyhHbs are listed in Table 1.
MetHb level of PolyhHb
Figure 4 displays the metHb levels of hHb, T-, and R-state
PolyhHb solutions. Both T- and R-state PolyhHbs display
metHb levels <10%, but higher than the metHb level of hHb
(0.88%?0.33%). Table 1 summarizes the biophysical prop-
erties of hHb and PolyhHb solutions.
CD spectroscopy of PolyhHb
hHb structure is determined by CD spectroscopy in the
operativity of PolyhHb solutions. (A) O2
equilibrium curves of native hHb and
PolyhHb solutions, where Y is the frac-
tion of heme sites saturated with O2. (B)
Regressed O2affinity (P50) of native hHb
and PolyhHb solutions. (C) Regressed
cooperativity coefficient (n) of native
hHb and PolyhHb solutions. Error bars
represent the standard deviation from
triplicate reactions. Color images avail-
able online at www.liebertonline
O2binding affinity and co-
POLYMERIZED HUMAN HEMOGLOBIN933
far-ultraviolet and near-ultraviolet regions. The influence of
hHb polymerization on the secondary structure of hHb is
determined by far-ultraviolet CD spectroscopy in the wave-
length range 200 to 250nm (Fig. 5A). In the far-ultraviolet
region, all hHb/PolyhHb spectra share a common peak at
*220nm with similar spectral intensity. In contrast, the in-
fluence of hHb polymerization on the heme environment is
determined by near-ultraviolet CD spectroscopy in the
wavelength range 250 to 500nm (Fig. 5B). In the near-
ultraviolet region, all hHb/PolyhHb spectra share common
peaks at *260 and 420nm, with slight differences in inten-
sity among all hHb and PolyhHb solutions.
Stopped-flow kinetics of PolyhHb and ligand reactions
Figure 6 shows a representative time course of CO binding
to deoxygenated Hb and the CO concentration dependence
of the apparent reaction rate constants for deoxygenated
native hHb and PolyhHb solutions. Unlike the slightly lower
CO binding rate constants of T-state PolyhHb solutions
compared to that of hHb, R-state PolyhHb solutions exhibits
much higher CO reactivities characterized by the large
increase (>20-fold) in bimolecular rate constants. Table 2
summarizes the CO binding (k’on,CO), O2dissociation (koff),
and NO binding/oxidation (k’ox,NO) rate constants as the
mean of a minimum of three separate measurements. The O2
dissociation rate constants of T- and R-state PolyhHb solu-
tions differ from that of native hHb by either a *30% in-
crease or reduction, respectively, whereas the NO reactivities
are very similar among all hHb/PolyhHb solutions in our
solutions. Error bars represent the standard deviation from
Methemoglobin levels of native hHb and PolyhHb
in the far-ultraviolet region ranging from 200 to 250nm. (B)
CD in the near-ultraviolet region ranging from 250 to 500nm.
Each curve represents the average of three measurements.
CD spectra of hHb and PolyhHb solutions. (A) CD
oxygenated hHb. The inset shows the CO concentration de-
pendence of the apparent reaction rate constants for native
hHb and PolyhHb solutions.
Representative time course of CO binding to de-
934 ZHANG ET AL.
Autoxidation kinetics of PolyhHb
The autoxidation rate constants of oxygenated hHb and
PolyhHb solutions are summarized in Table 3. The high O2
affinity PolyhHb solutions exhibit similar spontaneous oxi-
dation rate constants compared to that of unmodified hHb.
Conversely, the low O2 affinity PolyhHb solutions show
much elevated spontaneous oxidation rates under the same
experimental conditions. Additionally, PolyhHb solutions
show much less of a response compared to hHb in the
presence of the antioxidant enzymes superoxide dismutase
(SOD) and catalase.
Viscosity and COP of PolyhHb
Table 4 shows the viscosity and COP of hHb as well as
T- and R-state PolyhHb solutions. The viscosity of both
PolyhHbs in a distinct quaternary state increases with in-
creasing cross-link density, whereas the COP is virtually
unaffected by cross-link density. However, T-state PolyhHbs
exhibit a much lower COP versus R-state PolyhHbs.
Figure 7A shows the normalized O2consumption rate of
C3A hepatocytes as a function of the inlet pO2(pO2,in). The
value of the normalized O2flux starts at 15–20 at low pO2,ins
(*5mm Hg) and then decreases as the pO2,inincreases for all
HBOCs. At higher pO2,ins (*150mm Hg), O2transport is still
at least 2–3 times greater versus the case with no HBOC
The pO2profiles within the HF bioreactor (including lu-
men, membrane, and ECS) are shown in Figure 7B for all
HBOCs at varying concentrations. Each unit represents the
cross-sectional view of a single HF (Fig. 1C). The top hori-
zontal boundary represents the HF centerline, whereas the
left boundary represents the inlet of the lumen and the right
boundary represents the lumen exit. The maximum heme
concentration ([Heme]) in the HF bioreactor is normalized by
the average heme concentration in human blood (8800mM).
Without any HBOC supplementation in the HF bioreactor,
most of the ECS is hypoxic, with pO2levels below 20mm Hg.
As the HBOC concentration increases, O2transport in the HF
improves and the hypoxic region in the ECS gradually reduces
in size. Among all the HBOCs simulated, R-state PolyhHbs
show the least capacity to oxygenate the ECS versus hHb and
T-state PolyhHbs. However, T-state PolyhHbs are better at
oxygenating the ECS versus native hHb and R-state PolyhHb.
Similar oxygenation results are observed for the ECS
zonation breakdown plots (Fig. 7C). As mentioned previ-
ously, in the absence of HBOCs, the hypoxic region (<25mm
Hg) dominates the majority (*95%) of the ECS volume, and
only a small percentage of hepatocytes (*5%) are subjected
to in vivo pO2levels (25–70mm Hg). The hypoxic region gets
smaller in size when HBOCs are present in the cell culture
media. However, under the simulation conditions, only
T-state PolyhHbs successfully improve O2transport and re-
capitulated the in vivo pO2environment in the ECS with
virtually no part of the ECS experiencing hypoxic oxygena-
tion (<5%). In contrast, a significant volume of the ECS is
hypoxic with supplementation of hHb (*55%) and R-state
Light scattering, native-PAGE, and MALDI analysis of
both T- and R-state PolyhHb solutions show that they exhibit
large weight averaged MWs, without the presence of hHb
tetramers, trimers, ab dimers, and individual a/b subunits in
solution in approximately physiological conditions. On the
other hand, SDS-PAGE shows the presence of a small per-
centage of individual a/b subunits and ab/a-a/b-b dimers,
trimers, and tetramers in the PolyhHb solutions under de-
naturing conditions. This apparent contradiction is caused
by the different environmental conditions to which the
sample is exposed to during these measurements.
In SDS-PAGE, hHb/PolyhHb solutions are subjected to
harsh denaturing conditions, which should dissociate a/b
monomers and dimers that are not cross-linked to the
PolyhHb superstructure. However, these harsh sample pro-
cessing conditions actually chemically cross-link a/b sub-
units into higher order structures, that is, dimers, trimers,
and tetramers. Evidence of this phenomenon is found in the
SDS-PAGE of hHb, where only one band corresponding to
a/b subunits should be present in the gel. Instead, the SDS-
PAGE of hHb has an additional band corresponding to
dimers. There are no chemical cross-links between the indi-
vidual subunits of unmodified hHb. Therefore, SDS-PAGE of
Table 2. Kinetic Parameters of Gaseous Ligand
Reactions with Human Hemoglobin
and Polymerized Human Hemoglobin Solutions
40:1 T-state PolyhHb
50:1 T-state PolyhHb
20:1 R-state PolyhHb
30:1 R-state PolyhHb
Table 3. Autoxidation Rate Constants of Human Hemoglobin and Polymerized Human
Hemoglobin Solutions in the Presence and Absence of Superoxide Dismutase and Catalase
40:1 T-state PolyhHb
50:1 T-state PolyhHb
20:1 R-state PolyhHb
30:1 R-state PolyhHb
SOD, superoxide dismutase.
POLYMERIZED HUMAN HEMOGLOBIN935
hHb should only yield 1 band corresponding to a/b sub-
units. The fact that there are 2 bands indicates that the
sample preparation condition for SDS-PAGE induces che-
mical cross-links between subunits. This calls into question
the actual presence of cross-linked dimers, trimers, and tet-
ramers in PolyhHb solutions as determined by SDS-PAGE.
These cross-linked species are not present in solution when
PolyhHbs are analyzed by light scattering, native-PAGE, and
MALDI mass spectral analysis.
Therefore, it is safe to conclude that the final PolyhHb
products are free of a/b subunits, dimers, trimers, and tet-
ramers in solution. However, the PolyhHb products are
composed of some uncross-linked a/b subunits that are not
chemically integrated into the PolyhHb superstructure. At a
fixed quaternary state, SDS-PAGE, native-PAGE, light scat-
tering, and MALDI all show an increase in the MW of
PolyhHb with increased cross-link density. This result is
expected since the number of potential chemical cross-links
increases with increasing cross-link density.
Compared to other HBOCs reported in the literature,
T- and R-state PolyhHb solutions possess smaller MWs
(1.10–18.44 MDa) than 50:1 T- and 40:1 R-state PolybHb
(16.59–26.33 MDa)21,22and ZL-HbBv (42 MDa),17but larger
MWs than O-raffinose cross-linked hHb (O-R-polyHbA0, 64–
600kDa)31and Oxyglobin?(87.2–502.3kDa).32The two
commercially manufactured PolyHb products, Hemopure?
and PolyHeme?, possess average MWs of 250 and 150kDa,
respectively.11,13Hence, PolyhHb products are up to 70 times
larger than Hemopure?, and 120 times larger than Poly-
Heme?. In addition, Hemopure?and PolyHeme?both have
<5% unreacted hHb tetramers and ab dimers in solution.
Free hHb tetramers, ab dimers, and low-MW PolyHbs are
believed to elicit vasoconstriction and systemic hypertension
in vivo. All PolyhHbs are free of hHb, ab dimers, and low-
MW PolyHbs and should generate limited/no vasocon-
striction compared to current commercial PolyHbs.
T- and R-state PolyhHbs exhibit vastly different O2affin-
ities. T-state PolyhHbs possess P50s>35mm Hg, which are
(13.27?0.55mm Hg), hHb inside human red blood cells (26–
28mm Hg), PolyHeme?
(6.4mm Hg),17but comparable to Hemopure?(38mm Hg),12
O-R-polyHbA0 (50.9mm Hg),33and Oxyglobin?(35.1mm
Hg).32Since hHb is totally deoxygenated before, during and
after polymerization (but not during storage), the resultant
T-state PolyhHbs are conformationally frozen in the T-state
after polymerization. Hence, T-state PolyhHbs exhibit higher
P50s compared to hHb. In contrast, R-state PolyhHbs exhibit
P50s<2mm Hg, indicating their extremely high O2affinity.
This is because hHb is fully saturated with pure O2before,
during and after polymerization. Therefore, the resultant R-
state PolyhHbs are conformationally frozen in the R-state
after polymerization. Hence, R-state PolyhHbs exhibit lower
P50s compared to hHb. This pattern is consistent with
PolybHb (the P50is 41mm Hg for 50:1 T-state PolybHb and
0.66mm Hg for 40:1 R-state PolybHb).21,22
Both PolyhHbs show no cooperativity. Polymerization of
hHb freezes the structure of hHb in a well-defined quater-
nary state. Since, the individual subunits are cross-linked to
each other, quaternary structure changes, which would occur
during normal O2binding/offloading are hindered by the
presence of chemical cross-links. This results in the loss of
cooperative O2binding to the hHb tetramer for all PolyhHbs.
The two commercially produced PolyHbs, Hemopure?(n¼
1.4) and PolyHeme?(n¼1.7),12and other reported HBOCs,
PolybHb (n<1),21ZL-HbBv (n¼1.2),17O-R-polyHbA0(n¼
1),33and Oxyglobin?(n¼1.4),32also show reduced co-
operativity compared to native hHb (n¼2.59?0.12) or bHb
(n¼2.5).21These results indicate that the extent of poly-
merization of PolyhHb is significantly greater than that of
The metHb level of T- and R-state PolyhHbs is higher than
that of hHb. However, R-state PolyhHbs have a higher metHb
level than T-state PolyhHbs. This observation is consistent
with the fact that R-state PolyhHbs are maintained in an ox-
ygenated environment, which enhances autoxidation of the
heme prosthetic group. On the other hand, current HBOCs in
phase III trials possess similar metHb levels. For example,
Hemopure?has a metHb level <10%, and PolyHeme?has a
metHb level <8%.12Therefore, T- and R-state PolyhHbs ex-
hibit metHb levels that are acceptable for clinical evaluation.
Nonetheless, the metHb level of PolyhHb solutions could be
further reduced after polymerization by incubating these so-
lutions with additional NaCNBH3, a mild reducing agent.
The CD spectra of hHb and PolyhHb solutions show no
significant differences either in the far-ultraviolet region or in
near-ultraviolet region. This indicates that the influence of
polymerization on the secondary structure and heme envi-
ronment of hHb is negligible.
Fast kinetic analysis of gaseous ligand binding with
PolyhHb solutions shows that the O2dissociation rate con-
stant of PolyhHbs (49.7–51.7s?1for T-state PolyhHbs and
for R-state PolyhHbs) are comparable to
PolybHb (53s?1for 50:1 T-state PolybHb, and 22s?1for 40:1
R-state PolybHb)21and ZL-HbBv (27.4s?1),17but smaller
than the rate constants for O-R-polyHbA0 (130s?1)33and
Oxyglobin?(61.8s?1).32The CO association rate constants of
T-state PolybHb (0.18mM?1s?1),21ZL-HbBv (0.28mM?1s?1),17
and Oxyglobin?(0.19mM?1s?1).32In contrast, the CO associ-
ation rate constant of R-state PolyhHbs (4.76–4.88s?1) are
comparable to 40:1 R-state PolybHb (4.84s?1)21and O-R-
polyHbA0(1.2s?1).33Both T-state PolyhHbs possess O2dis-
sociation rate constants that are larger than that of hHb
(40.4s?1) and CO binding rates smaller than that of hHb
(0.214s?1). On the contrary, both R-state PolyhHbs possess O2
dissociation rate constants that are smaller than that of hHb,
and CO association rate constants that are much larger than
hHb. These results are consistent with the O2affinities of
PolyhHbs, which are in turn reflected by their P50 values
Table 4. Viscosity and Colloid Osmotic Pressure
of Human Hemoglobin and Polymerized Human
40:1 T-state PolyhHb
50:1 T-state PolyhHb
20:1 R-state PolyhHb
30:1 R-state PolyhHb
COP, colloid osmotic pressure.
936ZHANG ET AL.
of oxygenated PolyhHb solutions occurs very rapidly on the
order of 107M?1s?1, and remains largely unchanged after
glutaraldehyde polymerization. However, the kinetic param-
eters for O2dissociation and CO binding of PolyhHb solutions
indicate the reduced or increased O2affinities after T-state
or R-state PolyhHb modification, respectively. More specifi-
cilitated O2transport in a hepatic HF
bioreactor. (A) Normalized O2con-
sumption rate of HBOC supple-
mented HF bioreactors, normalized
by a control HF bioreactor (no
HBOC supplementation). (B) pO2
profiles within a single HF (lumen,
membrane, and ECS) for all HBOCs
at varying heme concentrations.
Each rectangle represents the region
depicted in Figure 1C. (C) ECS Zo-
nation within the HF bioreactor for
all HBOCs at [Heme]¼100%.
HBOCs, hemoglobin-based oxygen
carriers; ECS, extra capillary space.
Color images available online at
Simulation of PolyhHb fa-
POLYMERIZED HUMAN HEMOGLOBIN937
is mostly reflected in their increased CO binding rate constants,
whereas the increase in the P50values of T-state PolyhHb solu-
tions are in agreement with their O2dissociation rate constants.
These physicochemically characteristic changes are determined
oxygenated or deoxygenated conditions.
Hb autoxidation is an intrinsic characteristic of the heme
group, and is usually affected by chemical modification of
the Hb molecule. The autoxidation rate constant of low O2
affinity T-state PolyhHbs (0.00145–0.00134min?1) is higher
than that of R-state PolyhHbs (0.00050–0.00069min?1). The
elevated autoxidation rates, especially for low O2affinity
PolyhHb solutions, are consistent with that of previously
reported cross-linked and PolyHbs. For example, the autox-
idation rate constant of ZL-HbBv is three times greater than
that of bHb, whereas the rate of Oxyglobin?autoxidation is
1.3 times greater than that of bHb. Low O2 affinity 50:1
T-state PolybHb has an increased rate of autoxidation com-
pared to high O2affinity 40:1 R-state PolybHb and native
bHb.21This undesirable change is one of the biggest chal-
lenges facing HBOC development. The increased Hb oxida-
tion not only lowers its capacity to carry O2, but also
introduces enhanced toxicity that could elicit cell and tissue
damage. The weakened protection by SOD and catalase
against autoxidation of PolyhHb solutions underlines the
complexity of the issues faced by this chemical modification.
The viscosity of both types of PolyhHb solutions increases
with increasing cross-linking density and is higher than that
of whole blood (*3cp), Hemopure?(1.3cp at 13g/dL), and
PolyHeme?(2.1cp at 10g/dL),12but similar to that of
PolybHb (11.4cp for 50:1 T-state PolybHb and 7.8cp for 40:1
R-state PolybHb at a concentration of 10g/dL).21High-
viscosity HBOCs are preferred for transfusion, since they can
elicit the generation of endothelial-derived relaxing factors,
through flow-mediated endothelial mechanotransduction.34
This can potentially neutralize the vasoconstrictive effect
caused by NO scavenging or O2oversupply upon PolyHb
infusion, if it still exists. The COP of both PolyhHb solutions
is less than the COP of whole blood (27mm Hg).12However,
R-state PolyhHbs exhibit COPs comparable to Hemopure?
(25mm Hg) and PolyHeme?(23mm Hg).12T-state PolyhHbs
exhibit COPs comparable to PolybHb (1mm Hg for 50:1 T-
state PolybHb and 7mm Hg for 40:1 R-state PolybHb).21Low
COP fluids can facilitate the outward flow of intravascular
fluid across blood vessels into the tissue space, thereby re-
ducing the blood volume. However, this problem could be
remediated by supplementing human serum albumin to the
low COP PolyhHb solution, thereby increasing the PolyhHb
Among all the HBOCs we studied via simulation, the O2
equilibrium curves of R-state PolyhHbs are left-shifted cor-
responding to low P50values, whereas T-state PolyhHbs are
right-shifted with correspondingly high P50 values, com-
pared to native hHb. It is apparent from Figure 7A that all
PolyhHb solutions have slightly less capacity for improving
O2transport at lower inlet pO2s (<40mm Hg) compared to
native hHb. This is probably due to the low cooperativities
and large diffusion coefficients of these molecules that are
conferred upon polymerization. However, there is not much
difference between all PolyhHb solutions and native hHb in
the normalized O2consumption rate at higher inlet pO2s,
especially for T-state PolyhHbs, which possess higher P50s.
This becomes more evident in the pO2profile and ECS zo-
nation plots (Fig. 7B and C). Generally, T-state PolyhHbs
(high P50) enhance O2transport to the ECS in the HF biore-
actor. Under the simulated conditions (inlet pO2¼80mm
Hg), T-state PolyhHbs can release a considerable amount of
O2to the HF ECS especially as the local pO2drops below the
P50of the HBOC. However, for R-state PolyhHbs, which
have very high O2affinities, the pO2needs to be below 1mm
Hg to make these HBOCs offload their store of O2. At a pO2
below 1mm Hg, hepatocytes will suffer from extensive
hypoxia. In this case, R-state PolyhHbs perform like myo-
globin which primarily stores O2and only releases it under
extreme hypoxia in the tissue. Therefore, R-state PolyhHbs
are not suitable for tissue engineering applications where it is
important to recapitulate in vivo O2levels and gradients.
We have demonstrated that high-MW PolyhHbs can be
synthesized in a defined quaternary state without the pres-
ence of free hHb and low-MW PolyhHb species in solution.
Our results show that the PolyhHb MW and O2affinity can
be easily regulated by controlling the glutaraldehyde cross-
link density and hHb quaternary state. O2transport simu-
lations indicate that T-state PolyhHbs oxygenated the ECS of
a HF bioreactor to a greater extent versus R-state PolyhHbs.
Taken together, these results support the use of PolyhHbs as
efficacious O2carrying solutions with possible applications
in transfusion medicine and tissue engineering.
This work was supported by National Institutes of Health
Grants R01HL078840 and R01DK070862 to A.F.P. The au-
thors would like to thank David R. Harris for purifying the
hHb for these studies.
The findings and conclusions in this article have not been
formally disseminated by the Food and Drug Administration
and should not be construed to represent any agency de-
termination or policy.
No competing financial interests exist.
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Address correspondence to:
Andre F. Palmer, Ph.D.
William G. Lowrie
Department of Chemical and Biomolecular Engineering
The Ohio State University
Columbus, OH 43210
Received: June 14, 2010
Accepted: October 27, 2010
Online Publication Date: January 5, 2011
940ZHANG ET AL.