2 Materials and methods
2.1 Materials.
Sodium citrate anticoagulated whole blood was collected aseptically from
adult cattle (Quad Five, Ryegate, MT). Glutaraldehyde (70%), sodium
lactate, sodium dithionite
(Na2S2O4), calcium
chloride (CaCl2·2H2O), potassium
chloride (KCl), sodium cyanoborohydride (NaCNBH3),
sodium hydroxide (NaOH), sodium chloride (NaCl), sodium phosphate
monobasic (NaH2PO4), and sodium
phosphate dibasic (Na2HPO4) were
purchased from Sigma-Aldrich (St. Louis, MO). HF modules of 50 kDa, 100
kDa, 500 kDa, and 0.2 μm (MW cut offs) were obtained from Spectrum
Laboratories (Rancho Dominguez, CA). Acetonitrile
(C2H3N), α-cyano-4-hydroxycinnamic acid
(CHCA), trifluoroacetic acid (TFA), potassium cyanide (KCN), potassium
ferricyanide (K3Fe(CN)6) were procured
from Fisher Scientific (Pittsburgh, PA). Coomassie brilliant blue was
purchased from Bio-Rad Labs (Irvine, CA). 10-20%
NovexTM Tris-glycine gel, electrophoresis supplies and
0.2 µm syringe filters were purchased from Thermo Fisher Scientific
(Waltham, MA).
2.2 PolybHb Synthesis.
Bovine red blood cells (RBCs) were separated and washed by
centrifugation with 0.9% saline and lysed with phosphate buffer (PB)
(3.75 mM, pH 7.4). Tangential flow filtration (TFF) cartridges with
MWCOs of 500 kDa and 50 kDa were then used to purify and concentrate bHb
as described in the literature (Cabrales et al., 2010; A. F. Palmer et
al., 2009).
Initially, 30 g of bHb was diluted in 1.5 L phosphate buffer saline
(PBS, 0.1 M, pH 7.4) and placed into an airtight, amber-tinted reactor
vessel with continuous stirring. To produce T-state PolybHb, the bHb
solution was completely deoxygenated via continuous recirculation
through the liquid side of a 3M MiniModule gas/liquid exchange module
(Maplewood, MN) while the gas side was fed with pure nitrogen gas
(N2). The partial pressure of O2 in
solution (pO2) was measured using a RapidLab 248 Blood
Gas Analyzer (Siemens USA, Malvern, PA). When the pO2was lower than 20.0 mm Hg, 300 mg of sodium dithionite dissolved in
N2 purged PBS (0.1 M, pH 7.4) was injected into the bHb
solution via a needleless valve and allowed to mix for 15-20 minutes. To
ensure complete deoxygenation of the bHb solution, four additional 1 mL
injections of 50.0 mg sodium dithionite in N2 purged PBS
at pH 7.4 were delivered after the initial bolus injection. The
polymerization of bHb in the T-state was not initiated until the
pO2 attained a value of 0.0 mm Hg.
Deoxygenated bHb was maintained throughout the T-state polymerization
process by continuous purging of the reactor headspace with
N2. The same reactor vessel configuration was used to
synthesize R-state PolybHb via complete oxygenation of bHb. Instead of
using pure nitrogen on the gas side of the gas/liquid exchange membrane,
pure O2 was used. Polymerization was not initiated until
the pO2 was above 745 mm Hg. Oxygenation was maintained
throughout the R-state polymerization process by continuous purging of
the reactor headspace with O2.
T- and R-state PolybHb were both polymerized at glutaraldehyde:bHb molar
ratios of 25:1, 30:1, and 35:1. Glutaraldehyde was added dropwise to the
constantly stirring bHb solution at a rate of 2 mL/min to achieve a
total volume of 50.0 mL and then allowed to react for 2 hours at 37 °C.
To quench the polymerization reaction, a bolus addition of
NaCNBH3 was injected into the reactor vessel to reduce
the linkage of amine ligand and aldehyde (Schiff bases) and mixed for 30
minutes as the reactor temperature was cooled to ambient temperature (20
°C). NaCNBH3 was prepared at a 7:1 molar ratio to
glutaraldehyde in PBS (pH 7.4). The reactor vessel was then placed into
a refrigerator and maintained at 4°C overnight. A schematic of the
reactor setup, and timeline for reagent addition is shown inFigure 1 .
2.3 PolybHb Clarification and
Purification.
Both T- and R-state PolybHb were sterile filtered via TFF on a 0.2 μm HF
module as described previously (Cabrales et al., 2010; Elmer, Harris, &
Palmer, 2011). A schematic of this setup is shown in Figure 2.The PolybHb was then buffer exchanged into a modified lactated Ringer’s
solution (115 mM NaCl, 4 mM KCl, 1.4 mM
CaCl2.2H2O, 13 mM NaOH, 12.3 mM
N-acetyl-L-cysteine, 27 mM sodium lactate, pH 7.4) via diafiltration.
35:1 and 30:1 T-state PolybHb and 30:1 and 25:1 R-state PolybHb were
diafiltered and concentrated on a 500 kDa HF module. 35:1 R-state
PolybHb was diafiltered and concentrated on a 0.2 μm HF module. 25:1
T-state PolybHb was diafiltered and concentrated on a 100 kDa HF module
to retain most of the product.
2.4 Characterization of
PolybHb
2.4.1 Size
Quantification
2.4.1.1 Sodium Dodecyl Sulfate
Polyacrylamide Gel Electrophoresis
(SDS-PAGE).
bHb and PolybHb samples were diluted to ~1 mg/mL in
deionized (DI) water and mixed with tris-glycine buffer in a 1:1 v/v
ratio. 100 µL of 1M diothiothreitol (DTT) was added to run the samples
under reducing conditions. 20 µL of sample was loaded into a 10-20%
Novex™ tris-glycine gel and incubated for 40 mins. Gels were stained
with 1 × Coomassie brilliant blue and destained (3:1:6 v/v/v
methanol:acetic acid:DI mixture) overnight. Images were obtained using
an image scanner.
2.4.1.2 Dynamic Light Scattering
(DLS).
The hydrodynamic diameter of bHb
and PolybHb was measured using a BI-200SM Goniometer (Brookhaven
Instruments, Holtsville, NY) as described in literature (Zhou, 2011).
All bHb, T-state, and R-state PolybHb samples were diluted to
~ 1 mg/mL with deionized water.
2.4.1.3 Size Exclusion HPLC
(HPLC-SEC).
The molecular weight (MW) distribution of bHb and PolybHb was estimated
via a Thermo Scientific Dionex UltiMate 3000 UHPLC/HPLC system coupled
with an Acclaim SEC-1000 column. The samples were filtered through a 0.2
µm syringe filter before injection into the column. Each sample was run
at the flow rate of 0.35 mL/min in the mobile phase (PB, 50 mM, pH 7.4).
To represent the size of PolybHb molecules, the polymer order
(n0) is defined
as:
M = \(2^{n_{o}}\) (1)
where n0 is an integer ranging from 0-5, and M is the
total number of individual bHb molecules polymerized to yield one
PolybHb molecule. The MW of each polymer order species was estimated by
analyzing HPLC-SEC spectra with a deconvolution algorithm (Belcher,
Cuddington, Martindale, Pires, & Palmer, 2020; Cuddington, Moses,
Belcher, Ramesh, & Palmer, 2020). The quantification of the fraction of
each polymer order was performed based on the absorbance at 413 nm.
2.4.1.4 Transmission
Electron Microscopy (TEM)
Analysis.
The structural morphology of PolybHb was analyzed via transmission
electron microscopy (TEM) using a FEI Tecnai G2 Spirit TEM (FEI,
Hillsboro, OR). 1 mL of PolybHb sample at ~100 mg/ml was
first diluted to 0.2 mg/ml with DI water and vortexed to disaggregate
high MW PolybHb polymers. Then the PolybHb solution was filtered through
a 0.2 μm syringe filter to remove aggregates. 10 μL of the filtered
solution was placed on a CF300-CU TEM grid (EMS, Hatfield, PA), dried
overnight and imaged the next day. During imaging, the PolybHb grid was
initially placed on a specimen holder and then inserted into the airlock
cylinder. The condenser apertures was centered at a magnification of
~10000×, followed by direct alignment. The image of
PolybHb was recorded at a voltage of 80 kV and 39000× magnification.
2.4.2
Rheology.
The viscosity of bHb and PolybHb were measured in a DV3T-CP viscometer
with a cone spindle CPA-40Z (Brookfield AMETEK, Middleboro, MA) (Belcher
et al., 2018). 0.5 mL PolybHb solution (~50 mg/mL) was
loaded onto the cone spindle. All PolybHb samples were then measured at
a shear rate of 150 s-1.
2.4.3 Autoxidation
Experiments.
The initial metHb level (%) of all samples was lower than 5%.
Autoxidation kinetics at 37 °C was measured by incubating PolybHb
samples in PB (50 mM, pH 7.4) over a 24 h period. To determine the
autoxidation rate of bHb and PolybHbs (0.775 mM heme basis, the
concentration of HBOCs in the systemic circuit after intravenous
transfusion (Jahr, MacKenzie, Pearce, Pitman, & Greenburg, 2008)), the
absorbance was monitored from 350−700 nm in parafilm-sealed cuvettes
within a temperature-controlled HP 8452A diode array UV-visible
spectrometer (Olis, Bogart, GA). Extinction coefficients of oxyHb and
metHb were used to determine the molar concentration of the
corresponding species. First order autoxidation rate constants were
estimated by performing a biphasic linear regression on the natural log
of the normalized concentration of PolybHb/bHb.
2.4.4 Ligand-Binding
Kinetics.
Fast kinetic measurements of
gaseous ligand reactions with bHb, T-state, and R-state PolybHbs were
carried out via an Applied Photophysics SF-17 micro-volume stopped-flow
instrument (Applied Photophysics Ltd., Surrey, United Kingdom) (Belcher
et al., 2018; Jahr et al., 2008; Rameez & Palmer, 2011).
O2 dissociation kinetics were determined by rapidly
mixing 12.5 µM of bHb or PolybHbs (heme-based concentration) with sodium
dithionite (1.5 mg/mL). The absorbance changes at 437.5 nm were recorded
in 0.1 M PBS (pH 7.4, 25 °C). The O2 dissociation
kinetics traces were avergared and fit to monoexponential equations in
the Applied Photophysics program.
For the analysis of haptoglobin (Hp) binding to bHb/PolybHb, a Hp
mixture containing Hp2-1 and Hp2-2 was purified from human Cohn Fraction
IV. The kinetics of Hp binding to bHb/PolybHbs was recorded as described
in the literature (Baek et al., 2012; Meng et al., 2018b). To calculate
Hp-Hb binding biomolecular rate constants, the fluorescence emission was
measured by exciting at 285 nm and moniotring the emission at 310 nm.
The resulting fluorescence intensity was fit to a monoexponential
equation to determine the pseudo-first-order Hp-Hb binding rate
constant. To determine the bimolecular rate constant, a linear fit was
then performed on the pseudo first order rate constant as a function of
PolybHb/bHb concentration to regress the slope.
2.4.5 MetHb
Level.
The metHb level of cell-free bHb, T- and R-state PolybHb was determined
using the cyanomethemoglobin assay (Drabkin, DL ; Austin, 1935).
2.4.6 Circular Dichroism (CD)
Spectroscopy.
The CD spectra of bHb, T-state, and R-state PolybHb were measured using
an JASCO J‐815 CD (JASCO, Easton, MD) spectrometer. All samples were
first diluted to 0.5 mg/mL in PB (50 mM, pH 7.4). The CD specta was
recorded from 190 nm to 260 nm in a quartz cuvette (1 mm path length)
and analyzed by the JASCO software (Zhang et al., 2011).
2.4.7 MALDI-TOF
Analysis.
Protein samples were diluted to 0.5 mg/mL protein basis in DI water. A
saturated solution of α-cyano-4-hydroxycinnamic acid (CHCA) matrix
solution was prepared in a 50% v/v mixture of acetonitrile (99.9%) and
trifluoroacetic acid (0.1%). 1 µL of the mixture of the matirix and
protein solution was deposited on a matrix assisted laser
desorption/ionization (MALDI) plate and run in a Microflex MALDI-TOF MS
system (Bruker, Billerica, MA).
2.4.8 Statistical
Analysis.
Either t-test or one-way ANOVA was used to analyze the variance among
all data, and a p-value of <0.05 was considered significant. A
least-squares linear regression analysis was used to study the
correlation between procedural parameters and PolybHb biophysical
properties.