RSC
Chemical Biology
rsc.li/rsc-chembio

ISSN 2633-0679

PAPER
Teruyuki Komatsu et al.
Haemoglobin(K120C)–albumin trimer as an artificial O 2
carrier with sufficient haemoglobin allostery

Volume 1
Number 3
August 2020
Pages 81–158

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Chemical Biology
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Haemoglobin(bK120C)–albumin trimer as an
artificial O2 carrier with sufficient haemoglobin
allostery†
Yoshitsugu Morita,

Asuka Saito, Jun Yamaguchi and Teruyuki Komatsu*

The allosteric O2 release of haemoglobin (Hb) allows for eﬃcient O2 delivery from the lungs to thetissues. However, allostery is weakened in Hb-based O2 carriers because the chemical modifications of
the Lys- and Cys-b93 residues prevent the quaternary transition of Hb. In this paper, we describe thesynthesis and O2 binding properties of a recombinant Hb [rHb(bK120C)]–albumin heterotrimer thatmaintains sufficient Hb allostery. The rHb(bK120C) core, with two additional cysteine residues at thesymmetrical positions on its protein surface, was expressed using yeast cells. The mutations did not
influence either the O2 binding characteristics or the quaternary transition of Hb. Maleimide-activatedhuman serum albumins (HSAs) were coupled with rHb(bK120C) at the two Cys-b120 positions, yielding
the rHb(bK120C)–HSA2 trimer, in which the Cys-b93 residues were unreacted. Molecular dynamics
simulation demonstrated that the HSA moiety does not interact with the amino acid residues around thehaem pockets and the a1b2 surfaces of the rHb(bK120C) core, the alteration of which retards Hb allosReceived 29th April 2020,
Accepted 25th June 2020

tery. Circular dichroism spectroscopy demonstrated that the quaternary transition between the relaxed(R) state and the tense (T) state of the Hb core occurred upon both the association and dissociation of
O2. In phosphate-buffered saline solution (pH 7.4) at 37 1C, the rHb(bK120C)–HSA2 trimer exhibited a

DOI: 10.1039/d0cb00056f

sigmoidal O2 equilibrium curve with the O2 affinity and cooperativity identical to those of native Hb (p50 =rsc.li/rsc-chembiorHb(bK120C)–HSA2 trimer compared with naked Hb.

12 Torr, n = 2.4). Moreover, we observed an equal Bohr effect and 2,3-diphosphoglycerate response in the

Introduction
Allosteric regulation plays a crucial role in controlling theprotein function in biological systems through the binding ofa small-molecule eﬀector that induces a conformationalchange. Haemoglobin (Hb) is a well-known allosteric protein
encapsulated in red blood cells (RBCs),1–5 and its allostery has
been extensively studied to understand its mechanism.6–12 O2
binding converts the quaternary structure of Hb from a deoxy
tense (T) state with a low O2 affinity to a relaxed (R) state with ahigh O2 affinity.1–4 The homotropic allosteric effect, which is
known as cooperativity, allows Hb to efficiently transport O2
from the lungs to the peripheral tissues. As a heterotropic
allosteric effect, 2,3-diphospoglycerate (DPG) stabilizes the
T-state conformation by creating salt bridges between the twob subunits to reduce O2 affinity.13,14 Furthermore, O2 affinity

Department of Applied Chemistry, Faculty of Science and Engineering,
Chuo University, 1-13-27 Kasuga, Bunkyo-ku, Tokyo 112-8551, Japan.
E-mail: komatsu@kc.chuo-u.ac.jp† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0cb00056f

128 | RSC Chem. Biol., 2020, 1, 128--136

decreases with decreasing pH (the Bohr effect), shifting theequilibrium toward the T state.11,15,16
Artificial O2 carriers are currently required as RBC substitutes for transfusion therapy because of worldwide shortages inblood supply.17,18 However, the stroma-free Hb splits to an abdimer (a2b2 - 2ab) in the bloodstream and is immediately
expelled into urine. Moreover, Hb diﬀuses into the interstitialspaces between the endothelial cells and smooth muscle cells,causing hypertension by scavenging nitric oxide (NO;endothelial-derived vasodilator).19,20 Therefore, naked Hb is
not available for use in transfusion therapy. Since the 1980s,
several types of Hb-based O2 carriers have been developed,
including cross-linked Hb,21,22 polymerized Hb,23,24 and PEGylated Hb.25–27 However, these chemical modifications havemarkedly reduced cooperativity, the Bohr effect, and the DPG
effect,18 and a modified Hb that maintains the original allosteric effects of Hb has never been reported.18 We previously
synthesized a protein cluster as an RBC substitute consisting ofan Hb molecule wrapped covalently in human serum albumins(HSAs), called an Hb–HSAm cluster (m = 2–4, average m = 3.0 0.2).28,29 This protein cluster shows a long circulation lifetimein the bloodstream and a superior biocompatibility.30,31 The

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Lys-b120 is replaced with Cys at symmetrical positions on the
Hb surface, and we also present the rHb(bK120C)–HSA2 heterotrimer, in which MA-HSAs are linked at the Cys-b120 positions(Fig. 1B). Ultraviolet-Visible (UV-vis) and circular dichroism(CD) spectroscopies revealed that the quaternary structure of
the rHb(bK120C)–HSA2 trimer is reversibly convertible between
the R and T states. We also observed identical O2 affinity andallosteric effects in the rHb(bK120C)–HSA2 trimer comparedwith naked Hb.

Results and discussion
Expression and physicochemical properties of rHb(bK120C)

Fig. 1 (A) Recombinant Hb variant [rHb(bK120C)] with Lys-b120 residues
replaced by cysteines at the symmetrical positions. The mutation point
was designed to avoid the haem pockets and a1b2 interfaces. (B) Syntheticroutes of maleimide-activated HSA (MA-HSA) and the rHb(bK120C)–HSA2
trimer. The Cys-34 of the HSA was reacted with 1,6-bis(maleimido)hexane(BMH), resulting in MA-HSA. The two Cys-b120 residues at symmetrical
positions were reacted with MA-HSA to yield the rHb(bK120C)–HSA2
trimer.

quaternary structural analysis of the Hb–HSAm cluster revealed
that the chemical modification of the Lys and Cys-b93 residuesof Hb by N-succinimidyl 3-maleimidopropionate as a crosslinker prevents the R–T quaternary transition and maintains
the Hb core close to the R state.32 Thus, the Hb–HSAm cluster
demonstrated a low cooperativity, a low Bohr effect, and a low
DPG effect. If the HSA shells are linked at appropriate positions
on the Hb surface so that they do not affect the R–T quaternary
motion, the resulting Hb–HSAm conjugate maintains the original O2 binding properties of the native Hb. The geneticallyintroduced cysteine could act as a cross-linking point for
maleimide-activated HSA (MA-HSA). We assume that the mutation of Hb residues, excluding the mutation of (i) the a1b2
interfaces, including the N- and C-terminals (which play important roles in the R–T quaternary transition), and (ii) the haem
pockets (which directly affect O2 affinity), would allow theresulting Hb variant to maintain its original O2 binding capability.
Based on this hypothesis, the Lys-b120 residues were chosen as
the symmetrical mutation points (Fig. 1A). In this study, we
describe a novel recombinant Hb variant, rHb(bK120C), in which

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We first expressed rHb(bK120C) using Pichia yeast (Pichia pastoris
GS115) according to our previous reports.33,34 The expressedrHb(bK120C) was purified using cation- and anion-exchange
chromatographies (CEC and AEC). The sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis ofrHb(bK120C) depicted two clear bands corresponding to thea- and b-subunits (Fig. 2A). The a- and b-chain bands demonstrated near-identical mobility to those of native Hb. The
matrix-assisted laser desorption/ionization-time of flight(MALDI-TOF) mass spectrum analysis of rHb(bK120C) exhibited molecular-related ion peaks at 15 122 and 15 841 Da. These
were almost identical to the simulated masses of the aminoacid sequences (15 127 Da for the a-chain and 15 842 Da for theb-chain), indicating that Lys-b120 was replaced with cysteine.
The size-exclusion chromatography (SEC) chromatogram ofrHb(bK120C) exhibited a peak with the same elution volume
as that of native Hb, indicating that the tetramer (a1a2b1b2) was
formed in the rHb(bK120C) (Fig. 2B; elution volume = 1.9 mL).
Fortunately, the oligomeric rHb(bK120C) species created via
the disulfide bonds of Cys-b120 was not observed in the SEC
chromatogram. The fact that rHb(bK120C) and native Hb had
identical CD spectra (l = 200–250 nm) indicates that theirsecondary structures were equivalent (Fig. 2C). The isoelectric
point (pI) value of rHb(bK120C) was determined to be 6.8, which
was slightly lower than that of native Hb (pI = 6.9) because themutation (Lys-b120 - Cys) had negatively shifted the net surface
charge (Fig. S1, ESI†). The results of the sulfhydryl group assay
using 4,4 0 -dithiodipyridine (4,4 0 -DTP)35 revealed the number ofreactive sulfhydryl groups in rHb(bK120C) and native Hb as 4.0
and 2.0, respectively. This result strongly indicates that the two
Lys-b120 residues of the b1 and b2 chains were replaced by Cys
and that the introduced Cys-b120 residues were reactive with 4,40 DTP, as they are located at the molecular surface of rHb(bK120C).
Preparation of the rHb(bK120C)–HSA2 trimer
To prepare MA-HSA, we used 1,6-bis(maleimido)hexane (BMH)
as the cross-linking agent. One of the two maleimide groups in
BMH was reacted with the free sulfhydryl group of the Cys-34 in
HSA (which is the only reduced-form of cysteine in HSA), yielding
MA-HSA. The formation of the HSA dimer was not observed
because of electrostatic repulsion between the negatively charged
HSA surfaces present under our experimental conditions. The

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Fig. 2 (A) SDS–PAGE results of native Hb and rHb(bK120C). (B) SEC profiles of rHb(bK120C) and native Hb. (C) CD spectra of the rHb(bK120C) and native
Hb in PBS at 25 1C ([protein] = 0.2 mM).

sulfhydryl group assay using 4,4 0 -DTP revealed the complete
disappearance of the reduced form.
Capping of the Cys-b93 residues of native Hb with maleimide derivatives causes a decline in O2 binding cooperativity,
the Bohr eﬀect, and the influence of the allosteric eﬀectors.
The Cys-b93 residues of native Hb were able to react withmaleimide derivatives, such as N-ethyl maleimide (NEM) andN-succinimidyl 3-maleimidopropionate.32,36 We found that the
Cys-b93 residues had not reacted with MA-HSA because of thesteric hindrance between the native Hb and MA-HSA. In contrast, genetically introduced Cys-b120 located at the molecularsurface of rHb(bK120C) had reacted with MA-HSA. The SEC
chromatogram of the reaction mixture [rHb(bK120C) + MA-HSA]
revealed a new peak in the high-molecular-weight region (Fig. 3A;
elution volume = 1.4 mL), and native PAGE analysis also revealed

a new band above that of rHb(bK120C) (Fig. 3B). The highmolecular-weight component was purified using AEC followed
by gel filtration chromatography (GFC). The ion peak of theb-chain at 15.8 kDa in the MALDI-TOF MS disappeared after thereaction of rHb(bK120C) with MA-HSA. A new peak then appearedat 82.5 kDa, a value almost identical to the simulated mass of ab-chain + MA-HSA (82.3 kDa), indicating that the HSA moiety wascovalently linked to the b-chain of rHb(bK120C). Based on the[total protein]/[Hb unit] assays, the average HSA/Hb ratio of the
product was determined to be 2.0. The heme loss was notobserved during the preparation. We denoted this hybrid protein
as rHb(bK120C)–HSA2 (Mw: 198 kDa). The CD spectrum of therHb(bK120C)–HSA2 trimer coincided perfectly with the sum of therHb(bK120C) spectrum and a two-times-enlarged HSA spectrum(Fig. 3C). This result demonstrates that the secondary structures

Fig. 3 (A) SEC profiles of the resultant reaction mixture [rHb(bK120C) + MA-HSA] and the purified rHb(bK120C)–HSA2 trimer. (B) Native PAGE patterns ofrHb(bK120C), HSA, the reaction mixture, and the purified rHb(bK120C)–HSA2 trimer. (C) CD spectra of rHb(bK120C), HSA, rHb(bK120C), andrHb(bK120C)–HSA in PBS at 25 1C ([protein] = 0.2 mM). (D and E) The energy-minimized structure of the rHb(bK120C)–HSA2 trimer obtained by amolecular dynamics (MD) calculation using the Desmond software.

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Paperof the individual proteins were unaltered by the protein coupling.
The dynamic light scattering measurements revealed that the hydrodynamic diameter of the rHb(bK120C)–HSA2 trimer (12.4  2.8 nm,
P.I. = 0.010) was significantly larger than that of rHb(bK120C)(6.9 nm  1.9 nm, P.I. = 0.004). The pI value of the trimer(pI = 5.4) was close to that of HSA (pI = 4.9), which further
indicates the coupling of rHb(bK120C) (pI = 6.7) with HSAs
(Fig. S1, ESI†). The net negative surface charge and the large
molecular size could allow the rHb(bK120C)–HSA2 trimer tocirculate in the bloodstream for long periods of time and preventthe undesirable vasopressor response through NO depletion.19,20
Furthermore, the sulfhydryl group assay using 4,4 0 -DTP35 demonstrated that the number of reactive sulfhydryl groups in therHb(bK120C)–HSA2 trimer was 1.8, indicating that the Hb core
maintained the free Cys-b93 residues that are important for O2
binding ability, as mentioned above.36 We could not observe thedissociation of the a2b2 tetramer into two ab dimers during thesynthesis, purification, and measurements of rHb(bK120C)–HSA2,
implying that the a2b2 tetramer was sufficiently stable the same as
native Hb under our experimental conditions.
An MD simulation was used to predict the interactions
between the rHb(bK120C) and HSA moieties. The Cys-b1120
and Cys-b2120 residues exist at symmetrical positions of thecore Hb; therefore, a suﬃcient distance is maintained between
the two HSA parts in the rHb(bK120C)–HSA2 trimer. We performed MD simulation using a half model [ab(K120C)–HSA]
constructed using the crystal structures of native Hb (PDB ID:
2DN1)37 and HSA (PDB ID: 1AO6)38 (Fig. S2, ESI†). The main
chains (Ca and amide atoms) of the ab(K120C) moiety werefrozen for the duration of a 50 ns simulation. The root-meansquare deviation (RSMD) and the distances of the salt bridges
and hydrogen bonds between the ab(K120C) and HSA residues
reached a plateau within 25 ns (Fig. S3, ESI†). The MD simulation structure was represented in the rHb(bK120C)–HSA2 form
which was prepared by overlapping the two ab(K120C) moietiesof the models with the crystal structure of native Hb (a1b1a2b2;
Fig. 3D and Fig. S2B, ESI†). The distance between the sulfide
atoms of Cys-b120 [rHb(bK120C)] and Cys-34 (HSA) was measured to be 13.8 Å, meaning that the BMH was long enough tocross-link the Cys residues (Fig. 3E). We found that the following salt bridges and hydrogen bonding interactions betweenrHb(bK120C) and HSA helped stabilize the conformation of therHb(bK120C)–HSA2 trimer: (i) Glu-a23, Glu-a27, and His-a112
of rHb(bK120C)—Arg-81 of HSA and (ii) Ser-a49 and Asp-a47 ofrHb(bK120C)—Gln-94 of HSA. The HSA moiety did not interact
with the amino acid residues around the haem pockets and thea1b2 surfaces of the rHb(bK120C) core.
Quaternary structure analysis of the Hb core
We investigated the quaternary structure of the Hb core using
UV-vis absorption and CD spectroscopies. The UV-vis spectral
patterns and the absorbances of the oxy, deoxy, and carbonylrHb(bK120C) as well as the rHb(bK120C)–HSA2 trimer wereindistinguishable from those of native Hb in PBS solution(pH 7.4; Fig. 4A and Table S1, ESI†). Generally, the chemical
modification of the Lys and Cys-b93 residues of Hb decreases

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Fig. 4 (A) UV-vis absorption spectra of the carbonyl (gray), oxy (red), anddeoxy (blue) rHb(bK120C)–HSA2 trimer (solid line) and native Hb (dottedline). (B) CD spectra (240–320 nm) of the oxy (red) and deoxy (blue)
Hb components of the rHb(bK120C)–HSA2 trimer [rHb(bK120C)–HSA2 
twofold HSA] (solid line) and rHb(bK120C) (dotted line). The measurements
were performed in PBS solution (pH 7.4) at 25 1C ([protein] = 3 mM).

the Soret band absorption of the deoxy form, because the deoxy
Hb remains near the R-state quaternary structure.32 In contrast,
the deoxy rHb(bK120C) and the rHb(bK120C)–HSA2 trimer
revealed the same Soret band absorption as that of deoxy native
Hb, indicating that the identical T-state quaternary structures
were formed. CD spectroscopy was also used to analyse thequaternary structure of the Hb core in the rHb(bK120C)–HSA2
trimer. Generally, deoxy Hb (T state) exhibits a negative CDband at 287 nm, which is known as the T-state marker.39–41 At
the same time, this band is not usually observed in the R-state
quaternary structure of oxy Hb. The CD spectral patterns andintensities of oxy and deoxy rHb(bK120C) both resembled thoseof native Hb, implying that the oxy and deoxy forms of these
proteins are comparable (Fig. 4B).39 CD also revealed similar
spectral changes in the rHb(bK120C)–HSA2 trimer upon deoxygenation. CD spectra for the oxy and deoxy Hb cores in therHb(bK120C)–HSA2 trimer were obtained by subtracting thetwo-times-enlarged HSA spectrum from the correspondingrHb(bK120C)–HSA2 trimer spectra (Fig. 4B). The CD spectraof the oxy and deoxy Hb cores coincided with those ofrHb(bK120C). These spectral analyses revealed that the mutation
Lys-b120 - Cys and coupling of HSA at the Cys-b120 positions did

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not influence the quaternary structural transition between the R
and T states.

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O2 binding aﬃnity and cooperativity
Hb binds with O2 in the lungs, where there is a high O2 partialpressure (pO2), and releases O2 into the peripheral tissues,
where there is a low pO2. To evaluate the O2 binding properties,
O2 aﬃnity (p50; pO2 where Hb is half-saturated with O2) and theHill coeﬃcient (n; degree of cooperativity) of Hb were determined from the O2 equilibrium curve (OEC). As shown in
Fig. 5A, a sigmoidal OEC was observed for native Hb in PBS
solution (pH 7.4) at 37 1C (p50 = 12 Torr; n = 2.4). Upon therelease of O2, the quaternary structure of Hb converts fromthe R state with a high O2 aﬃnity to the T state with a low
O2 aﬃnity. The cooperative O2 release allows Hb to eﬃciently
deliver O2 from the lungs to the tissues. Nevertheless, chemicallyPaper
modified Hbs have demonstrated a low cooperativity because
the quaternary transition of the Hb core is prevented.32 Suchmodified Hbs include PEG-Hb (71% reduction),18,42 polymerized Hb (41% reduction),18,42 and Hb–HSAm clusters (71%
reduction).29,42 In contrast, the OECs of rHb(bK120C) and therHb(bK120C)–HSA2 trimer were found to have a sigmoidal
shape completely consistent with that of native Hb (Fig. 5A).
The O2 binding parameters of rHb(bK120C) ( p50 = 12 Torr,n = 2.3) and the rHb(bK120C)–HSA2 trimer ( p50 = 12 Torr,n = 2.4) were also equivalent to those of native Hb (Table 1).
Both rHb(bK120C) and the rHb(bK120C)–HSA2 trimer were able
to maintain identical O2 affinity and cooperativity because thequaternary structure of the Hb units was interconvertible
between the R and T states upon O2 association and dissociation, as in native Hb. The capping of Cys-b93 with NEM in
native Hb (NEM-Hb) reduced the p50 and n values (p50 = 8 Torr,n = 1.9). The NEM-rHb(bK120C)–HSA2 trimer demonstrated
O2 binding parameters similar to those of NEM-Hb, clearly
indicating that the Cys-b93 residues in the rHb(bK120C)–HSA2
trimer were in a reduced form. This result is supported by thesulfhydryl group assay mentioned above. We concluded thatthe selective coupling of MA-HSA occurred at the two Cys-b120
positions of the rHb(bK120C), whereas it did not take place at
the Cys-b93 residues because of steric hindrance. On the basisof these findings, we also concluded that the Lys-b120 - Cys
mutation and the selective conjugation of MA-HSA at the Cysb120 positions were impervious to O2 affinity and cooperativity.
To the best of our knowledge, this is the first example of achemically modified Hb showing an identical O2 affinity andcooperativity to those of native Hb.18
The autoxidation rate constant (kox) of the oxygenated Hbcore in the rHb(bK120C)–HSA2 trimer was measured in PBS
solution (pH 7.4) at 37 1C. The kox value of the rHb(bK120C)–
HSA2 trimer was ascertained as 0.027 h1, which was almost
similar to the data of native Hb (0.020 h1). The oxy form of theinternal Hb maintains good stability even after being covered
with HSAs.
Bohr eﬀect on rHb(bK120C)–HSA2 trimer
A high metabolic activity leads to an increase in the concentration of CO2 and a decrease in the pH value within the tissues.
Under low-pH conditions, the protonation of Hb occurs and the
T-state quaternary structure is stabilized, reducing O2 aﬃnity(the Bohr eﬀect). Thus, the OEC of native Hb was right-shiftedto a p50 value of 28 Torr at pH 6.2 compared with p50 = 12 Torr at
Table 1 O2 binding parameters and Bohr coeﬃcients of haemoproteinsin PBS solution at 37 1C

pH 7.4
Haemoprotein
Fig. 5 OECs and Bohr plots of Hb (dotted line) and the rHb(bK120C)–
HSA2 trimer (solid line) at 37 1C ([protein] = 5 mM). (A) OECs in PBS solutions
at pH 7.4 (black) and pH 6.2 (red). (B) The Bohr plots of Hb (black circle) andthe rHb(bK120C)–HSA2 trimer (red triangle) in PBS solutions (pH 6.2, 6.5,
7.0, 7.4, and 8.0). (C) OECs in 50 mM Tris–HCl buffer solution with 1 mM
DPG (blue) and without DPG (black).

132 | RSC Chem. Biol., 2020, 1, 128--136

pH 6.2p50 (Torr) n (—) p50 (Torr) n (—) Bohr coeﬃcienta

Hb
12
rHb(bK120C)
12
rHb(bK120C)–HSA2 12

2.4
2.3
2.4

28
29
28

1.9
1.9
1.9

0.29
0.29
0.29

a

The Bohr coeﬃcients were derived from the slopes obtained fromplotting Dlog p50/DpH between pH 6.2 and 8.0 (Fig. 5B).

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Table 2 Eﬀect of DPG on hemoproteins in 50 mM Tris–HCl buffer
solution (pH 7.4) at 37 1C containing 1 mM DPG

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Without DPG

With DPG

Hemoprotein

p50 (Torr)n (—)

p50 (Torr)

n (—)

Hb
rHb(bK120C)
rHb(bK120C)–HSA2

9
9
9

2.3
2.3
2.3

27
26
26

2.4
2.1
2.1

pH 7.4 (Table 1 and Fig. 5A). The Bohr eﬀect allows for Hb highO2-transport eﬃciency to the tissues. However, chemical modifications have tended to notably reduce the Bohr eﬀect, suchas in PEG-Hb (87% reduction)18 and polymerized Hb (75%
reduction).18 We observed that the O2 aﬃnities of rHb(bK120C)
and the rHb(bK120C)–HSA2 trimer were reduced under lower pH
conditions, similar to native Hb (p50: 12 - 29 Torr (pH 7.4 - 6.2)).
The p50 and n values of both rHb(bK120C) and the rHb(bK120C)–
HSA2 trimer were nearly identical to those of native Hb at pH 6.2(Table 1). The Bohr coefficients were derived from the slopes
obtained from plotting Dlog p50/DpH between pH 6.2 and 8.0(Fig. 5B and Table S2, ESI†). The Bohr coefficients of bothrHb(bK120C) and the rHb(bK120C)–HSA2 trimer were identicalto those of native Hb (Table 1). The value measured for native
Hb, however, was somewhat weaker than the result reported by
Ho et al.43 This difference is probably attributable to several
factors, such as the difference in temperature (37 1C vs. 29 1C)
and the difference in solvents (PBS with low phosphate andhigh NaCl concentrations vs. 0.1 M sodium phosphate buffer(PB)). Altogether, we conclude that neither the Lys-b120 - Cys
mutation nor the HSA conjugation affected the Bohr effect.
Eﬀects of DPG on O2 binding aﬃnity
Allosteric eﬀectors bind to proteins, altering their activity. The
most important allosteric regulator of Hb in RBCs is DPG,
which reduces the O2 binding aﬃnity (the DPG eﬀect). An X-ray
crystallography analysis has previously revealed that a single
DPG molecule binds to the cleft of Hb b chains, known as the
DPG binding site.13,14 The anionic groups of DPG form salt
bridges with the cationic groups of the b-chains, including theHis-b2, Lys-b82, and His-b143 residues, stabilizing the T-state
conformation and reducing O2 aﬃnity. The T - R conformational change induced by oxygenation leads to the closure of
the binding site and weakens the DPG binding aﬃnity to oxy
Hb. This DPG response allows Hb to eﬃciently deliver O2 fromthe lungs to the tissues. We measured the OEC of native Hb in
PBS (pH 7.4) solution containing 1 mM DPG at 37 1C, but areduction in O2 aﬃnity was not observed because of the factthat phosphate anions in the PBS solution interrupt the binding of DPG to Hb. Thus, we used 0.1 M Tris–HCl buffer for DPG
response experiments. Indeed, native Hb in Tris–HCl buffer
demonstrated a higher O2 affinity (p50 = 9 Torr) than in PBS
solution because of the exclusion of the Hb–phosphate anioninteractions (Table 2). The O2 affinity of the native Hb was also
reduced upon the addition of DPG to Tris–HCl buffer. Interestingly, rHb(bK120C) (p50 = 26 Torr) and the rHb(bK120C)–HSA2
trimer (p50 = 26 Torr) demonstrated identical DPG responses toThis journal is © The Royal Society of Chemistry 2020

native Hb (p50 = 27 Torr; Fig. 5C). These results indicate that therHb(bK120C)–HSA2 trimer maintained a DPG response similar tothat of native Hb and that its O2 affinity is controllable via theallosteric effector.

Conclusions
A novel Hb variant (Lys-b120 - Cys) was designed to prepare
the rHb(bK120C)–HSA2 trimer as an artificial O2 carrier. The
mutation did not affect the O2 binding properties of Hb. Two
MA-HSA molecules were selectively linked to Cys-b120 inrHb(bK120C), resulting in the rHb(bK120C)–HSA2 trimer inwhich Cys-b93 remained free because of steric repulsion
between the rHb(bK120C) core and the MA-HSA shell. The CD
spectra revealed that the R–T quaternary motion of the Hb corein the rHb(bK120C)–HSA2 trimer occurred upon O2 associationand dissociation. Thus, the rHb(bK120C)–HSA2 trimer demonstrated sufficient allosteric O2 binding properties with respect
to cooperativity, the Bohr effect, and the DPG effect. The
rHb(bK120C)–HSA2 trimer is a unique artificial O2 carrier with
sufficient allostery to be an RBC substitute.

Materials and methods
Materials and apparatus
1,6-Bis(maleimido)hexane (BMH) was purchased from Tokyo
Chemical Industry Co., Ltd. Human serum albumin (HSA,
albumin 25%, Benesis) was purchased from the Japan Blood
Products Organization. The other special-grade chemicals wereused without additional purification unless otherwise noted.
Water was deionized (18.2 MO cm) using two water purification
systems (Elix UV and Milli Q Reference; Millipore Corp.). The
SDS–PAGE and native PAGE analyses were performed using a
15% or 5–12% poly(acrylamide) precast gel (SuperSep Ace 15%;
SuperSep Ace 5–12%; Fujifilm Wako Pure Chemical Corp.).
Isoelectric focusing (IEF) was performed using a pH 3–10 IEF
protein gel (Novex; Thermo Fischer Scientific Inc.). The UV-vis
absorption spectra were obtained using a UV-Visible spectrophotometer (8543; Agilent Technologies Inc. or V-650; Jasco
Corp.). CD spectra were recorded using a CD spectrometer(J-820; Jasco Corp.) at 25 1C.
Expression and preparation of rHb(bK120C)
The expression plasmid for the rHb(bK120C) variant [pHIL-D2rHb(bK120C)] was constructed according to the standard protocol of the QuickChange XL Site-Directed Mutagenesis
Kit (Agilent Technologies Inc.) using a pHIL-D2-rHb(a- andb-chains) vector34 and an oligonucleotide primer set (forward0[5 0 -CTCGCGCATCACTTCGGCT]G TGAGTTTACACCGCCAGTTC-30
and reverse [5 -GAACTGGCGGTGTAAACTCAC
AGCCGAAGTGAT 
GCGCGAG-30 ]). The obtained pHIL-D2-rHb(bK120C) vector was
linearized with SalI and used to transform the GS115-strain
Pichia pastoris (Thermo Fisher Scientific K.K.) via electroporation.
Transformed clone cells were grown in a buﬀered mineral
glycerol complex (BMGY) medium (4 L total) in a shaking

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incubator (Bio-Shaker GBR-200; Taitec Corp.; 200 rpm, 30 1C)
and subsequently in a buﬀered mineral methanol complex(BMMY) medium (1.6 L total) containing 0.3 mM hemin for
5 days. During cultivation, 100% methanol at 1.5% of themedium volume was added every 24 h. The cells were harvested
by centrifugation at 3000g for 10 min. The obtained cells werethen washed with water (300 mL  2) and resuspended in
100 mL of sodium PB solution (10 mM, pH 6.0) containing
1 mM phenylmethanesulfonyl fluoride. After the addition ofglass beads (150 mL, d = 0.5 mm), the cells were lysed using a
BeadBeater (Biospec Products, Inc.) with four cycles of disruption (2 min) and incubated by cooling on ice (2 min). Aftercentrifugation at 12 000g for 1 h, the supernatant was equilibrated with CO atmosphere. The solution was loaded onto acation exchange chromatography column (SP Sepharose Fast
Flow, GE Healthcare UK Ltd), which was equilibrated in 10 mM
PB (pH 6.0). After washing with the same buﬀer solution, thetarget protein was eluted using 20 mM Tris–HCl buffer solution(pH 8.0). Next, the resulting protein solution was subjected to
AEC using a Q Sepharose Fast Flow column (GE Healthcare UKLtd) with 20 mM Tris–HCl (pH 8.0) as the running buffer. Afterwashing with 20 mM Tris–HCl (pH 8.0), rHb(bK120C) was
eluted using PBS solution (pH 7.4). The purity was checkedusing SDS–PAGE analysis and SEC on a high-performanceliquid chromatography (HPLC) system (AKTA purify; GE Healthcare UK Ltd). The system was equipped with an SEC column(Superdex 200 Increase, 5/150 GL; GE Healthcare UK Ltd), andPBS solution (pH 7.4) was used as the mobile phase. The
concentration of rHb(bK120C) was measured using a protein
assay kit (Pierce 660 nm; Thermo Fisher Scientific K.K.). The
obtained rHb(bK120C) had a concentration of approximately
100 mg L1 of media. The sulfhydryl group assay of rHb(bK120C)
was conducted by reaction with 4,4 0 -dithiodipyridine (4,4 0 -DTP).35
Preparation of MA-HSA
The 25% HSA solution (4 mL) was diluted with PBS solution(14 mL, pH 7.4). A DMSO solution of BMH (7.5 mM, 2 mL) was
then added dropwise to the HSA solution (8.4 mM, 18 mL).
After stirring for 3 h at 25 1C, the reactant was subjected to a
GFC column (Sephadex G25 superfine; GE Healthcare UK Ltd)
to remove the unreacted cross-linker. The protein concentration
was measured using a protein assay kit (Pierce 660 nm), whereas
the sulfhydryl group assay of MA-HSA was conducted by reaction
with 4,4 0 -DTP. The MA-HSA solution was stored at 80 1C.
Preparation of rHb(bK120C)–HSA2
The MA-HSA and rHb(bK120C) solutions were mixed andconcentrated to 6 mL ([MA-HSA] = 0.4 mM, [rHb(bK120C)] =0.1 mM). The reactant was stirred under dark conditions for
24 h at 4 1C. An aliquot of the reaction mixture was analysedusing SEC on an HPLC system (AKTA purify; GE Healthcare UKLtd) equipped with an SEC column (Superdex 200 Increase,
5/150 GL; GE Healthcare UK Ltd) and using PBS solution (pH 7.4)
as the mobile phase. The reaction mixture was purified using AEC
using a Q Sepharose Fast Flow column (GE Healthcare UK Ltd).
The mixture solution was diluted with the same volume of water134 | RSC Chem. Biol., 2020, 1, 128--136

Paperand loaded onto the column equilibrated with PBS solution. The
column was then washed using 10 mM PB (pH 7.4) solution. The
rHb(bK120C)–HSA2 trimer was eluted with a 10 mM PB (pH 6.0) +120 mM NaCl solution. Under this condition, the HSA dimer
remained in the column. The collected solution was concentrated
and subjected to GFC using a Superdex 200 pg column (GEHealthcare UK Ltd) to isolate the rHb(bK120C)–HSA2 trimer(yield: 50%). The total protein and Hb concentrations were
measured using a protein assay kit (Pierce 660 nm; Thermo
Fischer Scientific Inc.) and the extinction coefficient of cyanometHb (e541 = 4.4  104 M1 cm1), respectively. The average[HSA]/[rHb(bK120C) unit] ratio of the product was estimated tobe 2.0. The sulfhydryl group assay of MA-HSA was conducted
via the reaction with 4,4 0 -DTP.
Molecular dynamics simulations
MD simulations were performed using the Desmond application implemented in a Maestro graphical interface.44,45 We
used the optimized potentials for liquid simulations (OPLS_2005)
force field implemented in the Desmond software for all moleculesin the system.46 We constructed a half model of rHb(bK120C)[ab(K120C)–HSA model] because rHb(bK120C)–HSA2 has a C2
symmetry, and there has to be a sufficient distance between the
HSAs for their interactions to be negligible. The crystal structure ofnative Hb (PDB ID: 2DN1)37 was used for the ab(K120C) model. Lysb120 was replaced with Cys using the PyMOL software (PyMOL
Molecular Graphics System Version 2.0, Schrödinger, LLC.). For the
HSA moiety, we used the crystal structure of HSA (PDB ID: 1AO6).38
The missing atoms of the side chains in the HSA were added tothe model using the PyMOL software. The water molecules in themodel were removed. The bond orders were assigned, and thehydrogen atoms were added to the models using the Protein
Preparation Wizard tool47 in the Maestro software. Theab(K120C) and HSA models were covalently linked using theBMH cross-linker [Cys-b120(ab)-BMH-Cys-34(HSA)] and arranged
so that sufficient distance was maintained between them (Fig. S2A,
ESI†). The Cys-b120(Hb), Cys-34(HSA), and BMH moieties wereminimized using the 3D builder in the Maestro software. The
hydrogen networks and the protonated states were optimized to
pH 7 using the Protein Preparation Wizard for which the pKa valuesof the protein residues were predicted using PROPKA.48 The
ab(K120C)–HSA model was solvated using explicit TIP3P water49
with a buffering distance of 10 Å in an octahedral box. Sodium
counterions were added to neutralize the charges. The model was
relaxed using a default relaxation protocol implemented in the
Desmond software. An isothermal–isobaric MD simulation was
performed for 50 ns to maintain the 300 K temperature and1.01325 bar pressure. The main chains (Ca and amide atoms) of
the ab(K120C) moiety were frozen during the simulation. The time
step was 2 fs, and the information for analysis was printed every
100 ps. A 10 Å cut off was used for non-bonded interactions.
Preparation of oxy and deoxy forms of the rHb(bK120C)–HSA2
trimer
The oxy (O2 complex) rHb(bK120C)–HSA2 trimer solution (PBS,
pH 7.4, 3 mM, 3 mL) was prepared using our previously reported

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technique.29 The solution was transferred to an optical quartz
cuvette (10 mm path length) with a rubber septum cap. N2 gas
was blown into the oxy-form solution to yield the deoxyrHb(bK120C)–HSA2 trimer. The UV-vis absorption and CD
spectra of these species were recorded at 25 1C.

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O2 binding parameters
The O2 aﬃnity (p50; O2 partial pressure where Hb is halfsaturated with O2) and Hill coeﬃcient (n) were determinedusing an automatic recording system for the O2 equilibrium
curve (Hemox Analyzer; TCS Scientific Corp.) at 37 1C. The oxyform of the rHb(bK120C)–HSA2 trimer solution (PBS, pH 7.4,
approximately 5 mM, 4 mL) was used for the measurements.
The trimers in PBS solution (pH 7.4) were deoxygenated byflushing with N2 and oxygenated by increasing the O2 partialpressure.
O2 complex stability
The oxy form stability of the rHb(bK120C)–HSA2 trimer wasassessed using the first-order autoxidation rate constant (kox) of
the core Hb using our earlier described procedures.50

Conflicts of interest
There are no conflicts to declare.

Acknowledgements
This work was supported by a Grant-in-Aid for Challenging
Research (Exploratory) (No. 18K19007) and Early-Career Scientists (No. 19K20699) from JSPS, the Science Research Promotion Fund from Promotion and Mutual Aid Corporation forPrivate Schools of Japan, a Joint Research Grant from theInstitute of Science and Engineering, Chuo University, and a
Research Grant from Naito Foundation.

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