RESEARCH ARTICLE

Influence of Molecular Structure on O2p
Binding Properties
and Blood Circulation ofn A
Hemoglobin‒Albumin
Clusters
Kana Yamada1, Kyoko Yokomaku1, Risa Haruki1, Kazuaki Taguchi2, Saori Nagao3,
Toru Maruyama3, Masaki Otagiri2, Teruyuki Komatsu1*
1 Department of Applied Chemistry, Faculty of Science and Engineering, Chuo University, 1-13-27, Kasuga,
Bunkyo-ku, Tokyo, 112–8551, Japan, 2 Faculty of Pharmaceutical Sciences, Sojo University, 4-22-1, Ikeda,
Nishi-ku, Kumamoto, 860–0082, Japan, 3 Department of Biopharmaceutics, Graduate School of
Pharmaceutical Sciences, Kumamoto University, 5–1 Oe-Honmachi, Chuo-ku, Kumamoto, 862–0973, Japan* komatsu@kc.chuo-u.ac.jp

Abstract
OPEN ACCESS
Citation: Yamada K, Yokomaku K, Haruki R, Taguchi
K, Nagao S, Maruyama T, et al. (2016) Influence ofMolecular Structure on O2-Binding Properties andBlood Circulation of Hemoglobin‒Albumin Clusters.
PLoS ONE 11(2): e0149526. doi:10.1371/journal.
pone.0149526
Editor: Eugene A. Permyakov, Russian Academy of
Sciences, Institute for Biological Instrumentation,
RUSSIAN FEDERATION
Received: November 2, 2015
Accepted: February 2, 2016

A hemoglobin wrapped covalently by three human serum albumins, a Hb-HSA3 cluster, isan artificial O2-carrier with the potential to function as a red blood cell substitute. This paper
describes the synthesis and O2-binding properties of new hemoglobin‒albumin clusters (i)bearing four HSA units at the periphery (Hb-HSA4, large-size variant) and (ii) containing an
intramolecularly crosslinked Hb in the center (XLHb-HSA3, high O2-affinity variant).
Dynamic light scattering measurements revealed that the Hb-HSA4 diameter is greater thanthat of either Hb-HSA3 or XLHb-HSA3. The XLHb-HSA3 showed moderately high O2-affinity
compared to the others because of the chemical linkage between the Cys-93(β) residues in
Hb. Furthermore, the blood circulation behavior of 125I-labeled clusters was investigated byassay of blood retention and tissue distribution after intravenous administration into anesthetized rats. The XLHb-HSA3 was metabolized faster than Hb-HSA3 and Hb-HSA4.
Results suggest that the molecular structure of the protein cluster is a factor that can influence in vivo circulation behavior.

Published: February 19, 2016
Copyright: © 2016 Yamada et al. This is an open
access article distributed under the terms of theCreative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source arecredited.
Data Availability Statement: All relevant data arewithin the paper and its Supporting Information files.
Funding: This work was supported by a Grant-in-Aid
for Scientific Research on Innovative Area(“Coordination Programming” Area 2107, No.
21108013) from MEXT Japan, Chuo University Grant
for Special Research, and Joint Research Grant fromthe Institute of Science and Engineering, Chuo
University.

Introduction
In Japan, 85% of blood products are used for patients who are over 50 years old [1]. Japan’s continuously declining birthrate and aging society make it difficult to retain a stable blood transfusion system. The Japanese Red Cross Society forecasts a severe blood shortage equivalent to890,000 people per year in 2027 [2]. Consequently, a blood alternative, especially a red blood cell(RBC) substitute, is necessary as a medical measure to complement blood transfusion treatment.
In the last few decades, hemoglobin (Hb)-based O2-carriers (HBOCs) of various kinds, such as
glutaraldehyde-polymerized Hb [3–6] and poly(ethyleneglycol)-conjugated Hb (PEG-Hb) [7–11], have been developed [12,13]. However, the practical use of a safe and effective formulation
has not been realized [3,5,6,12,14,15]. A main side-effect is vasoconstriction, which elicits anacute increase in blood pressure. Many investigators have hypothesized that the vasopressor

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Hemoglobin‒Albumin Clusters

Competing Interests: The authors have declaredthat no competing interests exist.

response is attributable to rapid quenching of nitric oxide (endothelial-derived relaxing factor)
by a small fraction of free Hb leaked into the extravascular space [16–18].
Human serum albumin (HSA), the major plasma protein in the bloodstream (approximately 0.6 mM), fills two crucial roles of maintaining colloid osmotic pressure and of transporting hydrophobic metabolites and drugs [19]. We demonstrated previously that a covalentcore–shell structured protein cluster comprising Hb in the center and HSA at the periphery
functions as a unique HBOC [20–24]. The average binding number of HSA on one Hb is
3.0 ± 0.2. We designate this cluster as Hb-HSA3 (Fig 1A). HSA contains only one free sulfhydryl group at Cys-34. Consequently, a heterobifunctional crosslinker, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) was used for coupling of the Cys-34 of HSA
with surface amino groups of Lys residues of Hb [20,21]. The resultant cluster has satisfactorily
negative surface net charge (pI: 5.1) because of the surrounding HSA shells. As expected, intravenous transfusion of Hb-HSA3 into anesthetized rats (i) did not invoke unfavorable systemic
hypertension but (ii) showed reasonably long blood circulation [24]. Such superior properties
of Hb-HSA3 are a consequence of the electrostatic repulsion between the cluster surface andglomerular basement membrane around the endothelial cells. If one were able to engineer thebinding number of exterior HSAs and the chemical structure of interior Hb, then it mightengender unique variants with different molecular structure, molecular size, O2-affinity, andblood retention. In this study, we designed and synthesized new hemoglobin‒albumin clusters
bearing four HSA units at the periphery (Hb-HSA4, large-size variant) and including an intramolecularly crosslinked Hb (XLHb) in the center (XLHb-HSA3, high O2-affinity variant) (Fig1A). The influences of molecular structure on O2-binding properties and blood circulation of
the protein cluster have been investigated.

Materials and Methods
Materials and Apparatus
Human serum albumin (HSA) was purchased from the Japan Blood Products Organization.
Hemoglobin (Hb) was purified from bovine red blood cells purchased from Tokyo Shibaura
Zouki Co. Ltd. Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) was
purchased from Wako Pure Chemical Industries Ltd. 1,11-Bismaleimido-triethyleneglycol(BMTEG) was purchased from Thermo Fischer Scientific K.K. The water was deionized (18.2
MOcm) using water purification systems (Elix UV and Milli Q Reference; Millipore Corp.).
Native-PAGE and SDS-PAGE were conducted using an electrophoresis power supply (EPS
301; GE Healthcare) with precast 5‒20% polyacrylamide gel (Hi-QRAS Gel N 5‒20%: Kanto
Chemical Co. Inc.). Isoelectric focusing (IEF) was performed using an electrophoresis power
supply (EPS 601; GE Healthcare) with a pH 3–10 IEF protein gel (Novex; Thermo Fischer Scientific K.K.). The voltage was raised gradually to 500 V for 2 h.

Synthesis of Hb-HSA4 Cluster
DMSO solution of SMCC (40 mM, 2.0 mL) was added dropwise to the carbonyl Hb solution(0.1 mM, 20 mL in phosphate buffered saline solution, PBS) in a round-bottom flask (50-mL
volume). Then the mixture was stirred under dark conditions in CO atmosphere for 2 h at 4°C.
The resultant solution was loaded directly onto a gel filtration chromatograph (GFC) with aSephadex G25 (superfine) column to remove unreacted SMCC. The eluent was concentrated
to 20 mL ([Hb] = 0.1 mM) using a centrifugal concentrator (Vivaspin 20 ultrafilter, 10 kDa
MWCO; Sartorius AG). The obtained maleimide-activated-Hb (0.1 mM, 20 mL) was added
slowly into the HSA solution (2.0 mM, 20 mL in PBS). Then the mixture was stirred for 14 hunder dark conditions at 4°C. Part of the reactant was applied for size-exclusion

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Hemoglobin‒Albumin Clusters

Fig 1. Schematic illustrations of hemoglobin-albumin clusters. (A) Molecular structures of Hb-HSA3, Hb-HSA4, and XLHb-HSA3. (B) Structural model of
intramolecularly crosslinked Hb, in which sulfhydryl groups of Cys-93(β) residues (distance: 21.6 Å) are connected by BMTEG (molecular length: 18.3 Å,
shown in a space-filling representation) [25,26].
doi:10.1371/journal.pone.0149526.g001

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Hemoglobin‒Albumin Clusters

chromatography (SEC) on an HPLC system (LaChrom Elite; Hitachi High-Technologies
Corp.) with a column (Shodex Protein KW-803; Showa Denko K.K.) using phosphate buffered(PB) solution (50 mM, pH 7.4) as the mobile phase. The elution curve showed a new broad
peak at the high molecular weight region. Then the resultant solution was subjected to GFC(Superdex 200 pg; GE Healthcare UK Ltd.) using PBS as the running buffer. The eluent wasmonitored at 280 nm, and the cluster fraction was collected. The total protein concentrationand Hb concentration were measured, respectively, using a protein assay kit (Pierce 660 nm;
Thermo Fisher Scientific K.K.) and Hb assay kit (Nescoat Hemokit-N; Alfresa Pharma Corp.).
The cysteinyl thiol assay of Hb was conducted by reaction with 4,4’-dithiopyridine, whichbinds sulfhydryl group of the protein to give 4-thiopyridinone (λmax: 324 nm) [27]. The collected yield was ca. 80% based on Hb. The average HSA/Hb ratio of the product was 4.0 ± 0.2,
which is indicated as Hb-HSA4 with italicized subscript 4. The metHb ratio of Hb-HSA4 wasless than 1%.

Synthesis of XLHb-HSA3 Cluster
DMSO solution of 1,11-bismaleimido-triethyleneglycol (BMTEG) (10 mM, 1.0 mL) was added
dropwise to the carbonyl Hb solution (0.1 mM, 10 mL in PBS) in a round-bottom flask (30-mL
volume). After stirring for 1 h in CO atmosphere under dark conditions at 4°C, the reactant
was loaded onto GFC with a Sephadex G25 (superfine) column to remove unreacted BMTEG.
The eluent of ββ-crosslinked Hb (XLHb) was concentrated to 10 mL ([XLHb] = 0.1 mM) using
a centrifugal concentrator (Vivaspin 20 ultrafilter, 10 kDa MWCO). SDS-PAGE exhibited two
bands at 16 kDa and 32 kDa, which respectively correspond to the α-subunit monomer andcrosslinked ββ-subunit dimer. The cysteinyl thiol assay of XLHb using 4,4’-dithiopyridine
showed that both thiol groups of Cys-93(β) of Hb were bridged by BMTEG.
XLHb-HSA3 was prepared according to the same procedure used for Hb-HSA3 using XLHb
[21]. The collected yield was ca. 80% based on Hb. The average HSA/XLHb ratio of the cluster
mixture was 2.9 ± 0.2, which is denoted as XLHb-HSA3 with italicized subscript 3.

DLS Measurements
The molecular sizes of the protein clusters were ascertained using dynamic light scattering(DLS) measurements (Zetasizer Nano ZS; Malvern Instruments, Ltd.) at 25°C. A He-Ne laser(633 nm) was used and the scattering angle was 173°. Aqueous protein solutions with a concentration of 5 μM were prepared in Milli-Q water. The hydrodynamic diameters were estimatedby number from the particle size distribution.

O2-Binding Property
The visible absorption spectra were recorded using a UV–Visible spectrophotometer (8543;
Agilent Technologies Inc.) equipped with a temperature control unit (89090A; Agilent Technologies Inc.). To prepare the oxy Hb-HSA4, O2 gas was flowed to the PBS (pH 7.4) of carbonyl
Hb-HSA4 ([Hb]: ca. 10 μM) in an optical quartz cuvette (10 mm path length) sealed with a rubber septum under light (500 W halogen lamp) in an ice-water bath. Subsequently, N2 gas was
flushed to the O2 complex solution, yielding deoxy Hb-HSA4. The O2 affinity (P50: O2-partialpressure where Hb is half-saturated with O2) and Hill coefficient (n) were determined using an
automatic recording system for blood O2-equilibrium curves (Hemox-Analyzer; TCS Scientific
Corp.) using PBS (pH 7.4) at 37°C. The sample was oxygenated by an increasing O2-partialpressure and deoxygenated by flowing with N2. The visible absorption spectral changes, O2euqilibrium curves, P50, and n of XLHb-HSA3 were also measured using the same procedures.

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Hemoglobin‒Albumin Clusters

Animal Experiments
All animal experiments were approved by the Animal Care and Use Committee of Sojo University and Kumamoto University. Care and handling of the animals were performed in accordance with the guidelines, principles, and procedures for the care and use of laboratory animalsof Sojo University and Kumamoto University. All male Wistar rats were purchased from
Kyudo Co. Ltd. and were maintained under conventional housing conditions. The animals
were acclimated for 1 week before the experiments.

Blood Circulation Experiments
The 125-Iodinated Hb-HSA4 and XLHb-HSA3 were prepared using our previously reported
procedures [24,28]. The sample was diluted with non-labeled Hb-HSA4 and XLHb-HSA3 toadjust the protein concentration. Wistar rats were anesthetized with diethylether. The bodyweights were 253 ± 8 g for Hb-HSA4 group and 244 ± 7 g for XLHb-HSA3 group. Then thecluster solution was injected intravenously via a tail vein (Hb-HSA4, 50 mg/kg; XLHb-HSA3,
40 mg/kg) (0.2 mL/100 g, 1.5 × 106 cpm/rat, n = 6). At the time-points of 3, 10, 30 min, and 1,
3, 6, 12, and 24 h after the infusion, 200 μL of blood was collected from the lateral tail veinusing a heparinized syringe. After removal of the blood cell components by centrifugation(3000 rpm, 10 min), the 125I concentrations in the plasma were ascertained by measuring theirradioactivity using an automatic gamma counter (2480 WIZARD2; PerkinElmer Inc.). Acidprecipitability of the recovered radionuclide was confirmed using trichloroacetic acid. At theend of the experiments, the rats were anesthetized with diethylether and were euthanized byhemorrhage. Their vital organs (liver, kidney, spleen, lung, heart, and pancreas) were isolated,
and their weights were measured. Urine and feces were also collected at fixed intervals in a metabolic cage. The radioactivity of the excised organs, urine, and feces were determined using a
gamma counter as described above. As reference groups, identical experiments were conductedusing the 125I-labeled samples in the same protocols [Hb group (270 ± 9 g, 10 mg/kg infusion,n = 6), HSA group (260 ± 11 g, 30 mg/kg infusion, n = 6), HSA group (268 ± 7 g, 40 mg/kg
infusion, n = 4), and Hb-HSA3 group (269 ± 5 g, 40 mg/kg infusion, n = 6)]. The data of thesegroups were partially described in our previous report [24]. Furthermore, comparison experiments were conducted to measure the radioactivity of the vital organs at 1 h after the infusion[HSA group (262 ± 13 g, 30 mg/kg infusion, n = 6), HSA group (256 ± 11 g, 40 mg/kg infusion,n = 6), Hb-HSA3 group (274 ± 8 g, 40 mg/kg infusion, n = 6), Hb-HSA4 group (239 ± 8 g, 50
mg/kg infusion, n = 6), and XLHb-HSA3 group (243 ± 4 g, 40 mg/kg infusion, n = 6)].

Data Analysis
Pharmacokinetic analyses of the samples were conducted using a non-compartment model.
Pharmacokinetic parameters were calculated using the moment analysis program available
with Microsoft Excel (Microsoft Corp.). Data are shown as the mean ± SD for the indicatednumber of animals. Significant differences between groups were inferred using two-tailed
unpaired Student t-tests. Probability p < 0.05 was inferred as statistically significant.

Results and Discussion
Synthesis of Hb-HSA4 and XLHb-HSA3
Fundamentally, Hb-HSA4 was prepared using the same procedure as that used for Hb-HSA3
with some modifications [21]. First, 40-fold molar excess SMCC was reacted with Hb in PBS(pH 7.4). The maleimide-activated-Hb (MA-Hb) was added dropwise into the HSA solution(HSA/MA-Hb = 20, mol/mol), followed by stirring for 14 h at 4°C. Both the SMCC/Hb and

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Hemoglobin‒Albumin Clusters

HSA/MA-Hb ratios are double the synthesis of Hb-HSA3. Size exclusion chromatography(SEC) of the reaction mixture demonstrated an approximately single peak at the high molecular weight region (14‒19 min elution time) (Fig 2A). Unreacted Hb (ca. 22 min) was not detectable. The position of the peak top (16.3 min) coincided well with that of Hb-HSA4 hetero-Fig 2. SEC profiles of (A) Hb-HSA4 and (B) XLHb-HSA3. Dotted black lines are elution curves of the reaction mixture. Solid lines are elution curves ofpurified clusters.
doi:10.1371/journal.pone.0149526.g002

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Hemoglobin‒Albumin Clusters

pentamer [21]. The major product was purified by gel filtration chromatography (GFC); theexcess HSA was removed. Based on the protein assay and Hb assay, the average binding number of HSA on one Hb was found to be 4.0 ± 0.2.
The bovine Hb processes two sulfhydryl groups of Cys-93(β) residues in the central cavity(Fig 1B) [25]. Therefore, the site-specific reaction of α,ω-bismaleimide reagent with Hb would
enable us to make an intramolecular bridge in the protein. Because the distance of two Cys-93(β) residues is estimated as 21.6 Å based on crystal structural data [25], we choose 1,11-bismaleimido-triethyleneglycol (BMTEG) (18.3 Å molecular length). The SDS-PAGE of the resultant
mixture exhibited two bands with the same intensity at 16 kDa and 32 kDa, which respectively
correspond to the α-subunit monomer and crosslinked ββ-subunit dimer (S1 Fig). The numberof cysteinyl thiols per Hb decreased from 2.0 to 0.1 [27]. These results imply that two Cys-93(β) residues are connected intramolecularly by BMTEG, yielding ββ-crosslinked Hb (Fig 1B).
Isoelectric focusing (IEF) electrophoresis analysis showed that the isoelectric point (pI) of
XLHb was slightly lower than the value of naked Hb (pI: 7.0) (Fig 3B). Acharya et al. reported
that crosslinked Hbs [ββ-Hb with bis(maleimidophenyl)-PEG2000 and αα-Hb with bis(3,5-dibromosalicyl)fumarate] demonstrated the same electrophoretic mobility with unmodified Hb [7,10]. Presumably, site-specific crosslinking of Cys-93(β)s by BMTEG influences theorientations of ε-amino groups of Lys residues on the molecular surface, thereby reducing thepI value.
The XLHb-HSA3 was prepared using the same procedure for Hb-HSA3 using XLHb [21].
The SEC profile of XLHb-HSA3 resembled that of Hb-HSA3 (Fig 2B) [21]. The average binding
number of HSA on one XLHb was ascertained as 2.9 ± 0.2.

Fig 3. Diameters and IEF patterns of hemoglobin-albumin clusters. (A) Hydrodynamic diameters of Hb-HSA3, Hb-HSA4, and XLHb-HSA3 measured
using DLS. (B) IEF patterns of Hb-HSA3, Hb-HSA4, and XLHb-HSA3.
doi:10.1371/journal.pone.0149526.g003

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Molecular dimensions of these protein clusters were estimated from dynamic light scattering (DLS) measurements. The hydrodynamic diameters increased with the number of HSA
units: 8.9 nm for Hb-HSA3, 8.9 nm for XLHb-HSA3, and 11.6 nm for Hb-HSA4 (Fig 3A). The
Hb-HSA4 (molecular weight: ca. 332 kDa) is larger than the others having three HSA entities.
The IEF pattern revealed that the pI values of the clusters (pI: 5.0−5.1) were close to that of
HSA (pI: 4.9) (Fig 3B). It is noteworthy that the covalent wrapping of Hb with three-fold HSAs
is sufficient to mask the surface charge of the Hb core (pI: 7.0).

O2-Binding Properties
Visible absorption spectra of Hb-HSA4 and XLHb-HSA3 in PBS solution under N2, O2, and
CO atmospheres (deoxy, oxy, and carbonyl forms, respectively) were identical to the corresponding spectra of native Hb and Hb-HSA3 (S2 Fig and S1 Table) [29]. The O2 affinities (P50:O2-partial pressure where Hb is half-saturated with O2) and cooperativity coefficients (Hillcoefficient; n) were determined from the O2-binding equilibrium curves. The P50 of Hb-HSA4(9 Torr) was significantly lower than the value of Hb (P50 = 23 Torr) (Table 1). The Hill coefficient also decreased from 2.6 to 1.5. The reductions of P50 and n were equivalent to thoseobserved for Hb-HSA3. This report describes two feasible reasons for the high O2-affinity andlow cooperativity of Hb-HSA3, which were explained in earlier reports [20,23]. First is theblocking of a sulfhydryl group of Cys-93(β) in Hb by the maleimide terminal of free SMCC.
Chemical modification of Cys-93(β), which is located beside the proximal base (His-92) of theheme, is known to enhance the O2 affinity [9,11,30] and is known to restrict the quaternary
structure of Hb from the Tense (T)-state to a Relaxed (R)-state [30]. The second reason is themodifications of ε-amino groups of Lys residues on Hb by the succinimide terminal of SMCC.
They are necessary to create the cluster, whereas the chemical modification of Lys affects theO2 affinity and decreases the Hill coefficient of Hb [8,31,32]. Identical O2-binding parametersof Hb-HSA4 and Hb-HSA3 imply that the central Hb conformations are almost identical, independent of the binding number of HSA.
However, XLHb-HSA3 showed moderately low P50 and n (6 Torr, 1.0) with respect to HbHSA3 and Hb-HSA4. The Hill coefficient of 1.0 indicates a lack of cooperatively. The two sulfhydryl groups of Cys-93(β)s in Hb were bridged securely, thereby strongly constraining theavailable motion of quaternary structure of the XLHb core. The high O2-affinity variant may
be advantageous as a transfusion-related medication. Another possible reason of the side-effectby Hb derivatives is overoxygenation [33–36]. The small Hb injected into the bloodstream
facilitates the diffusive O2 transport to the blood vessel wall, initiating overoxygenation of thesurrounding tissues. An autoregulatory O2-sensing system responds by constricting the bloodvessel and reduces the available surface area for O2 transport. These physiological responses
engender vasoconstriction. The high O2-affinity (low P50) avoids early O2 offloading on thearterial vessels and may be beneficial for O2 release in the capillaries [34–36].
Table 1. O2-binding parameters of Hb-HSA3, Hb-HSA4, and XLHb-HSA3 in PBS solution (pH 7.4) at
37°C.
Hemoproteins

P50 (Torr)nkox (h-1)

Hb a

23

2.6

0.037

Hb-HSA3 a

9

1.5

0.035

Hb-HSA4

9

1.5

0.039

XLHb-HSA3

6

1.0

0.033

a

Ref. 21.

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Blood Circulation and Tissue Distribution
The 125I-labeled cluster solutions were injected into rats to observe the blood retention and tissue distribution. The 125I-labeled Hb was eliminated immediately from the circulation (Fig 4)[half-life (T50): 0.53 h] [24]. In contrast, time courses of plasma concentration of the clusters
demonstrated slower kinetics. Blood circulation parameters were ascertained using a non-compartment model (Table 2). The T50 of Hb-HSA3 (18.5 ± 1.7 h) was 1.7-fold longer than that of
HSA (T50 = 11.0 ± 0.67 h) [24]. This greater length is attributable to the decreases of clearance(CL) and volume of distribution in a steady state (Vdss). The negative surface net charge andlarge molecular size of Hb-HSA3 suppress the movement to the extravascular space and renal

Fig 4. Blood retention of hemoglobin-albumin clusters. Relative plasma concentrations of 125I-labeled Hb-HSA3, Hb-HSA4, XLHb-HSA3, and HSA after
intravenous administration to rats. Each data point represents the mean ± SD (n = 6). **p < 0.01 vs. HSA.
doi:10.1371/journal.pone.0149526.g004

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Table 2. Blood circulation parameters of 125I-labeled Hb-HSA3, Hb-HSA4, XLHb-HSA3, and HSA after intravenous administration to rats (n = 6).
Vdss (mL)

AUC (% of dose/mL h)

16.5 ± 2.4

12.0 ± 1.3

6.2 ± 0.9

0.95 ± 0.07

14.3 ± 1.4

105.5 ± 8.2

14.6 ± 1.1

0.94 ± 0.11

13.6 ± 1.1

107.9 ± 12.5

T50 (h)MRT (h)CLtot (mL/h)a0.53 ± 0.1

0.74 ± 0.14

HSA a,b

11.0 ± 0.67

15.0 ± 0.88

HSA c

10.8 ± 0.8

Hb18.5 ± 1.7**

26.3 ± 2.5**

0.50 ± 0.07**

13.0 ± 0.74

203.4 ± 23.2**

Hb-HSA4

19.1 ± 1.6**

27.1 ± 2.4**

0.48 ± 0.06**

13.0 ± 0.9

210.0 ± 26.9**

XLHb-HSA3

13.8 ± 0.4**

19.6 ± 0.5**

0.68 ± 0.03**

13.4 ± 0.6

146.4 ± 7.2**

Hb-HSA3a

T50, half-life; MRT, mean residence time; CLtotal, clearance; Vdss, volume of distribution in a steady state; AUC, area under the concentration-time curve.
Ref. 24.

ab

30 mg/kg dose.

40 mg/kg dose. Each value represents the mean ± SD (n = 6).
**p < 0.01 vs. HSAb.

cdoi:10.1371/journal.pone.0149526.t002

filtration. In view of these findings, we anticipated that Hb-HSA4 bearing four HSA units, alarge-size variant, might show longer blood retention. Nonetheless, the result fell short of our
expectations. All parameters of Hb-HSA4 were comparable to those of Hb-HSA3 (Table 2).
The kinetic profiles of HSA with a 30 mg/kg infusion and 40 mg/kg infusion were almost identical (S3 Fig and Table 2). This result shows that the quantity of the protein administrated does
not influence the blood circulation parameters in our experimental models. We concluded thatthe increase of HSA binding number on Hb (increase of molecular weight of the cluster) is ineffective to extend the circulation persistence. In other words, the three-fold HSA conjugation to
Hb is sufficient to retain the cluster molecules in the bloodstream for a long period.
In contrast, the T50 of XLHb-HSA3 (13.8 ± 0.4 h) was rather shorter than that of Hb-HSA3,
despite their molecular weights, diameters, and surface charges are nearly equal. It appears to
be attributable to different morphology of the cluster. The intramolecular crosslinking of Cys93(β)s of Hb might change the reactivity of ε-amino groups of Lys residues around the
BMTEG bridge. Results show that the binding positions of HSA units on XLHb become partially different from those of Hb-HSA3, yielding a different-shape variant. In fact, our image
processing and 3D reconstruction based on transmission electron microscopy data of HbHSA3 revealed that Lys-82(β1), which is located between the two Cys-93(β)s of Hb, is a potential binding partner of Cys-34 of HSA [20]. We presumed that the change of molecular shapeof the cluster might cause measurable alteration of the blood circulation kinetics.
The tissue distribution of the clusters in vital organs (liver, kidney, spleen, lung, heart, andpancreas) was determined at 1 h and 24 h after the administration (Fig 5). No correlation was
found among the three cluster groups and the HSA group at 1 h after infusion (Fig 5A). However, marked differences of the accumulated quantities were observed in the liver after 24 h(Fig 5B). The high hepatic distributions of Hb-HSA3, Hb-HSA4, and XLHb-HSA3 compared to
HSA are ascribed to their large molecular sizes. These high distributions are consistent withdata reported by Rennen et al. showing predominantly uptake of large proteins in the liver[37]. Among the three clusters, small distribution in liver was found in XLHb-HSA3 group. We
inferred that such a difference is also caused by the variation of the cluster molecular structure.
Total 125I activities of urine within 24 h were much higher than those of feces in all groups(Fig 6). The clusters are metabolized in the body and are excreted into urine, which is the same
excretion pathway of HSA. The excreted 125I amount in urine of XLHb-HSA3 group was somewhat higher than those of other groups. Results show that XLHb-HSA3 is metabolized faster

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Hemoglobin‒Albumin Clusters

Fig 5. Tissue distribution of hemoglobin-albumin clusters. Tissue (vital organs) distribution of 125I radioactivity (% of dose) (A) at 1 h and (B) at 24 h after
intravenous administration of 125I-labeled Hb-HSA3, Hb-HSA4, XLHb-HSA3, and HSA to rats. Each bar shows the mean ± SD (n = 6). **p < 0.01 vs. HSA.
doi:10.1371/journal.pone.0149526.g005

than Hb-HSA3 and Hb-HSA4, and suggest that the molecular structure influences the catabolism of protein clusters in the body.

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Hemoglobin‒Albumin Clusters

Fig 6. Urinary and fecal excretions. Urinary and fecal excretions of 125I radioactivity (% of dose) within 24 h after intravenous administration of 125I-labeled
Hb-HSA3, Hb-HSA4, XLHb-HSA3, and HSA to rats. Each bar shows the mean ± SD (n = 6). *p < 0.05 vs. Hb-HSA3, **p < 0.01 vs. Hb-HSA4.
doi:10.1371/journal.pone.0149526.g006

Conclusion
Covalent wrapping of Hb or XLHb with three or four HSAs generates various core–shell protein cluster conjugates. The Hb-HSA4 diameter was larger than those of the others enfolded bythree HSA units. Nevertheless, increasing of the HSA binding number has no marked influenceon the O2-binding property and blood circulation. The XLHb-HSA3 showed high O2-affinity(P50 = 6 Torr) because the Cys-93(β) residues of Hb are crosslinked intramolecularly. The

PLOS ONE | DOI:10.1371/journal.pone.0149526 February 19, 201612 / 15

Hemoglobin‒Albumin Clusters

superior blood circulation properties of all clusters compared to native Hb are attributed totheir negative surface net charges and larger molecular sizes. It is noteworthy that the circulation parameters of XLHb-HSA3 are close to those of HSA. This variant was metabolized fasterthan Hb-HSA3 and Hb-HSA4. Site-specific Cys-93(β) crosslinking of the Hb core may alter thechemical reactivity of Lys residues around the bridging site, thereby forming a different spatialconformation of the cluster and causing a useful reduction of the blood circulation. Our results
imply that the molecular structure is a factor that might affect the in vivo circulation behaviorof the cluster. The Hb-HSA4 and XLHb-HSA3 will become unique RBC substitutes that may
serve as a relay product enabling resuscitation of blood loss patients.

Supporting Information
S1 Fig. SDS-PAGE of XLHb.
(TIF)
S2 Fig. UV-vis. absorption spectral change of XLHb-HSA3. In PBS solution at 25°C.
(TIF)
S3 Fig. Blood retention of HSA. Relative plasma concentrations of 125I-labeled HSA after
intravenous 30 mg/kg infusion (n = 6) and 40 mg/kg infusions (n = 4) to rats. Each data point
represents the mean ± SD.
(TIF)
S1 Table. Visible absorption spectral data of Hb-HSA4 and XLHb-HSA3. In PBS solution(pH 7.4) at 25°C
(TIF)

Acknowledgments
The authors acknowledge Dr. Keitaro Sou, Waseda University, for kind supports with DLS
measurements and the members of the Radioisotope Center, Kumamoto University, forimportant contributions to blood circulation experiments.

Author Contributions
Conceived and designed the experiments: TK. Performed the experiments: K. Yamada K.
Yokomaku RH KT SN TM MO TK. Analyzed the data: K. Yamada K. Yokomaku RH KT SN
TM MO TK. Wrote the paper: K. Yamada K. Yokomaku TK.

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