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Process Biochemistry 42 (2007) 303–309
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The role of pH and its control on effective conjugation
of bovine hemoglobin and human serum albumin
Chunyang Zheng a,b, Guanghui Ma a,b, Zhiguo Su a,b,*
a
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering,
Chinese Academy of Sciences, P.O. Box 353, Beijing 100080, PR China
b
Graduate University of Chinese Academy of Sciences, Beijing 100049, PR China
Received 24 May 2006; received in revised form 21 August 2006; accepted 22 August 2006
Abstract
Human serum albumin (HSA) and bovine hemoglobin (Hb) conjugate is a promising candidate as a blood substitute. However, preparation of
the conjugate is problematic because both proteins tend to conjugate between themselves rather than crosslink each other. In this work, a facile
process for conjugation of Hb and HSA was developed through control strategy of the reaction. The reaction was carried out in a buffer containing
borax-borate and mannite. The borax-borate was used for pH buffering while mannite was used as a pH switch and a reaction promoter. As a result,
self-conjugation of Hb and self-conjugation of HSA were minimized. After the one-step conjugation reaction in aqueous solution, followed by the
one-step purification by ion-exchange chromatography, the conjugate of HSA and Hb was obtained with the total yield about 50%. The P50 and the
Hill coefficient for the product were 16.1 mmHg and 1.82, respectively.
# 2006 Elsevier Ltd. All rights reserved.
Keywords: Human serum albumin (HSA); Bovine hemoglobin (Hb); Borax-borate buffer–mannite system; Conjugate; Blood substitute
1. Introduction
Blood transfusion is a key method in emergency rescue and
in some medical operations such as open-heart surgery.
However, the shortage of available blood, the requirement of
blood-typing, and the danger of transmitting certain diseases
are serious problems. To circumvent these problems, blood
substitutes have been proposed to replace real human blood in
various medical treatments [1]. Among different blood
substitutes, hemoglobin-based blood substitute has been the
focus of research. Polymerized bovine hemoglobin has been
approved for medical use in South Africa [2]. Polyethylene
glycol modified hemoglobin is also under clinical trial [3].
Recently, a human serum albumin (HSA) and bovine
hemoglobin (Hb) conjugate was reported to be a good
candidate for blood substitute [4]. Considering the case of
severe blood loss, especially hemorrhage, HSA is also needed
[5], the HSA–Hb conjugate is theoretically assumed to be a
* Corresponding author at: National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, P.O.
Box 353, Beijing 100080, PR China.
E-mail address: [email protected] (Z. Su).
1359-5113/$ – see front matter # 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2006.08.011
better blood substitute than pure hemoglobin. Moreover, as a
hydrophilic macromolecule with anionic charge under physiologic conditions, HSA could also render the conjugate a
negative surface charge and make it away from the endothelial
cells lining the lumen of the circulatory system, which was
speculated to further prolong the half-time of the substitute [6].
However, in the trial of Bonhard and Boysen to couple
albumin and hemoglobin [7], a single-step conjugation reaction
using homobifunctional crosslinker resulted in self-conjugation
of HSA and/or Hb. When cross-linking two proteins, the
crosslinker may react initially with either one of the proteins,
forming an active intermediate. This intermediate further reacted
with a second protein through the free end of the crosslinker.
Because the reaction was not specific, the chance for reaction
with the same protein and with another protein would be the
same. To circumvent this problem, the two-step procedure was
established. First, one of the proteins to be conjugated is reacted
with the homobifunctional reagents, and then excess crosslinker
and by-products, e.g. self conjugates or polymers, are removed.
The downstream processing of the monomer-activated protein is
difficult because the physicochemical properties of the self
conjugates or polymers and the activated monomer are very
similar. In the second stage, the separated activated protein is
mixed with the other protein molecules to be conjugated often in
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reagent grade. Water used in the experiments was prepared with a RiOs ultrapure water system (Millipore, Bedford, MA).
2.2. Hemoglobin preparations
Bovine blood erythrocytes were obtained freshly from a local slaughterhouse, followed by osmotic hemolysis and stroma removal with centrifugation
at 30,000 g for 30 min. The stroma-free Hb solutions were purified by
selective DEAE cellulose adsorption, essentially as described by Cheung
et al. [12].
2.3. Determination of relationship between mannite content and pH
value
Fig. 1. Scheme of the coordinating mechanism of the Borax-borate buffer–
mannite system.
large amount to ensure reaction, and the final conjugation process
occurs between two proteins. A post-separation is also required
to remove the unreacted proteins. The two-step conjugation is
generally time-consuming and at low yield, which limits the
application of this method in the scale-up production [8].
Therefore, minimizing the ineffective self-polymerization without engineered properties during the conjugation process is a
challenge to the preparation and further investigation of this kind
of candidate blood substitute.
Borax-borate buffer–mannite system within the pH range
4.0–6.0 has once been applied to create pH gradient to study the
micro-heterogeneity of human serum albumin [9]. The original
pH value of the borax-borate buffer is about pH 9.0. By adding
appropriate mannite to the borax-borate buffer, the pH of the
borax-borate buffer system could be switched easily in the large
range from 4 to 9. On the other hand, as a polyhydroxy
compound, mannite forms easily dissociating complex with
boric acid as shown in Fig. 1 [10,11]. The formation of the
dissociating complex could influence the pKa of the available
groups and then microenvironment of the reactant proteins,
which is an important factor to facilitate the conjugation process.
In this work, the effect of pH of the reaction system on
conjugation process using homobifunctional glutaraldehyde as
crosslinker was investigated, and two separate pH ranges were
found to be effective to minimize the formation of the self
conjugates of HSA and self conjugates of Hb, respectively, which
made it possible to control the reaction by changing pH of the
reaction buffer. Therefore, Borax-borate buffer–mannite system
is utilized presumably to function both as a pH switcher and a
reaction promoter. This method offers means to facilely prepare
the conjugates of HSA and Hb while minimizing the formation of
self conjugates by-products of HSA and/or Hb. The yield is about
50%, and the production circle is about 6 h with only one-step ion
exchange chromatography separation involved.
2. Materials and methods
2.1. Chemicals and reagents
Human serum albumin (HSA, fraction V) and glutaraldehyde (50%, v/v,
grade I) were from Sigma. All other chemicals obtained were of analytical
The relationship between mannite content (w/v) and pH value of the boraxborate system with original pH 9 was investigated. After mannite was fully
dissolved, the pH of the Borax-borate buffer–mannite system was determined
and averaged of three repeated measurements.
2.4. Preparation of the conjugates
One hundred milligrams HSA was dissolved in the 10 ml of 20 mM, pH 9,
borax-borate buffer. Then, 480 ml of 5% (v/v) glutaraldehyde was added to the
activated HSA molecules. The activation reaction was carried out for 30 min at
4 8C, followed by the addition of mannite till the pH fell to 5.7. One milliliter of
100 mg ml 1 Hb solution was added, and the reaction was allowed to continue
for another 4 h at 4 8C, after which excessive Lysine was added to block further
reaction.
2.5. Purification of the conjugate
The reaction mixture was subjected to the ion-exchange chromatography
DEAE Sepharose Fast Flow. The column (10 cm 1.6 cm) was equilibrated by
10 mM PBS buffer, pH 7.0 (buffer A), and eluted with 0–0.1 M NaCl gradient in
buffer A. The flow rate was 0.5 ml min 1. The fractions containing the
conjugates were pooled and subjected to further analysis.
2.6. SDS-PAGE
SDS-PAGE was operated as the method of Laemmli [13]. The samples were
diluted with one volume of SDS sample buffer containing 0.1 M dithiothreitol
and boiled for 5 min, followed by electrophoresis for 1 h at 200 V. The gel was
stained in Coomassie Blue R-250 and then destained.
2.7. Oxygen equilibrium measurements
O2 equilibrium curves were measured on a Hemox Analyzer (TCS, PA) at
37 8C, using the Hemox buffer (pH 7.4). The analyzer measures the O2 pressure
with a Clark O2 electrode (Yellow Springs Instrument, OH) and simultaneously
calculates the Hb saturation via a dual-wavelength spectrophotometer. Values
for P50 (the O2 pressure at which Hb is half saturated) are obtained from the O2
equilibrium curves.
3. Results
3.1. Conventional single-step conjugation
Glutaraldehyde is a common crosslinker widely used in the
preparation of conjugates; however, as a result of high activity,
the self conjugates by-products plague its further precise
application, especially in the therapeutic field. Fig. 2 shows the
GF-HPLC profile of the resultant mixture prepared at pH 8
followed by the conventional single-step conjugation method.
The product was indeterminate. The reactant proteins were
C. Zheng et al. / Process Biochemistry 42 (2007) 303–309
Fig. 2. Characterization of the conjugation preparation using glutaraldehyde as
crosslinker by the conventional single-step method. The samples were loaded
on a Shodex gel filtration column, followed by elution at a flow rate of
0.5 ml min 1.
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of the reactant proteins, and therefore impact interaction of
them and the following reaction. As shown in Fig. 3, while
the pH of the reaction mixture was more than 9, the selfconjugation of HSA was inhibited, and the activated HSA was
in the monomeric formation (Fig. 4a), with trace HSA
dimmer that originally existed in the native HSA material;
while the pH of the reaction mixture was less than 6, the selfconjugation of Hb was inhibited, and the reaction of Hb and
glutaraldehyde was limited to the intra-crosslinking of Hb
(Fig. 4b). Hb has molecular weight of 64 kDa, and during
SDS-PAGE the four subunits dissociated and presented a
band with about 16 kDa, and after intra-molecularly crosslinked two additional bands appeared with the molecular
weight of about 32 and 48 kDa as shown in the inset (Fig. 4b).
The results demonstrated that whatever pH value was selected
as the working pH to couple HSA and Hb, the resultant
mixture was sure to include at least the ineffective byproducts of HSA polymer or Hb polymer.
highly crosslinked, and even insoluble precipitate was formed.
Therefore, proper control strategy of the reaction was needed to
minimize the formation of the self conjugate by-products and
ensure the production of the conjugates with engineered merits.
3.2. The effect of pH on the conjugation process
3.2.1. The effect of pH on the ineffective self conjugation of
HSA or Hb
The pH value plays an important role on the cross-linking
reaction of proteins. It is clear that high pH value could make
the reactive amino group less protonated. The de-protonated
amino group is the nucleophilic species and thus can facilitate
the reaction between the crosslinker and the functional
groups on the protein. On the other hand, the pH of the
reaction buffer also has an influence on the charge condition
Fig. 3. The effect of pH on the formation of polyhaemoglobin and polyalbumin.
The reaction samples under various pH values were determined using GFHPLC. The percentage of the uncoupled monomer was represented by the area
of the peak of monomer on the chromatogram. The content of polymer was
calculated by the formula of (100% monomer%). The results were obtained
from the average of three repeated measurements.
Fig. 4. The determination of the reaction samples of HSA (a) (detected by the
Shodex column) and Hb (b) (detected by the TSK3000sw column for the slight
change of elution behavior) under the selected pH condition using GF-HPLC.
After the modification of glutaraldehyde, HSA still remained as monomer, and
the subunits of Hb were crosslinked. The inset shows the characterization of the
intracrosslinked Hb using SDS-PAGE. Lane (1), native Hb; lane (2), standard
marker; lane (3), intracrosslinked Hb. The stacking and running gels were 5%
and 15% in acrylamide, respectively.
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Fig. 5. The conjugate and by-product yield as the function of pH. The
conjugation reaction was carried out at 4 8C for 1 h under different pH values,
and terminated by addition of excessive lysine solution. The components were
determined by TSK3000sw column. The yields of the by-products (crosslinked
products except effective conjugate of HSA and Hb) and conjugate were
estimated by integrating the chromatograms of the reaction products. The
results were obtained from the average of three repeated measurements.
Fig. 7. The plot of pH as the function of the content of mannite in borate buffer.
The results were obtained from the average of three repeated measurements.
conjugation, was reasonable to minimize the ineffective self
conjugation while promoting the effective conjugation between
HSA and Hb.
3.3. Acid-adjusted two-step conjugation
3.2.2. The effect of pH on the effective conjugation of HSA
and Hb
The effect of pH on the effective conjugation of HSA and Hb
was also investigated at different pH conditions. Fig. 5 indicates
that, the pH between two pIs of the reactant proteins (the pIs of
HSA and Hb are 4.7 and 6.8, respectively) promoted the
formation of the effective conjugation. Therefore, it was assumed
that, pH value influenced the conjugation process not only via the
protonated/de-protonated condition of the functional groups, but
via the net charge of reactant proteins. While the reactant proteins
were oppositely charged, the conjugation between them was
promoted by the electrostatic interaction.
Thus, a pH-switch strategy that first activating HSA with
glutaraldehyde at pH 9 and then changing pH to 5.7 (average of
the pI of HSA and Hb) by mannite and adding Hb for further
Fig. 6. Characterization of the resultant mixture prepared by acid-adjusted
conjugation reaction. The sample was loaded on the Shodex gel filtration
column, followed by elution of 50 mM PBS, plus 0.1 M Na2SO4.
Firstly, acid-adjustment was tried to change the pH of the
reaction system. However, after the addition of hydrochloric
acid reduced the pH of the reaction system to pH 5.7, as shown
in Fig. 6, compared with the conventional single-step method
(Fig. 2), the crosslinking degree was controled, but yield of the
conjugate of HSA and Hb was low.
3.4. Conjugation reaction in Borax-borate buffer–mannite
system
Borate has the properties of coordinating with polyhydroxyls resembling mannite, and the pH of borax-borate
buffer decreases as the increase of mannite added, as shown
in Fig. 7. The pH value of the borate buffer containing 3%
Fig. 8. Characterization of the resultant mixture prepared in the Borax-borate
buffer–mannite system. The sample was loaded on the Shodex gel filtration
column, followed by elution at a flow rate of 0.5 ml min 1 of 50 mM PBS, plus
0.1 M Na2SO4.
C. Zheng et al. / Process Biochemistry 42 (2007) 303–309
307
Fig. 9. The conjugate yield as the function of duration of reaction. The
conjugation reaction was carried out at 4 8C, and terminated by adding
excessive lysine.
(w/v) approximated 5.7, the working pH chosen to increase
the electrostatic interaction of the reactant proteins and then
improve the conjugation of them. Compared with the acidadjusted conjugation reaction (Fig. 6), the conjugate yield of
the conjugation reaction in the borate–mannite system was
markedly increased (Fig. 8). The effect of time duration of
the reaction over 30 h periods was also investigated to
achieve the best performance of the conjugation reaction.
Fig. 9 shows the kinetics of the conjugate yield as a
function of reaction time. After 6 h, the formation of 1:1
conjugate of HSA and Hb began to plateau, while the
accumulation of the conjugates of other ratio relations of
HSA and Hb continued to increase in another 12 h. Thus, the
duration of 6 h was used to ensure the maximal 1:1 conjugate
yield and limit the crosslinking degree of conjugates in other
stoichiometry.
Fig. 11. Characterization of the purified conjugates by the DEAE Sepharose
Fast Flow ion-exchange chromatography (a), native HSA (b), and native Hb (c).
The samples were loaded on the Shodex gel filtration column, followed by
elution at a flow rate of 0.5 ml min 1 50 mM PBS, plus 0.1 M Na2SO4.
3.5. Purification of the conjugates
The reaction mixture was loaded on a DEAE Sepharose Fast
Flow column. Peak (b) in Fig. 10 appeared by elution with 0–
0.1 M NaCl gradient in buffer A. The sample corresponding to
peak (b) was pooled and subjected to the gel filtration HPLC
determination. As presented in Fig. 11a, the result indicated that
the uncoupled Hb was removed. And most of activated HSA
was conjugated with Hb. Fig. 12 shows the SDS-PAGE profile
of the reaction mixture (lane 6), peak (a) (lane 1) and peak (b)
(lane 5) in Fig. 10. After the purification, the uncoupled Hb with
potential nephrotoxicity was removed in peak (a), and for peak
(b) (lane 5), the lack of the band with about 130 kDa appeared
in trace amount in lane 4 and band with 32 kDa appeared in lane
6 demonstrated that the both the self conjugate of HSA and that
of Hb were inhibited. The concentration of the preparation was
determined by Bradford method, and the total yield of the
conjugates was about 50%.
3.6. Bioactivity examination of the preparation
Fig. 10. Purification of the conjugate using ion-exchange chromatography on
DEAE Sepharose Fast Flow and monitored by the UV absorbance at 280 and
405 nm (the special absorbance wavelength of Hb). The column was equilibrated with buffer A. After the sample was loaded on the column, the column
was eluted with buffer A, followed by elution with 60 min, 0–0.1 M NaCl
gradient in buffer A. The flow rate was 0.5 ml min 1.
The O2 equilibrium curves of the conjugate and native
bovine Hb were shown in Fig. 13. It was evident that the curves
of the conjugate changed in position and shape. By comparison
with that of native bovine Hb (P50 = 27.1 mmHg, n = 2.76), the
P50 value and the Hill coefficient were 16.1 mmHg and 1.82,
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Fig. 12. Characterization of the conjugate using SDS-PAGE. Lane (1), peak (a)
in Fig. 10; lane (2), native Hb; lane (3), standard marker; lane (4), native HSA
(loaded in large amount for the trace HSA dimmer); lane (5), peak (b) in Fig. 10;
lane (6), the reaction mixture before separation. The stacking and running gels
were 5% and 15% in acrylamide, respectively.
control the conjugation process. It assumedly exerted two
functions in the conjugation process of HSA and Hb: (1) pH
switcher: in the original pH 9 borax-borate buffer, the HSA
was first activated by crosslinker glutaraldehyde, and the
conversion of functional groups on HSA to aldehyde blocked
further self-polymerization of the HSA molecules in the
following reaction. After mannite was added, the pH of the
system was switched to the pH range that could inhibit the
self-polymerization of Hb. In the Borax-borate buffer–
mannite system with pH 5.7, the hemoglobin was added to
the system and reacted with the activated HSA. Therefore,
both the ineffective by-products of HSA self-polymer and the
Hb self-polymer were inhibited in the Borax-borate buffer–
mannite system with mannite functioning as a pH switcher.
Moreover, (2) as a reaction promoter, when added to the
borax-borate buffer, mannite coordinated with the compounds
and formed a easily dissociating complex, which changed the
micro-environment of the reactant proteins and facilitated the
conjugation between HSA and Hb. On the other hand, the
addition of mannite also reduced the ion strength of the
system. Since the decreasing the ionic strength could increase
the attraction between the oppositely charged proteins and/or
molecules [14]. It was speculated that the reduction of ion
strength and the following increase of attraction between
HSA and Hb, oppositely charged under the given pH
condition, also contributed to the increase of conjugation
yield.
This might be also a practical and facile method to conjugate
other proteins or peptides with novel engineered merits.
Acknowledgement
The authors are grateful for the support from National
Natural Science Foundation of China (grant no. 20136020).
References
Fig. 13. O2 equilibrium curves of native bovine hemoglobin (a) and the
conjugates (b).
respectively. It showed that the coupling of HSA somewhat
impair the cooperativity of the subunits of hemoglobin.
Besides, the conjugate was saturated at the normal physiological range of O2 pressure (90–100 mmHg), indicating a good
performance of the conjugate for O2 delivery.
4. Discussion
The Borax-borate buffer–mannite system with flexible pH
range and special dissociation properties was first applied to
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