Hemoglobin Conjugated with a Band 3 N-terminus

Derived Peptide as an Oxygen Carrier

Yen-Lin Lin and Kuang-Tse Huang
Department of Chemical Engineering, National Chung Cheng University, Chia-Yi, Taiwan
Abstract: A peptide composed of 9 amino acids, 7 residues from N-terminus of human erythrocytic Band 3 protein (AcMEELQDD)
followed by cysteine and glutamic acids, was conjugated to hemoglobin (Hb) serving as an allosteric effector for oxygen release. The
activated polyethylene glycol (PEG), maleimide-PEG-N-hydroxysuccinimidyl, was used to crosslink Hb with the peptide. The putative
conjugation site on Hb for effective enhancement of oxygen release was characterized as Lys-b95 by liquid chromatography-tandem
mass spectrometry. In addition, the conjugated peptide causes a rightward shift of the oxygen dissociation curve as compared to that of
its parent Hb when the degree of oxygen saturation is higher than 50%. Furthermore, this conjugated peptide remains effective on
lowering Hbs oxygen affinity after Hb polymerization by another PEG crosslinker. The allosteric properties of the peptide-conjugated
Hb may provide a new aspect of Hb-based oxygen carriers.
Keywords: hemoglobin, blood substitutes, band 3, polyethylene glycol, oxygen carriers
INTRODUCTION
Free hemoglobin (Hb) has the function of carrying
oxygen molecules, but it still cannot directly be put into
the human body. The reason lies in that Hb in plasma
easily tends to dissociate from tetramer into dimers. The
excessive amount of Hb dimers will be transferred to
kidney, precipitate in the loop of Henle, and result in
acute renal toxicity [1]. In addition, Hb solution will cause
vasoconstriction due to its excess oxygen delivery to
tissues [2] or its extravasation into the spaces between
endothelial and smooth muscle cells, reacting with nitric
oxide (NO) therein [3,4]. Moreover, free Hb exhibits a
high oxygen affinity in the absence of allosteric effector
2,3- diphosphoglycerate (2,3-DPG) and would result in
inappropriate oxygen release in vivo [5].
To overcome the dimer formation of unmodified Hb,
crosslinking of Hb has been performed either chemically
or by genetically engineered a-a fused Hb [6]. Regarding
the oxygen affinity, the modification of Hb with pyridoxal
derivatives [7,8] was used to mimic the action of 2,3-DPG
to lower the oxygen affinity. To further prevent the
formation of Hb dimers, bis(3,5-dibromosalicyl) fumarate
was used to crosslink between Hb a chains under
anaerobic condition [9]. These modifications would,
however, still have a short in vivo half life or lack an
appropriate oxygen release due to the limited dynamic
behavior of Hb during the oxy-deoxy transition.
The N-terminal 11 residues of band 3 have been
shown to bind to Hb and shift the oxygen dissociation
curve for Hb to the right [10], which prompted us to have
Hb conjugated with the band 3 derived peptide as a
dynamic modulator for oxygen release. To create the site
for conjugation, the band 3-N terminal derived 7 residues
were added sequentially with cysteine and glutamic acid
at the C terminus. In this study, the activated polyethylene
glycol (PEG), maleimide-PEG-N-hydroxysuccinimidyl
(MAL-PEG-NHS) was used as a crosslinker and spacer
for Hb conjugation. Schematic representation of this
conjugation is shown in Figure 1. MAL is the functional
group to crosslink with cysteine of the peptide, whereas
NHS crosslinks with lysine exhibited on the outer surface
of Hb. This conjugated peptide was demonstrated to be
able to increase the oxygen release even after Hb is
polymerized with another activated PEG.
MATERIALS AND METHODS
Chemicals
Drugs were obtained from Sigma Chemical Co. (St.
Louis, MO), except as specifically stated otherwise.
Address correspondence to Kuang-Tse Huang, Department of Chemical Engineering, National Chung Cheng University, 168,
University Rd., Min-Hsiung, Chia-Yi 621, Taiwan. E-mail: chmkth@ccu.edu.tw
Artificial Cells, Blood Substitutes, and Biotechnology, 37: 3240
Copyright # 2009 Informa UK Ltd.
ISSN: 1073-1199 print / 1532-4184 Online
DOI: 10.1080/10731190802664684
32
Peptides were synthesized at DigitalGene Biosciences Co.
(Taipei, Taiwan). The PEG derivatives, MAL-PEG-NHS
and succinimidyl propionate-PEG-succinimidyl propio-
nate (SPA-PEG-SPA), were purchased from NOF Co.
(Tokyo, Japan). CM-sepharose was obtained from Gen-
eral Electric Co. (Fairfield, CT). The reagents for iso-
electric focusing (IEF) were from Bio-Rad Laboratories,
Inc. (Hercules, CA). The sequencing grade modified
trypsin was purchased from Promega Co. (Madison, WI).
Preparation of Hb Solutions
Hb solutions were prepared as described earlier [11].
Peptide Design
The first 7 residues of human erythrocytic band 3 protein,
AcM-E-E-L-Q-D-D, were used to serve as a dynamic
effector for oxygen release of Hb [10]. In order to
conjugate the peptide with Hb, the residues were added
sequentially with cysteine and glutamate at C terminus.
Cysteine in the peptide is the site for crosslinking with
maleimide in MAL-PEG-NHS and glutamate is for
increasing the surface probability of cysteine according
to the Emini method in Seqweb (version 3.1, Accelrys,
Madison, WI).
Preparation of Peptide-PEG-Hb
The following procedures were maintained in anaerobic
conditions by purging reagents with argon. 0.383 mg of
peptide in 0.2 ml HEPES buffer (5 mM HEPES, pH 7)
reacted with 1.13 mg MAL-PEG-NHS (molar ratio of
peptide to MAL-PEG-NHS1:1) for 3 min at room
temperature and then cysteine was added to a final
concentration of 1 mM to consume the residual MAL
function group. The resulting peptide-PEG-NHS solution
was immediately injected into 1 ml of 277 mM deoxyHb
(molar ratio of peptide-PEG-NHS to deoxyHb1.2:1) in
HEPES buffer (8.3 g/L NaCl and 20 mM HEPES, pH 8)
using a gas-tight syringe and the reaction was performed
at room temperature for 1 h. The reaction mixture was
dialyzed 3 times with 10 mM pH 6.5 potassium phosphate
buffer and fractionated by a CM-sepharose column (0.7
14.5 cm) using 10 ml of 100-190 mM NaCl gradient and
15 ml 200 mM NaCl. The Hb concentration in each
fraction (0.5 ml) was measured by a UV/Vis spectro-
photometer at 542 nm (Cary 50, Varian, Palo Alto, CA).
Similarly, peptide-PEG-cysteine was prepared using this
protocol except MAL-PEG-NHS reacting exclusively
with cysteine instead of peptide.
Polymerization of Peptide-PEG-Hb with SPA-PEG-
SPA
The peptide-PEG-Hb solution was mixed with unmodi-
fied Hb in a molar ratio of 1:1 at room temperature for 1 h
and then this mixture was dialyzed 3 times with HEPES
buffer (8.3 g/L NaCl and 20 mM HEPES, pH 8). 0.342
mg of SPA-PEG-SPA dissolved in 100 mL deoxygenated
HEPES buffer (5 mM HEPES, pH 7) was injected into the
argon-purged Hb mixture using a gas-tight syringe in a
molar ratio 8:1 and the reaction was carried out at room
temperature for 1 h. The degree of Hb polymerization was
accessed using SDS-polyacrylamide gel electrophoresis.
SDS-polyacrylamide Gel Electrophoresis
Each lane of gel was loaded with 3 mg of Hb and the
electrophoresis was carried out using 5 and 15% of
polyacrylamide in the stacking and separating gels,
respectively.
Mass Spectrometry of Peptide-PEG-Hb
The matrix-assisted laser desorption/ionization time of
flight mass spectrometry (MALDI-TOF MS) was
N
O
O
O C
O
PEG N
O
O
NHS-PEG-MAL
+ SH
pH 7
N
O
O
O C
O
PEG N
O
O
H
S peptide
pH 8
C
O
PEG N
O
O
H
S peptide
N
O
O
OH
Hb-CH
2
-CH
2
-CH
2
-CH
2
-NH
+
peptide
Hb-CH
2
-CH
2
-CH
2
-CH
2
-NH
2
Figure 1. Schematic representation of peptide-PEG-Hb synth-
esis. Peptide in HEPES buffer (5 mM HEPES, pH 7) reacted to
MAL-PEG-NHS for 3 min at room temperature. The molar ratio
is 1:1 in anaerobic conditions. Using a 1.2:1 molar ratio of
peptide-PEG solution to deoxyhemoglobin in HEPES buffer (8.3
g/L NaCl and 20 mM HEPES, pH 8) was incubated in room
temperature for 1 h. MAL and NHS are the functional groups in
PEG for crosslinking with cysteine in the peptide and lysine in
the outer surface of hemoglobin, respectively.
Peptide-hemoglobin Conjugate 33
equipped with the linear mode of a nitrogen laser (VSL-
337, 337 nm, 3 ns pulse) and the accelerating voltage in
the positive-ion source was 25 kV. The MS spectra were
secured by a Voyager DE-PRO Biospectrometry Work-
station (Applied Biosystems). The Hb solutions were
dialyzed with deionized water 3 times to remove salts.
1 mL of 14 mM (in heme) Hb solution was mixed with
10 mL of sinapinic acid (44.6 mM in 70% (v/v)
acetonitrile buffer) and the mixture was measured by
MALDI-TOF MS after air-drying at room temperature
[12].
Two Dimensional Gel Electrophoresis (2D-GE)
The first dimensional of 2D-GE was carried out in 7 cm of
pH 3-10 linear gradient IEF gel. The gel was loaded with
10 mg of desalted Hb protein and rehydrated in a
rehydration buffer (8 M urea, 2% 3-[(3-cholamidopro-
pyl)dimethylammonio]-1-propanesulfonate (CHAPS), 50
mM dithiothreitol (DTT) and 0.2% Bio-Lyte ampholytes)
at 50 V, 20 8C for 1216 h. After rehydration, the IEF gel
was run with 250 V for 15 min, 2504000 V for 2 h, and
40005500 V-h. After isoelectric focusing, the strip was
removed and sequentially equilibrated 10 min with buffer
I (6 M urea, 0.375 M Tris, pH 8.8, 2% SDS, 20%
glycerol, 2.5% (w/v) DDT) and 10 min with buffer II (6
M urea, 0.375 M Tris, pH 8.8, 2% SDS, 20% glycerol,
2.5% (w/v) iodoacetamide). 15% polyacrylamide gel was
used for the second dimension of 2D-GE. After electro-
phoresis, the gel was stained with Coomassie brilliant
blue R-250.
Liquid Chromatography-tandem Mass Spectrometry
(LC-MS/MS)
50 mL of 3.5 mM (use 11.28 mg) desalted Hb was digested
at 378C for 16 h using 5 mg of modified trypsin in 25 mM
ammonium bicarbonate. 20 mL of the resulting peptide
was mixed with 20 mL of 5% ACN/0.1% formic acid and
provided to MS analysis for peptide identification. Nano-
HPLC-ESI-MS/MS was performed to determine the site
of peptide cross-linking in Hb. C
18
microcapillary column
(75 mm15 cm) was used in the nano-HPLC system (LC
Packings, Netherlands) and the mobile phase consisted of
buffer A (5% v/v ACN/0.1% v/v formic acid) and buffer
B (80% v/v ACN/0.1% v/v formic acid) with 40 min
linear gradient from 100% to 40% buffer A and 0% to
60% buffer B, 10 min 0% to 100% buffer A, and 20 min
100% buffer A at 200 nL/min flow rate. An ion trap mass
spectrometer (LCQ DECA XP Plus, ThermoFinnigan,
San Jose, CA) was coupled to the nano-HPLC system,
which was equipped with an ESI source. The performing
parameters were 1.3 kV of spraying voltage and 2008C of
heating capillary temperature [13].
Oxygen Dissociation Curve
The oxygen dissociation curve was obtained by the
protocatechuic acid (PCA)/ protocatechuic acid 3,4-
dioxygenase (PCD) system proposed by Vandegriff
[14]. Solution conditions were 10 mM (in heme) of
various Hbs, 8.3 g/L NaCl, 20 mM HEPES pH 7.4, and
1 mM EDTA. The data were analyzed using singular
value decomposition (SVD) algorithm against oxy-,
deoxy-, and metHb standard spectra to estimate the
amount of oxy-, deoxy-, and metHb in the solution.
PCA and PCD were added to the Hb solution at final
concentrations of 1.4 mM and 0.06 units/mL, respec-
tively.
RESULTS
Synthesis of Peptide-PEG-Hb
When the N-acetyl peptide MEELQDDYC was synthe-
sized, corresponding to the first 8 residues of band 3 and
an added cysteine at the C-terminus for the crosslinking
purpose, the cysteine was not able to react with MAL-
PEG-NHS, due likely to the lower surface probability of
cysteine in solution. To increase the reactivity of cysteine
in peptide, we estimated the surface probability of
cysteine by the Emini method in Seqweb (version 3.1,
Accelrys, Madison, WI). The surface probability of
cysteine in peptide can be increased from 0.672 for
MEELQDDYC to 0.716, 0.935, and 0.970 for
MEELQDDC, MEELQDDCD, and MEELQDDCE, re-
spectively. Therefore, the N-acetyl peptide
MEELQDDCE was selected as the Hb oxygen modulator
in our following studies due to its higher surface
probability.
Figure 2A shows the elution profile of the Hb
conjugates from a CM-sepharose column. The peaks
were analyzed using SDS-polyacrylamide gel and the first
two peaks were due to conjugated Hb and followed by an
unmodified Hb peak as shown in Figure 2B. The ratio of
peptide conjugation to Hb was about 10% in the
unpurified Hb mixture. The peptide-PEG conjugated Hb
a or b chain in Peak 1 and 2 exhibited a diffused band at
molecular weight around 26 kD. Notably, the ratios of
conjugated to unmodified Hb monomer in Peak 1 and 2
are, respectively, 0.9490.04 and 0.6890.02, indicating
that the Hbs eluted from the CM-sepharose column were
34 Yen-Lin Lin and Kuang-Tse Huang
primarily dimers. Here, we denoted Peak 1 and 2 as
peptide-PEG-Hb-1 and peptide-PEG-Hb-2.
Characterization of Peptide-PEG Conjugated Hb
To characterize the conjugation of peptide-PEG to Hb, the
molecular weights of Hb fractions were first analyzed
using MALDI-TOF MS. The molecular weight of MAL-
PEG and the peptide are 3,500 Da and 1,153 Da,
respectively. Therefore, the theoretical molecular weights
of peptide-PEG-conjugated a and b chain become
19,710 Da and 20,500 Da, respectively. Figure 3A
shows the MS spectrum of unmodified Hb, which
contains two major peaks corresponding to the a chain
at 15,068.46 Da and the b chain at 15,810.63 Da and three
minor peaks around 30,901 Da corresponding to the Hb
dimers. Peptide-PEG-Hb-1 has the peaks of unmodified
Hb but the relative intensity of b chain with respect to a
chain is lower as compared with unmodified Hb (Figure
3A and B), indicating that the peptide-PEG was primarily
linked to the b chain of Hb in peptide-PEG-Hb-1.
0 10 20 30 40
0
0.4
0.8
1.2
1.6
2.0
Fraction
O
D
,

5
4
2
n
m
peak1
(A)
(B)
peak2
peak3
72 kD
55 kD
43 kD
34 kD
26 kD
17 kD
170 kD
130 kD
95 kD
Hb m-Hb peak1 peak2 peak3
Figure 2. CM-sepharose elution prole and SDS-polyacryla-
mide gel electrophoresis of the peptide-PEG-Hb mixture. (A)
Peptide-PEG-Hb was separated by ion exchange chromatogra-
phy on a CM-sepharose column and monitored at 542 nm. The
CM-sepharose column was eluted with each 1 mL, 100190 mM
NaCl difference 10 mM gradient, and later was eluted with 15
mL, 200 mM NaCl and collected 0.5 mL in each fraction. The
fraction 1013, fraction 1821, and fraction 2628 were
collected as peak1, peak2, and peak3, respectively. (B) 15%
SDS-polyacrylamide gel was used to characterized various Hbs.
Hb: unmodied Hb; m-Hb: unpuried peptide-PEG-Hb; peak1-
3: same as Figure 2A.
20490.08
15054.16
15847.89
30937.07
35722.75
12000
8000
4000
0
I
n
t
e
n
s
i
t
y
I
n
t
e
n
s
i
t
y
Mass (m/z)
16000
(A)
(B)
(C)
12000
8000
4000
0
15068.46
15810.63
30901.12
30128.67
10000 20000 30000 40000
Mass (m/z)
10000 20000 30000 40000
Mass (m/z)
10000 20000 30000 40000
12000
8000
4000
0
I
n
t
e
n
s
i
t
y
15055.47
15889.55
20044.88
30974.20
35689.65
Figure 3. MALDI-TOF mass spectra of various Hbs. Various
Hbs were dialyzed against deionized water 3 times to remove
salt before analyzing by a MALDI-TOF mass spectrometer. (A):
unmodied Hb; (B): peptide-PEG-Hb-1; (C): peptide-PEG-Hb-
2.
Peptide-hemoglobin Conjugate 35
In addition, there is a significant extra broader peak at
20,490.08 Da, which is close to the theoretical molecular
weight of peptide-PEG-conjugated b chain 20,500 Da,
further confirming the primary b chain conjugation. On
the other hand, the broader peak in peptide-PEG-Hb-2 is
at 20044.88 Da, which represents a Hb mixture with a
similar amount of a and b chain conjugated with the
peptide (Figure 3C).
In Figure 4A, the pI of unmodified Hb a and b chain
are 9.5 and 8.0, respectively. The synthetic peptide
contains five acidic amino acids. When conjugated to
Hb, the peptide shifts the pI of Hb a and b chain to a
lower pH range with a greater change in peptide-PEG-Hb-
1 than in peptide-PEG-Hb-2 as shown in Figure 4B
and C.
Identification of the Conjugation Site of Hb
To identify the sites of peptide-PEG conjugation on Hb,
peptide-PEG-Hb-1 and peptide-PEG-Hb-2 were trypsi-
nized and analyzed by LC-MS/MS equipped with a C
18
microcapillary column. Peak 2 and 9 in the LC profile of
trypsinized unmodified Hb (Figure 5A) were shifted to the
left as compared to that for peptide-PEG-Hb-1 (Figure 5B),
pI 3 10
(A)
pI 3 10
(B)
pI 3 10
(C)

Figure 4. Two dimensional gel electrophoresis of various Hbs.

The rst dimensional was carried out in 7 cm, pH 3-10 linear
gradient IEF gel and the second dimensional was performed
using 15% polyacrylamide gels and followed by staining with
the Coomassie brilliant blue R-250. (A): unmodied Hb; (B):
peptide-PEG-Hb-1; (C): peptide-PEG-Hb-2.
Time (min)
(C)
1
3
4
5
6
7
Time (min)
(B)
1
3
4
5
6
7
R
e
l
a
t
i
v
e

A
b
o
u
n
d
a
n
c
e
Time (min)
8
1
2
3
4
5
6
7
8
8
9
10
10
10
9
(A)
0
20
40
60
80
100
R
e
l
a
t
i
v
e

A
b
o
u
n
d
a
n
c
e
0
20
40
60
80
100
R
e
l
a
t
i
v
e

A
b
o
u
n
d
a
n
c
e
0
20
40
60
80
100
I
I
2
9
20 25 30 35 40
20 25 30 35
20 25 30 35 40
Figure 5. LC-MS/MS chromatographic proles of the digestion
products of various Hbs using TPCK-treated trypsin. (A):
unmodied Hb; (B): peptide-PEG-Hb-1; (C): peptide-PEG-Hb-
2. The peptide sequences for the corresponding peaks are listed
in Table 1.
36 Yen-Lin Lin and Kuang-Tse Huang
suggesting their conjugation with hydrophilic peptide-
PEG. On the other hand, the chromatographic profile for
peptide-PEG-Hb-2 fragments does not show any apparent
peak shift (Figure 5C). The amino acid sequences of the
peaks in the LC profiles were determined according to b
and y ion series in tandem MS as shown in Table 1. In
particular, Peak 2 and 9 represent for Hb b
83-95
and a
6190
,
indicating that the sites of peptide-PEG conjugation in
peptide-PEG-Hb-1 probably locate, at least, at Lys-b95 and
Lys-a61.
Polymerization of Peptide-PEG-Hb
One drawback of Hb solution as an oxygen carrier is the
cause of kidney damage by Hb dimers. To lower down
the formation of Hb dimer, SPA-PEG-SPA was used to
crosslink intra- and inter-Hb molecules. Peptide-PEG-Hb
after CM-sepharose purification was primary dimers, so
we first incubated peptide-PEG-Hb-1 and peptide-PEG-
Hb-2 with unmodified Hb in a molar ratio of 1:1 and then
polymerized the Hb mixture with SPA-PEG-SPA, form-
ing SPA-peptide-PEG-Hb-1 and SPA-peptide-PEG-Hb-2,
respectively. The degree of inter-molecule crosslink was
analyzed by 15% SDS-polyacrylamide gel as shown in
Figure 6. The percentage of proteins with molecular
weight higher than Hb dimers in the polymerization
mixture in peptide-PEG-Hb-1 and peptide-PEG-Hb-2 are
around 22%.
Oxygen Dissociation Curve of Peptide-PEG-Hb
To test the function of the conjugated peptide in the
modulation of Hb oxygen release, oxygen dissociation
curves for various Hb solutions were obtained by the
PCA/PCD system with the simultaneous measurements of
the Hb spectra and dissolved oxygen concentration
(Figure 7) [14]. Peptide-PEG-Hb-1/unmodified Hb (1:1)
and SPA-peptide-PEG-Hb-1 show a significant rightward
shift of their oxygen dissociation curves in the range of
the oxygen partial pressure higher than 10 mmHg (Figure
7A) and have a lower Hill coefficient (Table 2). On the
Table 1. The peptide sequences of the chromatographic peaks in LC proles of trypsinized Hbs
Peaks Ions
a
Residue Sequence
b
1 658.2 b
18-30
VNVDEVGGEALGR
690.06 b
121-132
EFTPPVQAAYQK
2 711.47 b
83-95
GTFATLSELHCDK
3 466.96 b
9-17
SAVTALWGK
4 612.4 a
41-56
TYFPHFDLSHGSAQVK
5 627.2 a
128-139
FLASVSTVLTSK
6 536.7 a
32-40
MFLSFPTTK
I 843.96 b
83-104
GTFATLSELHCDKLHVDPENFR
7 638.2 b
31-40
LLVVYPWTQR
835.67 b
67-82
VLGAFSDGLAHLDNLK
8 1030.6 b
41-59
FFESFGDLSTPDAVMGNPK
9 782.5 a
61-90
KVADALTNAVAHVDDMPNALSALSDLHAHK
10 999.9 a
62-90
VADALTNAVAHVDDMPNALSALSDLHAHK
a
Observed mass-to-charge ratios.
b
Elucidated from observed mass-to-charge ratios b- and y-ions of tandem MS
spectra.
170 kD
130 kD
95 kD
72 kD
55 kD
43 kD
34 kD
26 kD
17 kD
SPA-1 SPA-2
Figure 6. SDS-polyacrylamide gel of polymerization products
of peptide-PEG-Hb with SPA-PEG-SPA. Peptide-PEG-Hb and
unmodied Hb was mixed in a molar ratio of 1:1 for 1 h. SPA-
PEG-SPA in HEPES buffer (5 mM HEPES, pH 7) were then
added to the Hb mixture in a molar ratio of 8:1. SPA-1 and SPA-
2: polymerization of peptide-PEG-Hb-1/Hb and peptide-PEG-
Hb-2/Hb with SPA-PEG-SPA, respectively.
Peptide-hemoglobin Conjugate 37
other hand, peptide-PEG-Hb-2 and SPA-peptide-PEG-
Hb-2 do not show any significant change in oxygen
affinity and co-operativity (Figure 7A insert and Table 2).
Using cysteine instead of peptide as an allosteric effector
can also decrease the oxygen affinity of Hb, but the extent
is smaller than peptide-PEG-Hb-1 (Figure 7B).
DISCUSSION
In this study, we described a novel approach to Hb
modification by conjugation with a synthetic peptide
corresponding to the first N-terminal 7 residues of red cell
membrane band 3 protein and two additional amino acids
followed, cysteine and glutamine, using a bifunctional
PEG crosslinker. The synthetic peptide and PEG are,
respectively, served as an allosteric effector and a spacer
arm for dynamically modulating Hbs oxygen affinity.
The putative conjugation sites on Hb are characterized as
Lys-b95 and Lys-a61 by LC-MS/MS. Furthermore, this
conjugated 9-residue synthetic peptide remains effective
on lowering Hbs oxygen affinity after Hb polymerization
by another PEG crosslinker. Taken together, our results
demonstrate that the equipment of Hb with a flexible
allosteric effector is practicable and may exhibit a better
oxygen release behavior.
Previous studies identified that the N-terminal 5 to 7
residues of band 3 being the binding site for deoxyHb can
lower Hb oxygen affinity [10]. To study effects of
conjugated peptide on Hb oxygen dissociation, we chose
cysteine and lysine as the conjugation sites for peptide
and Hb, respectively. Notably, cysteine is generally
considered to be hydrophobic and is uncommonly found
on the surface of a protein or peptide. Therefore, it is often
necessary to increase the surface probability of cysteine
on the peptide design to enhance its reactivity with the
crosslinker. In our study, we found that when monitored
by Ellmans reaction, the extent of reaction between the
synthetic peptide and MAL-PEG-NHS at room tempera-
ture (pH 7.0) could reach 80% within 3 min after
optimizing the surface probability of cysteine in the
peptide. On the other hand, the potential sites on Hb for
conjugation using NHS are the 4 N termini, 11 lysines in
a chain, and 11 in b chain. Due to a low pKa relative to
the o-amino group, the N termini have a higher reactivity
with crosslinkers than lysine [15]. However, the usage of
the N termini of b chain may hinder the binding of
allosteric effector and reduce the stabilization of T state.
To reduce the reactivity of N termini with NHS in
S
a
t
u
r
a
t
i
o
n
0 20 40 60 80 100
0.0
0.2
0.4
0.6
0.8
1.0
unmodified Hb
peptide-PEG-Hb-1
SPA-peptide-PEG-Hb-1
Hb + 5mM 2,3-DPG
0 20 40 60 80 100
0.0
0.2
0.4
0.6
0.8
1.0
S
a
t
u
r
a
t
i
o
n
unmodified Hb
peptide-PEG-Hb-2
SPA-peptide-PEG-Hb-2
0 20 40 60 80 100
0.0
0.2
0.4
0.6
0.8
1.0
Po
2
(mmHg)
unmodified Hb
peptide-PEG-Hb
cysteine-PEG-Hb
S
a
t
u
r
a
t
i
o
n
(A)
Po
2
(mmHg)
Po
2
(mmHg)
(B)
Figure 7. Oxygen dissociation curves for various Hbs. The
oxygen dissociation curve was obtained using the PCA (1.4
mM)/PCD (0.06 units/ml) enzyme system. 10 M (in heme) of
various Hbs was dissolved in the buffer containing 8.3 g/L NaCl,
20 mM HEPES pH 7.4, and 1 mM EDTA. The data were
analyzed using SVD algorithm against oxy-, deoxy-, and metHb
standard spectra. (A) unmodied Hb (squares), peptide-PEG-
Hb-1/Hb (molar ratio 1:1) (circles), SPA-peptide-PEG-Hb-1
(triangles), and unmodied Hb5 mM 2,3-DPG (inverse
triangles). The inset shows unmodied Hb (squares), peptide-
PEG-Hb-2/Hb (molar ratio 1:1) (circles), SPA-peptide-PEG-Hb-
2 (triangles). (B) unmodied Hb (squares), peptide-PEG-Hb-1/
Hb (molar ratio 1:1) (circles), cysteine-PEG-Hb/Hb (molar ratio
1:1) (triangles).
Table 2. Hill coefcients of various Hbs
Hemoglobin n
max
a
Hb 2.91
peptide-PEG-Hb-1 2.25
peptide-PEG-Hb-2 3.02
SPA-peptide-PEG-Hb-1 2.14
SPA-peptide-PEG-Hb-2 2.97
Hb5mM 2,3-DPG 3.30
cysteine-PEG-Hb 2.97
a
n
max
, the max value of Hill coefficient.
38 Yen-Lin Lin and Kuang-Tse Huang
peptide-PEG-NHS, the conjugation reaction was per-
formed under anaerobic condition.
Peptide-PEG-Hb-1 eluted earlier than peptide-PEG-
Hb-2 from a CM-sepharose column as shown in Figure
2A, indicating a higher exposure probability of the
negative charges of the conjugated peptide in peptide-
PEG-Hb-1 than in peptide-PEG-Hb-2. This result may
explain the effectiveness of the conjugated peptide in
peptide-PEG-Hb-1 on modulation of Hbs oxygen affinity
and is consistent with the lower average pI value for
peptide-PEG-Hb-1 as compared to that for peptide-PEG-
Hb-2 (Figure 4).
The ratio of conjugated to unconjugated Hb a or b
chain was close to 1 in peptide-PEG-Hb-1 (Figure 2B),
demonstrating that peptide-PEG-Hb-1 exists primarily as
dimers. This result is consistent with other PEGylated Hbs
[15,16]. The molecular basis for enhancing the formation
of Hb dimers upon purification of PEGylated Hb using a
CM sepharose column is still unclear. Hu et al. proposed
that the hydrated PEG around Hb may shield the charge
of a and b chains, decrease the electrostatic attractions for
the a1b2 interface, and result in the formation of Hb
dimmers [16]. On the other hand, evidence shows that
binding of band 3 to oxyHb promotes dimer formation
[17]. In the case of peptide-PEG-Hb-1, the acidic peptide
derived from band 3 N terminus may bind to positively
charged a subunits [18] in oxy state and in turn increase
dimer formation.
Different from free PEG, the conjugated PEG
increases the oxygen affinity of Hb [15,19]. Colombo
et al. calculated from the data of p
50
and osmotic pressure
that the number of bound water molecules in oxyHb is
60 more than that in deoxyHb. The existence of free
PEG favors the release of water during R0T transition
and results in the increase in p
50
. In contrast, the water
molecules liberated during R0T transition in PEGylated
Hb may require doing more work on pushing the
conjugated PEG away. In the absence of allosteric
effector, this work seems greater than the energy released
by the osmotic work of conjugated PEG and the
formation of salt bridges in T state. However, when
additional ionic interactions formed between the acidic
peptide and the positively charged group within the 2,3-
DPG binding site during R0T transition such as in
peptide-PEG-Hb-1, this will overcome the hindrance of
conjugated PEG and stabilize the Hb in T state.
The effective length of PEG in the random coil
conformation is defined by the Flory dimension, R
F

aN
3/5
, where a3.5 A

is the length of an oxyethylene

unit and N is the degree of polymerization [20]. The
molecular weight of the PEG we used is around 3400 Da,
which corresponds to N 77 and R
F
47 A

. The linear
distances from Lys 82b
2
to Lys 95b
2
, Lys 95b
1
, Lys
61a
2
, and Lys 61a
1
are 24.1, 32.5, 37.8, and 42.4 A

,
respectively (Figure 8). As the average diameter of Hb
containing the above residues is 52.5 A

, so the arc
distances from Lys 82b
2
to Lys 95b
2
, Lys 95b
1
, Lys
61a
2
, and Lys 61a
1
become 25.0, 35.0, 42.2, and 49.4 A

,
respectively. Svergun et al. estimated the length of the
PEG chains on the Hb with two PEG grafted as 60% of
their full Flory dimension [21]. If we apply Sverguns
observation, the length of our PEG on the Hb would
become 28 A

. Therefore, the peptide conjugated to Lys

b95 is more likely the allosteric effector for Hb oxygen
release than that to Lys a61.
In summary, we have demonstrated that when
conjugated to Hb by an activated PEG, a synthetic
peptide derived from the first N-terminal 7 residues of
erythrocytic band 3 protein can serve as an allosteric
effector for Hb oxygen release. The putative conjugation
site on Hb for effective reduction of oxygen affinity is
characterized as Lys-b95 by LC-MS/MS. The conjugated
synthetic peptide remains able to help the Hb oxygen
release after Hb polymerized by another PEG crosslinker.
This study has provided an alternative choice to solve the
poor oxygen release from Hb solution and further
suggests the potential target site for direct mutagenesis
of Hb to specifically conjugate the peptide-derived
allosteric effector.
Figure 8. The distances of possible conjugation locations of the
peptide to the 2,3-DPG binding site. This graph and measure-
ments were generated using The PyMOL Molecular Graphics
System [22] based on the deoxyHb structure (3HHB).
Peptide-hemoglobin Conjugate 39
ACKNOWLEDGEMENT
We are grateful to the National Center for High-
performance Computing, Taiwan, for computer time and
facilities. This work was supported by grants NSC 92-
2320-B-194-003, NSC 93-2320-B-194-003, and NSC 96-
2221-E-194-037 from the National Science Council,
Taiwan.
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This paper was first published online on iFirst on 8 January 2009.
40 Yen-Lin Lin and Kuang-Tse Huang