36561380 blood

Hemoglobin Conjugated with a Band 3 N-terminusDerived Peptide as an Oxygen Carrier

Yen-Lin Lin and Kuang-Tse HuangDepartment 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-tandemmass 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]. Regardingthe 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 underanaerobic 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: [email protected]

Artificial Cells, Blood Substitutes, and Biotechnology, 37: 3240Copyright # 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 roomtemperature 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) inHEPES 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 (0.714.5 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 deoxygenatedHEPES 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 theelectrophoresis 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

ONHS-PEG-MAL

+ SHpH 7

N

O

O

O C

O

PEG N

O

O

H

S peptidepH 8

CO

PEG N

O

O

H

S peptide

N

O

O

OHHb -CH2-CH2-CH2-CH2-NH+

peptide

Hb -CH2-CH2-CH2-CH2-NH2

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 with10 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 arehydration 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 gelwas run with 250 V for 15 min, 2504000 V for 2 h, and40005500 V-h. After isoelectric focusing, the strip wasremoved 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 digestedat 378C for 16 h using 5 mg of modified trypsin in 25 mMammonium bicarbonate. 20 mL of the resulting peptidewas mixed with 20 mL of 5% ACN/0.1% formic acid andprovided to MS analysis for peptide identification. Nano-

HPLC-ESI-MS/MS was performed to determine the site

of peptide cross-linking in Hb. C18 microcapillary column

(75 mm15 cm) was used in the nano-HPLC system (LCPackings, 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 ofheating 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) ofvarious 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 atmolecular 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, indicatingthat 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 3Ashows the MS spectrum of unmodified Hb, which

contains two major peaks corresponding to the a chainat 15,068.46 Da and the b chain at 15,810.63 Da and threeminor 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 achain 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

OD,

542

nm

peak1

(A)

(B)

peak2

peak3

72 kD55 kD43 kD

34 kD

26 kD

17 kD

170 kD130 kD

95 kD

Hb m-Hb peak1 peak2 peak3

Figure 2. CM-sepharose elution profile 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 mMNaCl 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 werecollected as peak1, peak2, and peak3, respectively. (B) 15%

SDS-polyacrylamide gel was used to characterized various Hbs.

Hb: unmodified Hb; m-Hb: unpurified peptide-PEG-Hb; peak1-

3: same as Figure 2A.

20490.08

15054.16

15847.89

30937.0735722.75

12000

8000

4000

0

Inte

nsity

Inte

nsity

Mass (m/z)

16000(A)

(B)

(C)

12000

8000

4000

0

15068.46

15810.63

30901.1230128.67

10000 20000 30000 40000

Mass (m/z)10000 20000 30000 40000

Mass (m/z)10000 20000 30000 40000

12000

8000

4000

0

Inte

nsity

15055.47

15889.55

20044.88 30974.2035689.65

Figure 3. MALDI-TOF mass spectra of various Hbs. VariousHbs were dialyzed against deionized water 3 times to remove

salt before analyzing by a MALDI-TOF mass spectrometer. (A):

unmodified Hb; (B): peptide-PEG-Hb-1; (C): peptide-PEG-Hb-

2.


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. Onthe 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 thepeptide (Figure 3C).

In Figure 4A, the pI of unmodified Hb a and b chainare 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 alower 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 C18microcapillary 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 first 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): unmodified Hb; (B):

peptide-PEG-Hb-1; (C): peptide-PEG-Hb-2.

Time (min)

(C)

1

3

45

67

Time (min)

(B)

1

34 5 6

7R

elat

ive

Abou

ndan

ce

Time (min)

8

1

2 34 5 6

7

8

8

910

10

109

(A)

0

20

40

60

80

100

Rel

ativ

e Ab

ound

ance

0

20

40

60

80

100

Rel

ativ

e Ab

ound

ance

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 profiles of the digestionproducts of various Hbs using TPCK-treated trypsin. (A):

unmodified Hb; (B): peptide-PEG-Hb-1; (C): peptide-PEG-Hb-

2. The peptide sequences for the corresponding peaks are listed

in Table 1.


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 band y ion series in tandem MS as shown in Table 1. Inparticular, Peak 2 and 9 represent for Hb b83-95 and a6190,indicating that the sites of peptide-PEG conjugation in

peptide-PEG-Hb-1 probably locate, at least, at Lys-b95 andLys-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 profiles of trypsinized Hbs

Peaks Ionsa Residue Sequenceb

1 658.2 b18-30 VNVDEVGGEALGR690.06 b121-132 EFTPPVQAAYQK

2 711.47 b83-95 GTFATLSELHCDK3 466.96 b9-17 SAVTALWGK4 612.4 a41-56 TYFPHFDLSHGSAQVK5 627.2 a128-139 FLASVSTVLTSK6 536.7 a32-40 MFLSFPTTKI 843.96 b83-104 GTFATLSELHCDKLHVDPENFR7 638.2 b31-40 LLVVYPWTQR

835.67 b67-82 VLGAFSDGLAHLDNLK8 1030.6 b41-59 FFESFGDLSTPDAVMGNPK9 782.5 a61-90 KVADALTNAVAHVDDMPNALSALSDLHAHK10 999.9 a62-90 VADALTNAVAHVDDMPNALSALSDLHAHK

a Observed mass-to-charge ratios.b Elucidated from observed mass-to-charge ratios b- and y-ions of tandem MS

spectra.

170 kD130 kD

95 kD72 kD55 kD43 kD

34 kD

26 kD

17 kD

SPA-1 SPA-2

Figure 6. SDS-polyacrylamide gel of polymerization productsof peptide-PEG-Hb with SPA-PEG-SPA. Peptide-PEG-Hb and

unmodified 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.


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, thisconjugated 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 tothe o-amino group, the N termini have a higher reactivitywith crosslinkers than lysine [15]. However, the usage of

the N termini of b chain may hinder the binding ofallosteric effector and reduce the stabilization of T state.

To reduce the reactivity of N termini with NHS in

Satu

ratio

n

0 20 40 60 80 1000.0

0.2

0.4

0.6

0.8

1.0unmodified Hbpeptide-PEG-Hb-1SPA-peptide-PEG-Hb-1Hb + 5mM 2,3-DPG

0 20 40 60 80 1000.0

0.2

0.4

0.6

0.8

1.0

Satu

ratio

n

unmodified Hbpeptide-PEG-Hb-2SPA-peptide-PEG-Hb-2

0 20 40 60 80 1000.0

0.2

0.4

0.6

0.8

1.0

Po2(mmHg)

unmodified Hbpeptide-PEG-Hbcysteine-PEG-Hb

Satu

ratio

n

(A)

Po2(mmHg)

Po2(mmHg)

(B)

Figure 7. Oxygen dissociation curves for various Hbs. Theoxygen 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) unmodified Hb (squares), peptide-PEG-

Hb-1/Hb (molar ratio 1:1) (circles), SPA-peptide-PEG-Hb-1

(triangles), and unmodified Hb5 mM 2,3-DPG (inversetriangles). The inset shows unmodified Hb (squares), peptide-

PEG-Hb-2/Hb (molar ratio 1:1) (circles), SPA-peptide-PEG-Hb-

2 (triangles). (B) unmodified Hb (squares), peptide-PEG-Hb-1/

Hb (molar ratio 1:1) (circles), cysteine-PEG-Hb/Hb (molar ratio

1:1) (triangles).

Table 2. Hill coefficients of various Hbs

Hemoglobin nmaxa

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 3.30cysteine-PEG-Hb 2.97

a nmax, the max value of Hill coefficient.


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 bchain 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 forthe a1b2 interface, and result in the formation of Hbdimmers [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 increasedimer formation.

Different from free PEG, the conjugated PEG

increases the oxygen affinity of Hb [15,19]. Colombo

et al. calculated from the data of p50 and osmotic pressurethat the number of bound water molecules in oxyHb is

60 more than that in deoxyHb. The existence of freePEG favors the release of water during R0T transitionand results in the increase in p50. In contrast, the watermolecules liberated during R0T transition in PEGylatedHb 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 inpeptide-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, RFaN3/5, where a3.5 A is the length of an oxyethyleneunit 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 RF47 A. The lineardistances from Lys 82b2 to Lys 95b2, Lys 95b1, Lys61a2, and Lys 61a1 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 82b2 to Lys 95b2, Lys 95b1, Lys61a2, and Lys 61a1 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% oftheir 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 Lysb95 is more likely the allosteric effector for Hb oxygenrelease 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 conjugatedsynthetic 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).


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.


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