Biochimica et Biophysica Acta 1844 (2014) 1201–1207

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Biochimica et Biophysica Acta

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Reversible protection of Cys-93(β) by PEG alters the structural andfunctional properties of the PEGylated hemoglobin

Qingqing Wang a,b, Lijing Sun a, Shaoyang Ji a, Dawei Zhao a,b, Jiaxin Liu c, Zhiguo Su a,d,⁎, Tao Hu a,⁎⁎a National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, Chinab Graduate University of Chinese Academy of Sciences, Beijing 100049, Chinac Institute of Blood Transfusion, Chinese Academy of Medical Sciences, Chengdu 610052, Sichuan Province, Chinad Collaborative Innovation Center of Chemical Science and Engineering, Tianjin, China

Abbreviations: Hb, hemoglobin; PEG, polyethylene gwith polyethylene glycol; PDPH, 3-(2-pyridyldithio)propibuffered saline; TFA, trifluoroacetic acid; HBOC, hemoglopartial oxygen pressure at half saturation; SEC, size exHPLC, reverse phase high performance liquid chromatogrHill coefficient; 4-PDS, 4,4′-dithiopyridine; TCEP, tris(2-cSC, PEG-succinimidyl carbonate⁎ Correspondence to: Z. Su, National Key Laborator

Institute of Process Engineering, Chinese Academy of SciHaidian District, Beijing 100190, China. Tel.: +86 10 6256⁎⁎ Correspondence to: T. Hu, National Key LaboratorInstitute of Process Engineering, Chinese Academy of SciHaidian District, Beijing 100190, China. Tel.: +86 10 6255

E-mail addresses: zgsu@home.ipe.ac.cn (Z. Su), thu@ip

http://dx.doi.org/10.1016/j.bbapap.2014.04.0051570-9639/© 2014 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 16 January 2014Received in revised form 2 April 2014Accepted 7 April 2014Available online 15 April 2014

Keywords:PEGylationHemoglobinAutoxidationTetramer stability

As a potential hemoglobin (Hb)-based oxygen carrier (HBOC), the PEGylated Hb has receivedmuch attention forits non-nephrotoxicity. However, PEGylation can adversely alter the structural and functional properties of Hb.The site of PEGylation is an important factor to determine the structure and function of the PEGylated Hb.Thus, protection of some sensitive residues of Hb from PEGylation is of great significance to develop thePEGylated Hb as HBOC. Here, Cys-93(β) of Hb was conjugated with 20 kDa polyethylene glycol (PEG20K)through hydrazone and disulfide bonds. Then, the conjugate was modified with PEG5K succinimidyl carbonate(PEG5K-SC) using acylation chemistry, followed by removal of PEG20K Hbwith hydrazone hydrolysis and disul-fide reduction. Reversible conjugation of PEG20K at Cys-93(β) can protect Lys-95(β), Val-1(α) and Lys-16(α) ofHb from PEGylation with PEG5K-SC. The autoxidation rate, oxygen affinity, structural perturbation and tetramerinstability of the PEGylated Hb were significantly decreased upon protection with PEG20K. The present study isexpected to improve the efficacy of the PEGylated Hb as an oxygen therapeutic.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Hemoglobin (Hb)-based oxygen carriers (HBOCs) can overcome theshortage of blood supply and avoid pathogen-borne diseases throughblood transfusion (e.g., AIDS and hepatitis C) [1–3]. As a potentialHBOC, the PEGylated Hb has recently received much attention for itsnon-nephrotoxicity, one of the major obstacles for the therapeutic ap-plication of HBOC [4,5]. In addition, the PEGylated Hb can act as plasmaexpander for its unique property of high colloid osmotic pressure. Sev-eral PEGylated Hbs have been developed for clinical trials, such asMP4 (Sangart, USA) [6,7], Euro PEG-Hb [8] and PEGylated bovine Hb(Enzon, USA) [9].

lycol; PEGylation, conjugationonyl hydrazide; PBS, phosphatebin based oxygen carrier; P50,clusion chromatography; RP-aphy; CD, circular dichroism; n,arboxyethyl) phosphine; PEG-

y of Biochemical Engineering,ences, No. 1 Bei-Er-Tiao Street,1817; fax: +86 10 62561813.y of Biochemical Engineering,ences, No. 1 Bei-Er-Tiao Street,5217; fax: +86 10 62561813.e.ac.cn (T. Hu).

However, PEGylation, conjugation of polyethylene glycol (PEG)chain, can adversely alter themolecular, structural, and functional prop-erties of Hb [10]. For example, PEGylation can increase the oxygen affin-ity and decrease the Hill coefficient and Bohr effect of Hb, therebydecreasing the oxygen unloading ability of Hb [11]. PEGylation causesstructural perturbation to Hb, such as tetramer instability and exposureof the heme environment [12]. PEGylation also increases the autoxida-tion rate and the heme instability of Hb, followed by production of reac-tive oxygen species and nonfunctional metHb [13]. Thus, it is necessaryto minimize these adverse alterations in the development of thePEGylated Hb as HBOC.

The site of PEGylation is an important factor to determine themolec-ular, structural, and functional properties of the PEGylated Hb. For ex-ample, Cys-93(β) is adjacent to proximal His-92(β) that coordinateswith the center iron of heme [14]. PEGylation at Cys-93(β) can alterthe Fe-proximal His residue stretching frequency and significantly influ-ence the heme pocket of the β globin [15]. It also significantly increasesthe oxygen affinity and the autoxidation rate of Hb [15]. PEGylation atthe residue proximal to His-92(β), such as Lys-95(β) should have sim-ilar effects on the PEGylated Hb. Val-1(α) is topologically located atthe αα end of the central cavity of HbA [16]. Val-1(α) is involved in anumber of well-known functions, such as the transition from R stateto T state, the cooperative release or uptake of oxygen and the uniqueelectrostatic interactions with allosteric effectors [16]. PEGylation atVal-1(α) can lead to an extensive tetramer-dimer dissociation of Hb

1202 Q. Wang et al. / Biochimica et Biophysica Acta 1844 (2014) 1201–1207

[17]. This unfavorably alters the heme environment and quaternarystructure and decreases the Hill coefficient, the Bohr effect of Hb andthe sensitization to the presence of the allosteric effectors [17]. Thus,protection of these amino acid residues from PEGylation is an effectiveapproach to improve the molecular, structural, and functional proper-ties of the PEGylated Hb.

Historically, PEGylation of deoxy-Hb is an approach to protect Cys-93(β) from PEGylation [8]. Reversible protection of Cys-93(β) with4,4′-dithiopyridine (4-PDS) during PEGylation of oxy-Hb is an alterna-tive approach [18]. However, these two approaches cannot protect thevicinity region (e.g., Lys-95(β)) around His-92(β) from PEGylation.Propylation or propylbenzmethylation at Val-1(α) can structurallystabilize the PEGylated Hb and avoid PEGylation at Val-1(α) [19]. How-ever, these methods cannot simultaneously protect these sensitive res-idues (e.g., Val-1(α) and Lys-95(β)) from PEGylation.

In the present study, Cys-93(β) of Hb was conjugated with 20 kDaPEG (PEG20K) through the hydrazone and disulfide bonds, followedby PEGylation with 5 kDa PEG succinimidyl carbonate (PEG5K-SC)using acylation chemistry. The two PEG20K chains conjugated at Cys-93(β) could act as a scaffold and create a large shell of water moleculesaround Hb. This can shield some sensitive residues of Hb fromPEGylationwith PEG5K-SC. Briefly, 3-(2-pyridyldithio)propionyl hydra-zide (PDPH) was allowed to react with Cys-93(β) to form a disulfidebond (Fig. 1). The hydrazide moiety was then reacted with PEG20Kpropionaldehyde to form a hydrazone bond, followed by conjugationwith PEG5K-SC. Finally, PEG20K was released from Cys-93(β) of Hb byhydrazone hydrolysis at pH 5.0 and disulfide reduction with tris(2-carboxyethyl) phosphine (TCEP) (Fig. 1). Our present study suggestedthat Val-1(α), Lys-95(β), and Lys-16(α)were protected fromPEGylationwith PEG5K-SC. The autoxidation rate, structure and function of the re-sultant PEGylated Hb were investigated in details.

2. Experimental procedures

2.1. Preparation of the PEG-Hb samples

2.1.1. Purification of HbAHuman whole blood was kindly provided by the Beijing Red Cross

Blood Center (China). Human adult hemoglobin (HbA) was purifiedfrom erythrocytes as previously described [20].

2.1.2. Protection of Cys-93(β) of HbA with PEG20KHbA (0.5 mM as tetramer) in PBS buffer (pH 7.4) was allowed to

reactwith 10mM3-(2-pyridyldithio)propionyl hydrazide (PDPH, Ther-mo, USA) at 4 °C for 4 h. In order to remove the excess PDPH, the resul-tant samples were centrifuged at 6000 g using Centricon with 50 kDacutoff membrane (Millipore, USA) against 20 mM trisodium citrate

Hb-SH + NH2NH-C(CH2)2-S-S-

O

N

Hb-S-S-(CH2)2CNHNH2

O

PEG20K-CHO+

Hb-S-S-(CH2)2CNH-N=CH-PEG20K

O

+ PEG5

Hb-S-S-(CH2)2CNH-N=CH-PE

O

(PEG5K)n-

Hb-SHHb-SH + NH2NH-C(CH2)2-S-S-

O

NNH2NH-C(CH2)2-S-S-

O

N

Hb-S-S-(CH2)2CNHNH2

O

Hb-S-S-(CH2)2CNHNH2

OO

-CHO+

Hb-S-S-(CH2)2CNH-N=CH-PEG20K

O

Hb-S-S-(CH2)2CNH-N=CH-PEG20K

OO

+ PEG5

Hb-S-S-(CH2)2CNH-N=CH-PE

O

Hb-S-S-(CH2)2CNH-N=CH-PE

OO

(PEG5K)n-

Fig. 1. Schematic representat

buffer (pH 6.0). The sampleswere refilledwith 20mMtrisodium citratebuffer (pH6.0) and centrifuged at 6000 gfive times. The PDPH-modifiedHb (0.25 mM as tetramer) was allowed to react with 1 mM PEG20Kpropionaldehyde (PEG20K-ald, National Research Center for Biotech-nology, Beijing, China) in 20 mM trisodium citrate buffer (pH 6.0)at 4 °C for 10 h. The PEGylated Hb (PEG20K-Hb) was separatedfrom the reaction mixture on a Superdex 200 column (1.6 cm × 60 cm)(GE Healthcare, Uppsala, Sweden). The column was equilibrated andeluted with 20 mM sodium phosphate buffer (pH 7.4).

2.1.3. Preparation of PEG5K-Hb with protectionPEG20K-Hb (0.25 mM tetramer) was reacted with 5 mM PEG5K-SC

(National Research Center for Biotechnology, Beijing, China) in PBS buffer(pH 7.4) at 4 °C overnight. The reactionmixturewas loaded on aDEAE Se-pharose Fast Flow column (1.6 cm × 20 cm, GE Healthcare, Uppsala,Sweden). The column was equilibrated with 50 mM Tris-Ac buffer,pH 8.5 (buffer A). The unreacted PEG5K-SC was removed by elution withbuffer A. The PEGylated Hb was eluted from the column with 50 mMTris-Ac buffer (pH7.0, buffer B). The resultant productwas dialyzed exten-sively against 20 mM trisodium citrate buffer (pH 5.0) to release PEG20Kthrough hydrazone hydrolysis. Afterwards, 30-fold molar excess of TCEPwas added to release PEG20K through the disulfide reduction [21].

2.1.4. Preparation of PEG5K-Hb without protectionHbA (0.25 mM tetramer) was reacted with 5 mM PEG5K-SC in PBS

buffer (pH 7.4) at 4 °C overnight. The reaction mixture was loaded onthe DEAE Sepharose Fast Flow column (1.6 cm × 20 cm). The columnwas equilibrated with buffer A. The unreacted PEG5K-SC was removedby elution with buffer A. The PEGylated Hb without protection waseluted from the column with buffer B.

2.1.5. Preparation of PEG5K-Hb intermediatePEG20K-Hb (0.25 mM tetramer) was reacted with 5 mM PEG5K-SC

in PBS buffer (pH 7.4) at 4 °C overnight. The unreacted PEG5K-SC wasremoved according to the procedure in Section 2.1.3. The resultantproduct (PEG5K-Hb-PEG20K) was the one obtained before the finalreduction to remove the PEG20K moiety. The PDPH-modified Hb(0.25 mM as tetramer) was reacted with 5 mM PEG5K-SC in PBS buffer(pH 7.4) at 4 °C overnight. The resultant product (PEG5K-Hb-PDPH)and PEG5K-Hb-PEG20K were used for the next functional study ofPEG-Hb.

2.2. Analytical methods

Size exclusion chromatography (SEC) analysis was carried out on aSuperdex 200 column (1 cm×30 cm, GEHealthcare, USA) at room tem-perature. The column was equilibrated and eluted with 20 mM sodium

Hb-S-S-(CH2)2CNHNH2

O

Hb-S-S-(CH2)2CNH-N=CH-PEG20K

O

K-SC

G20KTCEP

pH 5.0Hb-SH(PEG5K)n-

Hb-S-S-(CH2)2CNHNH2

O

Hb-S-S-(CH2)2CNHNH2

OO

Hb-S-S-(CH2)2CNH-N=CH-PEG20K

O

Hb-S-S-(CH2)2CNH-N=CH-PEG20K

OO

K-SC

G20KG20KTCEP

pH 5.0Hb-SH(PEG5K)n- Hb-SH(PEG5K)n-

ion of PEGylation of Hb.

1203Q. Wang et al. / Biochimica et Biophysica Acta 1844 (2014) 1201–1207

phosphate buffer (pH 7.4) at a flow rate of 0.5 ml/min. The effluentwasmonitored at 540 nm.

Reverse-phase HPLC analysis was performed using a Proteonavi C4column (4.6 mm × 250 mm, SHISEIDO, Tokyo, Japan) equilibratedwith 5% (v/v) acetonitrile containing 0.1% (v/v) trifluoroacetic acid(TFA). The samplewas eluted using a linear gradient of 35–50% acetoni-trile containing 0.1% TFA in 100min at a flow rate of 1.0 ml/min. The ef-fluent was monitored at 214 nm.

SDS-PAGE analysis was carried out on a 15% polyacrylamide gel. Thegel was stained with Coomassie Blue R-250. The bands were analyzedby densitometry (Image Scanner, BioRad).

2.3. Tryptic peptide mapping

The Hb samples were pretreated with cold acid acetone to removethe heme and obtain the globin [10]. Tryptic digestion of the globinwas performed in 100 mM NH4HCO3 at an enzyme to substrate ratioof 1:50 (w/w) for 8 h at 37 °C. The tryptic peptides were analyzed byRP-HPLC on a C18 column (0.46 cm × 25 cm, Agilent, USA). Percentagemodification of the peptides in the PEGylated Hb was calculated by di-viding the peak area of each peptide with the corresponding peak forHbA. The recovery of the peptide βT4 was used as an internal standard.

2.4. Autoxidation

Autoxidation experiments were carried out by incubation of 25 μMHb samples in PBS buffer (pH 7.4) containing 300 μM EDTA at 37 °C insealed tubes. Aliquots of the Hb samples were taken out at varioustime intervals. Absorbance changes in 500–700 nmdue to the autoxida-tion of Hb were recorded in a spectrophotometer (Hitachi Instruments,Inc., San Jose, CA). The first-order autoxidation rate constant (kox) of theHb sample was measured and calculated as described previously [13].

2.5. Circular dichroism spectroscopy

Circular dichroism (CD) spectra were recorded on a J-810 spec-tropolarimeter (Jasco, Japan) using a 1 mm path length cuvette at25 °C [22,23]. The spectra of buffer blanks were measured beforethe samples and subtracted from the spectra of the samples. TheCD spectra (250–480 nm) were obtained using 26.0 μM Hb samples(in tetramer). Far UV CD spectra (200–250 nm) were obtainedusing 1.3 μM Hb samples (in tetramer). The molar ellipticity (θ) wasexpressed in deg cm2/dmol on a heme basis.

2.6. Dynamic light scattering

Dynamic light scattering for molecular radius measurement wasconducted using a DynaPro Titan TC instrument (Wyatt, USA) at25 °C. The Hb samples were at a protein concentration of 1.0 mg/ml in20 mM phosphate buffer (pH 7.2). The Hb samples were centrifugedat 15,700 g for 15 min before analysis.

2.7. Analytical ultracentrifugation

All experiments were conducted at 25 °C in a ProteomeLab XL-1 an-alytical ultracentrifuge (Beckman, U.S.A.) equipped with absorbanceoptics and an An60-Ti rotor. Boundary movement or concentration dis-tributions were followed at 280 nm using the centrifuge's absorptionoptics.

Sedimentation velocity analysis of the Hb samples was conducted at55,000 rpm using double sector cells at 20 °C. Datawere collected at thenominal concentration (A280 = 0.740) in PBS buffer (pH 7.4) for eachsample. The g(s*) distributions were determined using ν of 0.74 ml/gfor Hb and 0.806ml/g for the PEGylated Hb [16]. The sedimentation co-efficient (S) and the ratio of frictional coefficient (f/f0)were determined.

These values were normalized to standard conditions by correcting forbuffer density and viscosity [24].

Sedimentation equilibrium experiments were conducted at 10,000and 15,000 rpm using six channel centerpieces [17]. Three protein con-centrations (A280=0.1, 0.4 and 0.8)were analyzed for each sample. Theidentity of scans taken at 24 h confirmed that the samples had reachedequilibrium. The best-fit values and joint confidence intervals werereported.

2.8. Oxygen affinity measurements

Oxygen equilibrium measurements of the Hb samples were carriedout using a Hemox analyzer (TCS Corp., PA, USA) in Hemox buffer(pH 7.4) at 37 °C. The analyzer measured the oxygen pressure with aClark oxygen electrode (Yellow Springs Instrument, OH) and simulta-neously calculated theHb saturation via a dual-wavelength spectropho-tometer. The oxygen equilibrium curves for each sample were recordedto obtain the values of P50 (the oxygen pressure at which Hb is half sat-urated) and Hill coefficient (n). Measurement of each sample was per-formed three times. The Hb samples were at a protein concentrationof 16 mg/ml.

3. Results

3.1. Characterization of the Hb samples

3.1.1. Size exclusion chromatography analysisThe Hb samples were characterized by size exclusion chromatogra-

phy (SEC). As shown in Fig. 2a, HbAwas eluted as a single and symmet-ric peak on a Superdex 200 column. The elution peak of HbA was left-shifted upon conjugation with two PEG20K chains at Cys-93(β) ofHbA. The elution peak of PEG20K-Hb was left-shifted upon furtherPEGylation with PEG5K-SC. PEG5K-SC modified PEG20K-Hb was right-shifted under acid condition (pH 5.0) and TCEP reduction. This sug-gested that PEG20K was released from Cys-93(β) of HbA uponhydrazone hydrolysis and disulfide reduction. As reflected by the titra-tion with 4,4′-dithiopyridine (4-PDS) [18], PEG5K-Hb with protectionhad two free thiol groups per tetramer. This suggested that PEG20Kwas entirely released from Cys-93(β). PEG5K-Hb without protectionwas eluted much earlier than the one with protection. This suggestedthat protection with PEG20K retarded the conjugation of PEG5K onHbA.

3.1.2. SDS-PAGE analysisThe SDS-PAGE pattern of HbA showed a band corresponding to its

dissociated globin (~16 kDa) (Lane 2, Fig. 2b). As compared with HbA,PEG5K-Hb without protection showed five additional bands with slowmobility, along with the disappearance of the band corresponding toHbA (Lane 3). According to a previous report by Vandegriff et al. [25],the five additional bands indicated that each globin was conjugatedwith 1, 2, 3, 4 and 5 PEG5K chains. Densitometric analysis showedthat percentages of total for Bands 1, 2, 3 and 4–5 (from bottom totop, Lane 3) were 7.7%, 26.8%, 24.5% and 41.0%, respectively. PEG5K-Hb with protection (Lane 4) showed three additional bands, alongwith the presence of the dissociated globin. The three additionalbands indicated that each globin was conjugated with 1, 2, and 3PEG5K chains. Densitometric analysis showed that percentages oftotal for the dissociated globin, Bands 1, 2, and 3 (from bottom to top,Lane 4) were 36.6%, 37.7%, 21.6% and 4.1%, respectively. This furtherconfirmed that protection of PEG20K at Cys-93(β) can protect some res-idues of Hb from conjugation with PEG5K-SC.

3.1.3. Dynamic light scattering analysisThemolecular radii of the Hb samples were determined by dynamic

light scattering. As shown in Table 1, PEG5K-Hb without protectionshowed a molecular radius of 6.17 nm, which was much higher than

0 10 20 30 40 50

a5

4

3

2

1Ab

sorb

ance

at

540

nm

Time (min)

20 40 60 80 100

PEG5K-Hb withprotection

c

8

PEG5K-Hb withoutprotection

HbA

7

6

76

5

4

5

3

3

21

21

Ab

sorb

ance

at

214

nm

Time (min)0 1 2 3 4 5 6

0

4

8

12

16 d

Met

Hb

(%

)

Time (h)

HbAWith protectionWithout protection

200.0116.097.2

1 2 3 4kDa

66.4

44.3

20.114.3

b

β

β

β

α

α

Fig. 2. Characterization of the Hb samples. Size exclusion chromatography analysis (a) of HbA (1), PEG20K-Hb (2), PEG5K modified PEG20K-Hb (3), PEG5K-Hb with protection (4), andPEG5K-Hbwithout protection (5)was conducted on a Superdex 200 column at room temperature. SDS-PAGE analysis (b)was carried out on a 15% Tris-glycine gel. Lane 1, Marker; Lane 2,HbA; Lane 3, PEG5K-Hb with protection; Lane 4, PEG5K-Hb without protection. RP-HPLC analysis (c) of the Hb samples was conducted on a Proteonavi C4 column. Fig. 2d indicated thekinetics of metHb formation of the Hb samples in PBS buffer (pH 7.4) at 37 ºC.

1204 Q. Wang et al. / Biochimica et Biophysica Acta 1844 (2014) 1201–1207

that of HbA (3.35 nm). This indicated that PEGylation can significantlyincrease themolecular radius of HbA. On the other hand, PEGylation de-creased the diffusion coefficient of HbA. This indicated the slowerBrownianmovement of the PEGylatedHbdue to its increasedmolecularvolume. In contrast, PEG5K-Hb with protection showed a molecular ra-dius of 5.16 nm, which is lower than that of PEG5K-Hb without protec-tion (6.17 nm).

3.1.4. RP-HPLC analysisThe HPLC pattern of HbA showed two well separated elution peaks

corresponding to α and β globins, respectively (Fig. 2c). Upon directmodification with PEG5K-SC, the two globins of HbA essentially disap-peared and several new elution peaks appeared. The new elutionpeaks suggested that α globin was conjugated with 1, 2, 3, and 4PEG5K chains (Peaks 1–4), and β globin was also conjugated with 1, 2,

Table 1The molecular radius and volume of the Hb samples.a

Sample Radiusb(nm) Volumec(nm3) Diffusion coefficient(cm/s 10−7)

HbA 3.35 157.4 6.86PEG5K-Hb withprotection

5.16 575.2 5.15

PEG5K-Hb withoutprotection

6.17 983.4 5.32

a Each sample was measured three times.b The samples were at a protein concentration of 1.0 mg/ml and measured at 25 °C.c The molecular volume (V) was calculated with the equation V = 4πr3/3, where r is

the molecular radius.

3, and 4 PEG5K chains (Peaks 5–8). However, Peaks 4 and 8 disappearedand unmodified α and β globins appeared upon protection withPEG20K, along with decrease in Peaks 1, 2, 3, 5, 6 and 7. This suggestedthat protection with PEG20K can partially inhibit conjugation of PEG5Kon HbA.

3.2. Autoxidation

Autoxidation of Hb involves production of reactive oxygen speciesand nonfunctional metHb, which is a parameter that reflects thein vivo toxicity of Hb. Fig. 2d showed the metHb formation of the Hbsamples as a function of incubation time. PEG5K-Hb with protectionshowed metHb contents higher than that of HbA and lower than thatof PEG5K-Hb without protection. The first-order autoxidation rate con-stants (kox) of Hb and PEG5K-Hbwithout protection were calculated as0.013 h−1 and 0.030 h−1, respectively. This suggested that PEGylationwithout protection significantly increased the autoxidation of Hb. Incontrast, PEG5K-Hbwith protection showed a kox of 0.017 h− This indi-cated that protectionwith PEG20K could significantly reduce the autox-idation of PEG5K-Hb.

3.3. Characterization of the sites of PEGylation

Tryptic peptide mapping was used to characterize the sites ofPEGylation in PEG5K-Hb with and without protection. As shown inFig. 3, several tryptic peptides appeared upon trypsin digestion of theHb samples. As compared to those of HbA, the areas of the peaks corre-sponding to the peptidesαT1,αT1+ 2,αT3,αT5,αT9,αT13, βT1, βT2,βT10 + 11 and βT13 decreased for PEG5K-Hb without protection.

20 40 60 80

*

**

* *

*

T8+9

T13

*

***

T10+11

T5

T8+9

T9

T5

T4

T6T2

T3 T11

T14

T13T4

T1

T1+2T3

T1

3

2

1

Ab

sorb

ance

at

214

nm

Time (min)

Fig. 3. Characterization of the sites of PEGylation by tryptic peptide mapping. The analysisof HbA (1), PEG5K-Hb without protection (2) and with protection (3) was conductedusing a C18 column. Themodifiedpeptides for PEG5K-Hbwithout protection are indicatedwith an asterisk. The peptide βT4 was used as an internal standard.

200 210 220 230 240 250-5

-4

-3

-2

-1

0

1

2

3

32

1

a

(10

-6 d

eg c

m2 /d

mo

l)

Wavelength (nm)

280 320 360 400 440 480

0

2

4

6

8

10

31

23

3 2 1

b

(10

-4 d

eg c

m2 /d

mo

l)

Wavelength (nm)

θθ

Fig. 4. Circular dichroism spectra of theHb samples. CD spectra of HbA (1), PEG5K-Hbwithprotection (2), and PEG5K-Hbwithout protection (3)were recorded at room temperaturein the near-UV regions of 200–250 nm (a) and 250–480 nm (b).

1205Q. Wang et al. / Biochimica et Biophysica Acta 1844 (2014) 1201–1207

Particularly, the peaks corresponding to the peptides αT1,αT1+ 2 andβT10 + 11 essentially disappeared. This suggested that Val-1(α) andLys-95(β) was essentially completely PEGylated with PEG5K. In con-trast, these disappearing peaks could be fully recovered upon protectionwith PEG20K. This suggested that PEG20K can fully protect some impor-tant residues from PEGylation, due to the steric shielding effect ofPEG20K on Hb.

The PEGylation extents for the specific amino acid residues are sum-marized in Table 2. For PEG5K-Hbwithout protection, Val-1(α) and Lys-95(β) were essentially completely PEGylated by PEG5K-SC. In addition,Val-1(β), Lys-8(β), Lys-16(α), Lys-40(α), Lys-90(α), Lys-139(α), andLys-120(β) were partially PEGylated by PEG5K-SC. In contrast, Val-1(α), Lys-16(α) and Lys-95(β) can be fully protected from PEGylationwith PEG20K. Moreover, Lys-8(β) and Lys-16(α) were partiallyprotected by PEG20K. By calculation of the PEGylation extents, PEG5K-Hb with and without protection were deduced to conjugation of 3.90and 8.34 copies of PEG5K with one Hb tetramer, respectively. Thus,PEG5K-Hb with and without protection were tetra- and octa-PEGylated Hbs, respectively. Because some modification sites wereshielded by the protective PEG20K in the PEGylation process, PEG5K-Hb without protection was twice as PEGylated as the one withprotection.

3.4. Structure analysis of the Hb samples

3.4.1. CD analysisThe structural properties of the Hb samples were investigated using

CD spectroscopy. No changes were observed in the far-UV (200–250 nm) CD spectra for the Hb samples (Fig. 4a). This indicated thatthe secondary structure of HbA was not influenced by PEGylation withor without protection.

Table 2The sites of PEGylation for the Hb samples.a

Residues PEG5K-Hb with protection (%) PEG5K-Hb without protection (%)

Val-1(α) 0 92Val-1(β) 24 44Lys-8(β) 11 25Lys-16(α) 0 16Lys-40(α) 24 18Lys-90(α) 38 35Lys-139(α) 75 64Lys-95(β) 0 90Lys-120(β) 25 29

a The sites of PEGylation in the Hb samples were determined by tryptic peptide map-ping as described in Experimental procedures.

The CD spectra of 250–480 nmwere shown in Fig. 4b. The CD regionin the range of 260–300 nm has been recently investigated as a markerof conformational (tertiary and quaternary) transition, using gel-immobilized Hb [26,27], Hb mutants [28] and other experiments.PEGylation without protection markedly decreased the CD ellipticityof HbA in the range of 260–300 nm, indicating the conformational tran-sition of HbA. In contrast, PEG5K-Hbwith protection showed the CD el-lipticity comparable to HbA in this region. Thus, protection of Hb withPEG20K can increase the conformation stability of PEG5K-Hb.

The Soret band (around 420 nm) of Hb is informative of the interac-tions of the heme prosthetic group with the surrounding aromatic resi-dues [29]. HbA showed a maximal ellipticity at 419 nm. PEGylationwithout protection led to an apparent increase in the Soret band ofHb, along with a maximal ellipticity shifted from 419 nm to 415 nm(Fig. 4b). This indicated a strong structural perturbation in the heme en-vironment of PEG5K-Hb without protection. Interestingly, protectionwith PEG20K can decrease the ellipticity of PEG5K-Hb and make themaximal ellipticity shift back to 419 nm. This indicated that protectionwith PEG20K can decrease the structural perturbation in the heme envi-ronment of PEG5K-Hb.

3.4.2. Analytical ultracentrifugationThe Hb samples were structurally analyzed by both sedimentation

velocity and sedimentation equilibrium analytical ultracentrifugation.During sedimentation equilibrium, the proteins were characterized

in their native state, whereas the SEC method for the tetramer–dimerequilibrium involved interactions between matrices and proteins.Thus, sedimentation equilibrium was used to measure the tetramer–

Table 4Oxygen-delivery properties of the Hb samples.

Sample P50 (mm Hg)a Hill coefficient

HbA 13.8 ± 0.4 2.8 ± 0.1PEG5K-Hb with protection 9.4 ± 0.5 2.0 ± 0.2PEG5K-Hb without protection 5.2 ± 0.3 1.5 ± 0.2PEG5K-Hb-PDPH 4.6 ± 0.1 1.3 ± 0.1PEG5K-Hb-PEG20K 5.3 ± 0.2 1.5 ± 0.2MP4b 6.0 ± 2.0 1.2 ± 0.5(SP-PEG5K)6-Hbc 6.5 2.2Euro-PEG-Hbd 5.83 ± 0.19 1.57 ± 0.09

Oxygen equilibrium curves of the samples were measured at 37 °C.a The partial oxygen pressure at 50% saturation that is expressed in mm Hg.b The data obtained from Vandegriff et al. [7].c The data obtained from Manjula et al. [31].d The data obtained from Portoro et al. [8].

1206 Q. Wang et al. / Biochimica et Biophysica Acta 1844 (2014) 1201–1207

dimer equilibrium of the Hb samples. Fitting the concentration distribu-tions to the dimer-tetramer association model, the best-fit dissociationconstant (Kd) of PEG5K-Hbwas 6.1-fold of that of HbA (Table 3). This in-dicated that PEGylation without protection significantly increased thetetramer–dimer dissociation of HbA. As compared with HbA, PEG5K-Hb with protection showed 2.1-fold increase in the best-fit Kd. Thissuggested that protection with PEG20K can structurally stabilize thetetramers of PEG5K-Hb.

Sedimentation velocity analysiswas carried out to obtain the confor-mational information of the Hb samples. As shown in Table 3,PEGylation without protection led to a significant decrease in the sedi-mentation coefficient (S020,W) and an increase in the ratio of frictionalcoefficient (f/f0) of HbA. Thus, the PEG chains could behave as a para-chute to decrease the S020,W and alter the hydrodynamic behavior ofHbA. The increased f/f0 suggested that PEG can endow the asymmetricmolecular shape of HbA [30]. In contrast, protection with PEG20K canincrease the S020,W and decrease the f/f0 of PEG5K-Hb. This is due tothe synergy of the less conjugated PEG5K and increased tetramer stabil-ity of PEG5K-Hb with protection.

3.5. Oxygen affinity

As shown in Table 4, HbA showed a P50 of 13.8 mmHgwith the Hillcoefficient (n) within the normal range of 2.8. PEG5K-Hb without pro-tection showed a P50 of 5.2 mm Hg with the Hill coefficient of 1.5.This indicated that PEGylation without protection resulted in a strongperturbation in the quaternary transition of HbA (R state to T state)and a significant decrease in the subunit cooperativity. In contrast,PEG5K-Hbwith protection showed a P50 of 9.4mmHgwith the Hill co-efficient of 2.0. This indicated that protectionwith PEG20K can structur-ally stabilize the T state of PEG5K-Hb and improve its subunitcooperativity.

The two intermediates (PEG5K-Hb-PDPH and PEG5K-Hb-PEG20K)were functionally characterized. As shown in Table 4, PEG5K-Hb-PDPH showed lower P50 and n than PEG5K-Hb without protection.This indicated that initial derivatization with PDPH altered the quater-nary conformational equilibrium of Hb. Interestingly, PEG5K-Hb-PEG20K showed higher P50 and n than PEG5K-Hb-PDPH. This sug-gested that the conjugated PEG20K can improve the functional propertyof PEG-Hb by protection of some sensitive residues of HbA.

4. Discussion

MP4 (SP-PEG5K)6-Hb and Euro-PEG-Hb were hexa-PEGylated Hbgenerated based on the extension arm facilitated PEGylation. As forMP4, ε-amino groups of oxy-HbA were reacted with iminothiolane(IT) to introduce extrinsic thiol groups [7]. Then, the extrinsic thiolgroups and two intrinsic thiol groups of Cys-93(β) in oxy HbA wereallowed to react with maleimide activated PEG5K (PEG5K-mal) [7]. Asfor (SP-PEG5K)6-Hb, IT and PEG5K-mal were simultaneously added tothe oxy-Hb solution, where the introduced extrinsic thiol groupswere immediately modified with PEG5K-mal [31]. As for Euro-PEG-Hb, IT and PEG5K-mal were simultaneously added in the inositolhexaphosphate bound deoxy-Hb solution, where Cys-93(β) was fullyburied [8]. In contrast, Enzon Inc. developed a PEGylated bovine Hb

Table 3The analytical ultracentrifugation analysis of the Hb samples.

Sample S20,Wa f/f0b Kd (μM)c

HbA 4.14 ± 0.22 1.14 6.02PEG5K-Hb with protection 2.49 ± 0.21 2.03 12.93PEG5K-Hb without protection 1.96 ± 0.18 2.29 36.90

a The sedimentation coefficient.b The ratio of frictional coefficient.c The best-fit value of the dissociation constant determined from the dimer-tetramer

association model.

carrying ten PEG5K chains using acylation chemistry, where ε-aminogroups of Lys and α-amino groups of Val-1(α) and Val-1(β) of HbAwere randomly modified [9]. In the present study, PEG20K wasreversibly conjugated at Cys-93(β) of HbA, which protected Lys-95(β),Val-1(α) and Lys-16(α) of Hb from acylation chemistry basedPEGylation. Accordingly, the resultant tetra-PEGylated Hb showed a de-creased autoxidation rate and improved structural and functionalproperties.

PEG is flexible with an extensive degree of hydration and can dy-namically occupy a large volume in an aqueous environment [32].Upon conjugation at Cys-93(β) of HbA, the PEG20K chains can formloosely organized domains like an umbrella and sterically shield the re-active functional residues over large areas of theHbA surface. The acces-sibility of Lys-95(β) to PEG5K can be extensively hindered by thePEG20K chains, due to the proximity of Lys-95(β) to Cys-93(β). SinceVal-1(α) is close to the α1β2 interface of HbA, PEG20K conjugated atCys-93(β) can topologically shield Val-1(α) from PEGylation withPEG5K. In addition, it is well known that Cys-93(β) can be spared byPEGylation by working under anaerobic conditions, as the T state ex-poses different reactive residues with respect to the R state. Thus, an al-ternative explanation would be that the initial derivatization withPDPH,without further reactionwith PEG20K, alters the quaternary con-formational equilibrium, thus affecting the subsequent PEGylationpattern.

Lys-95(β) and Cys-93(β) are adjacent to proximal His-92(β) that co-ordinates with the center iron of heme. PEGylation of Lys-95(β) andCys-93(β) perturbed the heme environment and made the heme ex-posed to the surrounding environment, as reflected by the high autoxi-dation rate of PEG5K-Hb without protection. The extensive tetramerdissociation by PEGylation at Val-1(α) contributed the autoxidationrate of PEG5K-Hb. Thus, protection of Lys-95(β) and Val-1(α) fromPEGylation can significantly decrease the autoxidation rate of PEG5K-Hb. (SP-PEG5K)6-Hb and MP4 also showed higher autoxidation rate,possibly related with PEGylation at Cys-93(β) [34]. In addition, theincreased autoxidation rate might result from a shift of the quaternarytoward the R state, which oxidizes faster than the T state and not neces-sarily to a short range interaction between PEG and the heme.

The α1β2 interface is a highly polar and dynamic region of Hb, un-dergoing large structural changes as the Hb molecule transitions fromthe R to T state [33]. Val-1(α) and Lys-16(α) are topologically close tothe α1β2 interface of Hb. PEGylation at these residues may structurallyperturb the α1β2 interface, restrict the quaternary transitions of Hbfrom the R to T state, and reduce the subunit cooperativity of Hb. In ad-dition, PEGylation of Lys-95(β) and Cys-93(β) adversely altered the in-teraction of heme and globin [34]. This can, in turn, decrease the P50and cooperativity of Hb. Cys-93(β) was PEGylated both in MP4 [25]and (SP-PEG5K)6-Hb [18]. Lys-95(β) was PEGylated both in MP4 [25]and Euro-PEG-Hb [35]. As shown in Table 4, PEG5K-Hb with protectionshowed higher P50 than MP4 (SP-PEG5K)6-Hb and Euro-PEG-Hb. Thiswas partially due to the free Lys-95(β) and Cys-93(β) in PEG5K-Hb

1207Q. Wang et al. / Biochimica et Biophysica Acta 1844 (2014) 1201–1207

with protection. PEG5K-Hb with protection also showed highercooperativity than MP4 and Euro-PEG-Hb, due to the protection effectof PEG20K. However, PEG5K-Hb with protection also showed lowercooperativity than (SP-PEG5K)6-Hb. The reason was not clear to us.

Previously, Cys-93(β) was reversibly protected as a mixed disulfidewith thiopyridine during extension arm facilitated PEGylation [18].In the present study, Cys-93(β) and its vicinity region (Lys-95(β), Val-1(α) and Lys-16(α)) were efficiently protected with PEG20K. PEG20Kwas reversibly conjugated with Cys-93(β) and was entirely releasedfrom Cys-93(β) through hydrazone hydrolysis and disulfide reduction.However, hydrazone hydrolysis or disulfide reduction alone cannot en-tirely release PEG20K from Cys-93(β) (data not shown).

In summary, reversible conjugation and release of PEG20K can pro-tect Lys-95(β), Val-1(α) and Lys-16(α) from acylation chemistry basedPEGylation. The structural perturbation, tetramer instability and the au-toxidation rate of PEG5K-Hbwere significantly reduced upon protectionwith PEG20K. TheHill coefficient of PEG5K-Hbwas improved and its ox-ygen affinity decreased upon protection with PEG20K. The presentstudy was expected to improve the efficacy of the PEGylated Hb as anoxygen therapeutic.

Acknowledgments

Ms. Xiaoxia Yu from the Institute of Biophysics, Chinese Academy ofSciences is appreciated for the analytical ultracentrifugation experi-ments. This study was financially supported by the 863 High-Tech Pro-ject of China (Grant No. 2012AA021903-3), the National Key BasicResearch Program of China (Grant No. 2013CB733600) and the BeijingNatural Science Foundation (7142104).

References

[1] M. Samaja, L. Terraneo, Impact of hemoglobin concentration and affinity for oxygenon tissue oxygenation: the case of hemoglobin-based oxygen carriers, Artif. Organs36 (2012) 210–215.

[2] A. Mozzarelli, L. Ronda, S. Faggiano, S. Bettati, S. Bruno, Haemoglobin-based oxygencarriers: research and reality towards an alternative to blood transfusions, BloodTransfus. 8 (Suppl. 3) (2010) s59–s68.

[3] C.L. Varnado, T.L. Mollan, I. Birukou, B.J. Smith, D.P. Henderson, J.S. Olson, Develop-ment of recombinant hemoglobin-based oxygen carriers, Antioxid. Redox Signal.18 (2013) 2314–2328.

[4] R.H. Cole, K.D. Vandegriff, MP4, a vasodilatory PEGylated hemoglobin, Adv. Exp.Med. Biol. 701 (2011) 85–90.

[5] T.A. Silverman, R.B. Weiskopf, Hemoglobin-based oxygen carriers: current statusand future directions, Anesthesiology 111 (2009) 946–963.

[6] R.M. Winslow, MP4, a new nonvasoactive polyethylene glycol-hemoglobin conju-gate, Artif. Organs 28 (2004) 800–806.

[7] K.D. Vandegriff, A. Malavalli, J. Wooldridge, J. Lohman, R.M. Winslow, MP4, a newnonvasoactive PEG-Hb conjugate, Transfusion 43 (2003) 509–516.

[8] I. Portoro, L. Kocsis, P. Herman, D. Caccia, M. Perrella, L. Ronda, S. Bruno, S. Bettati, C.Micalella, A. Mozzarelli, A. Varga, M. Vas, K.C. Lowe, A. Eke, Towards a novelhaemoglobin-based oxygen carrier: Euro-PEG-Hb, physico-chemical properties, va-soactivity and renal filtration, Biochim. Biophys. Acta 1784 (2008) 1402–1409.

[9] C.D. Conover, R. Linberg, K.L. Shum, R.G. Shorr, The ability of polyethylene glycolconjugated bovine hemoglobin (PEG-Hb) to adequately deliver oxygen in both ex-change transfusion and top-loaded rat models, Artif. Cells Blood Substit. Immobil.Biotechnol. 27 (1999) 93–107.

[10] T. Hu, B.N. Manjula, D. Li, M. Brenowitz, S.A. Acharya, Influence of intramolecularcross-links on the molecular, structural and functional properties of PEGylatedhaemoglobin, Biochem. J. 402 (2007) 143–151.

[11] T. Hu, D. Li, F. Meng, M. Prabhakaran, S.A. Acharya, Increased inter dimeric interactionof oxy hemoglobin is necessary for attenuation of reductive pegylation promoted dis-sociation of tetramer, Artif. Cells Blood Substit. Immobil. Biotechnol. 39 (2011) 69–78.

[12] K.D. Vandegriff, A. Malavalli, C. Minn, E. Jiang, J. Lohman,M.A. Young, M. Samaja, R.M.Winslow, Oxidation and haem loss kinetics of poly(ethylene glycol)-conjugated

haemoglobin (MP4): dissociation between in vitro and in vivo oxidation rates,Biochem. J. 399 (2006) 463–471.

[13] T. Hu, D. Li, B.N. Manjula, S.A. Acharya, Autoxidation of the site-specificallyPEGylated hemoglobins: role of the PEG chains and the sites of PEGylation in the au-toxidation, Biochemistry 47 (2008) 10981–10990.

[14] H. Muirhead, J.M. Cox, L. Mazzarella, M.F. Perutz, Structure and function ofhaemoglobin. 3. A three-dimensional fourier synthesis of human deoxyhaemoglobinat 5.5 Angstrom resolution, J. Mol. Biol. 28 (1967) 117–156.

[15] B.N. Manjula, A. Tsai, R. Upadhya, K. Perumalsamy, P.K. Smith, A. Malavalli, K.Vandegriff, R.M. Winslow, M. Intaglietta, M. Prabhakaran, J.M. Friedman, A.S.Acharya, Site-specific PEGylation of hemoglobin at Cys-93(beta): correlation be-tween the colligative properties of the PEGylated protein and the length of the con-jugated PEG chain, Bioconjug. Chem. 14 (2003) 464–472.

[16] M.F. Perutz, A.J. Wilkinson, M. Paoli, G.G. Dodson, The stereochemical mechanism ofthe cooperative effects in hemoglobin revisited, Annu. Rev. Biophys. Biomol. Struct.27 (1998) 1–34.

[17] T. Hu, D. Li, B.N. Manjula, M. Brenowitz, M. Prabhakaran, S.A. Acharya, PEGylation ofVal-1(alpha) destabilizes the tetrameric structure of hemoglobin, Biochemistry 48(2009) 608–616.

[18] D. Li, T. Hu, B.N. Manjula, S.A. Acharya, Extension arm facilitated pegylation ofαα-hemoglobin with modifications targeted exclusively to amino groups:functional and structural advantages of free Cys-93(β) in the PEG-Hb adduct,Bioconjug. Chem. 20 (2009) 2062–2070.

[19] T. Hu, D. Li, J. Wang, Q. Wang, Y. Liang, Y. Su, G. Ma, Z. Su, S. Wang,Propylbenzmethylation at Val-1(alpha) markedly increases the tetramer stabilityof the PEGylated hemoglobin: a comparison with propylation at Val-1(alpha),Biochim. Biophys. Acta 1820 (2012) 2044–2051.

[20] B.N. Manjula, S.A. Acharya, Purification and molecular analysis of hemoglobin byhigh-performance liquid chromatography, Methods Mol. Med. 82 (2003) 31–47.

[21] R. Liu, J. Wang, R. Qiu, Q. Lin, G. Zhang, G. Ma, Z. Su, T. Hu, Preparation, characteriza-tion and in vitro bioactivity of N-terminally PEGylated staphylokinase dimers, Pro-cess Biochem. 47 (2012) 41–46.

[22] C. Li, Q. Lin, J. Wang, L. Shen, G. Ma, Z. Su, T. Hu, Preparation, structural analysis andbioactivity of ribonuclease A-albumin conjugate: tetra-conjugation or PEG as thelinker, J. Biotechnol. 162 (2012) 283–288.

[23] S. Liu, J. Wang, G. Ma, Z. Su, T. Hu, Mono-PEGylation of ribonuclease A: highPEGylation efficiency by thiolation with small molecular weight reagent, ProcessBiochem. 47 (2012) 1364–1370.

[24] X. Xue, D. Li, J. Yu, G. Ma, Z. Su, T. Hu, Phenyl linker-induced dense PEG confor-mation improves the efficacy of C-terminally monoPEGylated staphylokinase,Biomacromolecules 14 (2013) 331–341.

[25] K.D. Vandegriff, A. Malavalli, G.M. Mkrtchyan, S.N. Spann, D.A. Baker, R.M. Winslow,Sites of modification of Hemospan, a poly(ethylene glycol)-modified human hemo-globin for use as an oxygen therapeutic, Bioconjug. Chem. 19 (2008) 2163–2170.

[26] M. Nagai, S. Nagatomo, Y. Nagai, K. Ohkubo, K. Imai, T. Kitagawa, Near-UV circulardichroism and UV resonance Raman spectra of individual tryptophan residues inhuman hemoglobin and their changes upon the quaternary structure transition,Biochemistry 51 (2012) 5932–5941.

[27] S. Nagatomo, M. Nagai, T. Ogura, T. Kitagawa, Near-UV circular dichroism and UVresonance Raman spectra of tryptophan residues as a structural marker of proteins,J. Phys. Chem. B 117 (2013) 9343–9353.

[28] Y. Jin, H. Sakurai, Y. Nagai, M. Nagai, Changes of near-UV CD spectrum of human he-moglobin upon oxygen binding: a study of mutants at alpha 42, alpha 140, beta 145tyrosine or beta 37 tryptophan, Biopolymers 74 (2004) 60–63.

[29] M.C. Hsu, R.W. Woody, The origin of the heme Cotton effects in myoglobin and he-moglobin, J. Am. Chem. Soc. 93 (1971) 3515–3525.

[30] C. Dhalluin, A. Ross, L.A. Leuthold, S. Foser, B. Gsell, F. Muller, H. Senn, Structural andbiophysical characterization of the 40 kDa PEG-interferon-alpha(2a) and its individ-ual positional isomers, Bioconjug. Chem. 16 (2005) 504–517.

[31] B.N. Manjula, A.G. Tsai, M. Intaglietta, C.H. Tsai, C. Ho, P.K. Smith, K. Perumalsamy, N.D.Kanika, J.M. Friedman, S.A. Acharya, Conjugation of multiple copies of polyethyleneglycol to hemoglobin facilitated through thiolation: influence on hemoglobin struc-ture and function, Protein J. 24 (2005) 133–146.

[32] L. Wu, S.V. Ho, W. Wang, J. Gao, G. Zhang, Z. Su, T. Hu, N-terminal mono-PEGylationof growth hormone antagonist: correlation of PEG size and pharmacodynamic be-havior, Int. J. Pharm. 453 (2013) 533–540.

[33] B. Vallone, A.E. Miele, P. Vecchini, E. Chiancone, M. Brunori, Free energy of buryinghydrophobic residues in the interface between protein subunits, Proc. Natl. Acad.Sci. U. S. A. 95 (1998) 6103–6107.

[34] F.E. Lui, P. Dong, R. Kluger, Polyethylene glycol conjugation enhances the nitrite re-ductase activity of native and cross-linked hemoglobin, Biochemistry 47 (2008)10773–10780.

[35] R. Iafelice, S. Criston, D. Caccia, R. Russo, L. Rossi-Bernardi, K.C. Lowe, M. Perrella,Identification of the sites of deoxyhaemoglobin PEGylation, Biochem. J. 403(2007) 189–196