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Bioconjugate Chem. 2003, 14, 464−472
Site-Specific PEGylation of Hemoglobin at Cys-93(β): Correlation between the Colligative Properties of the PEGylated Protein and the Length of the Conjugated PEG Chain 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,† and A. S. Acharya† Albert Einstein College of Medicine, Bronx, New York, University of CaliforniasSan Diego, La Jolla, California, BioAffinity Systems, Rockford, Illinois, Sangart, Inc., San Diego, California, and Structural BioInformatics, San Diego, California.. Received September 5, 2002; Revised Manuscript Received December 13, 2002
Increasing the molecular size of acellular hemoglobin (Hb) has been proposed as an approach to reduce its undesirable vasoactive properties. The finding that bovine Hb surface decorated with about 10 copies of PEG5K per tetramer is vasoactive provides support for this concept. The PEGylated bovine Hb has a strikingly larger molecular radius than HbA (1). The colligative properties of the PEGylated bovine Hb are distinct from those of HbA and even polymerized Hb, suggesting a role for the colligative properties of PEGylated Hb in neutralizing the vasoactivity of acellular Hb. To correlate the colligative properties of surface-decorated Hb with the mass of the PEG attached and also its vasoactivity, we have developed a new maleimide-based protocol for the site-specific conjugation of PEG to Hb, taking advantage of the unusually high reactivity of Cys-93(β) of oxy HbA and the high reactivity of the maleimide to protein thiols. PEG chains of 5, 10, and 20 kDa have been functionalized at one of their hydroxyl groups with a maleidophenyl moiety through a carbamate linkage and used to conjugate the PEG chains at the β-93 Cys of HbA to generate PEGylated Hbs carrying two copies of PEG (of varying chain length) per tetramer. Homogeneous preparations of (SP-PEG5K)2-HbA, (SPPEG10K)2-HbA, and (SP-PEG20K)2-HbA have been isolated by ion exchange chromatography. The oxygen affinity of Hb is increased slightly on PEGylation, but the length of the PEG-chain had very little additional influence on the O2 affinity. Both the hydrodynamic volume and the molecular radius of the Hb increased on surface decoration with PEG and exhibited a linear correlation with the mass of the PEG chain attached. On the other hand, both the viscosity and the colloidal osmotic pressure (COP) of the PEGylated Hbs exhibited an exponential increase with the increase in PEG chain length. In contrast to the molecular volume, viscosity, and COP, the vasoactivity of the PEGylated Hbs was not a direct correlate of the PEG chain length. There appeared to be a threshold for the PEG chain length beyond which the protection against vasoactivity is decreased. These results suggest that the modulation of the vasoactivity of Hb by PEG could be a function of the surface shielding afforded by the PEG, the latter being a function of the disposition of the PEG chain on the protein surface, which in turn is a function of the length of the PEG chain. Thus, the biochemically homogeneous PEGylated Hbs described in the present study, surface-decorated with PEG chains of appropriate size, could serve as potential candidates for Hb-based oxygen carriers.
INTRODUCTION
Several chemically and genetically modified acellular hemoglobins (Hbs)1 have been investigated to date as oxygen carrying therapeutic agents (2-4). One of the two major toxicities of Hb, namely, nephrotoxicity, has been overcome by intramolecular cross-linking (2, 5). The current major focus in the field is to overcome the vasoactivity of Hb (6, 7). The vasoactivity of acellular Hb has been attributed to its extravasation into the interstitial spaces and subsequent scavenging of nitric oxide (6-16) and may involve a second mechanism related to * Corresponding author. Address: Department of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. Phone: 718-430-2062. Fax: 718-430-8819. E-mail:
[email protected]. † Albert Einstein College of Medicine. ‡ UCSD. § BioAffinity Systems. | Sangart. ⊥ Structural BioInformatics.
the autoregulation of capillary blood flow (17). Increasing the molecular size of Hb has been advocated as one of the approaches to prevent the extravasation of acellular Hb into the interstitial spaces. Intertetrameric crosslinking and conjugation to monofunctional PEG have been sought as approaches to increase the molecular size of Hb. PEGylation of proteins and peptides has been shown to increase their molecular size, resulting in an increased circulatory half-life and also to eliminate or reduce their antigenicity (18, 19). Recent studies by Winslow and co-workers (17, 20, 21) employing intratetramerically cross-linked Hbs, polymerized Hbs, and PEGylated Hbs have shown that, despite similar in vitro NO binding activity, the modified Hbs differ significantly 1 Abbreviatons: Hb, hemoglobin; PEG, poly(ethyleneglycol); SP, succinimidophenyl; IEF, isoelectric focusing; SEC, size exclusion chromatography; RPHPLC, reverse-phase highperformance liquid chromatography; PBS, phosphate-buffered saline; Tris, tris(hydroxymethyl) amino methane; COP, colloidal osmotic pressure; FCD, functional capillary density.
10.1021/bc0200733 CCC: $25.00 © 2003 American Chemical Society Published on Web 02/14/2003
PEGylation of Hemoglobin at Cys-93(β)
in their extent and duration of induced vasoactivity in a rat model. All hemoglobin tetramers studied were found to be vasoactive to different degrees, depending upon their oxygen affinity. Hemoglobin oligomers were less vasoactive, and their effect was transient. Of the two PEGylated Hbs examined, PEG-Hb (bovine Hb carrying ∼10 copies of PEG5000 per tetramer - Enzon product) exhibited no pressor response, whereas PHP-Hb (PEGylated pyridoxalated Hb carrying ∼10 copies of PEG3000 per tetramer-Ajinomoto product) exhibited a reduced and transient pressor response. Although the viscosity of the two PEGylated Hbs were nearly the same, PEGHb had a much higher colloidal osmotic pressure than PHP-Hb and a much higher molecular volume. Thus, the pressor effect of the PEGylated Hbs exhibited a correlation with their molecular size and solution properties. Thus, these studies suggested that PEGylation of Hb is an efficient way to reduce its vasoactivity. The PEG-Hbs described above are generated by attachment of PEG to the surface amino groups of the protein using the succinimidyl chemistry. The activated PEG reagent is labile and reacts with lysine in competition with hydrolysis, necessitating the use of a large excess of reagent for the reaction. Hence, specificity of this reaction is low. Furthermore, the amino groups of Hb are randomly located on its surface. Thus, the stoichiometry of the PEG conjugated to the protein and the sites of conjugation cannot be precisely controlled, resulting in products that are not biochemically homogeneous. In addition, the linkage of the PEG to the Hb is through isopeptide bonds. As a result, the original positive charge of the surface amino group is lost during this modification. Thus, the altered surface electrostatic potential of the PEGylated Hb may also be contributing for the neutralization of the vasoactivity. We have been exploring new and novel, site-specific chemical strategies which could overcome these limitations and enable the generation of chemically welldefined, molecularly homogeneous preparations of PEGylated-Hbs with varying colligative properties (controllable by the synthetic protocol) so that the same could be used for understanding the relation between the colligative properties of acellular Hb and its vasoactivity (22, 23). In the present study, we have taken advantage of the unusual chemical reactivity of Cys-93(β) of oxyHbA and the high specificity of maleimide for the protein -SH groups for the surface decoration of HbA with PEG chains. A new series of maleimide based monofunctional PEG reagents have been developed for these studies. In the commercially available PEG-maleimide reagents, the maleimide moiety is linked to the terminal -OH of the PEG either through an ethylene linker or through an amide bond with a flexible aliphatic linker. In contrast, in our PEG maleimide reagents, we have introduced a rigid phenyl ring coupled to the PEG via a polar carbamate linkage, as the spacer between the maleimide and the PEG chain of the desired length (Figure 1). The choice of this stereochemistry for the PEG-maleimide reagent is based on our observation on the unusually high quantitative intramolecular crosslinking of HbA by bis-maleidophenyl PEG2000 (23). Reaction of HbA with monofunctional maleidophenyl PEG results in the conjugation of the PEG chains on to the intrinsic β93 -SH groups of Hb via a succinimidophenyl linkage (2 PEG chains/Hb tetramer). The molecular size of Hb has been varied by varying the chain length of the PEG used for conjugation. The physical and functional properties of these PEGylated Hbs have been compared to understand the relationship of their colli-
Bioconjugate Chem., Vol. 14, No. 2, 2003 465
Figure 1. Structure of the poly(ethylene glycol)-based monofunctional maleimide reagents.
gative properties and vasoactivity with the mass and chain length of PEG conjugated on Cy-93(β). MATERIALS AND METHODS
Hemoglobin. Human HbA was purified from the erythrocyte lysate as described earlier (24). Synthesis of Maleidophenyl Polyethylene Glycol (MalPhePEG) Reagents. Mono functional maleidophenyl derivatives of PEG5000, PEG10000, and PEG20000 were synthesized according to the procedures described earlier (22). The synthesis essentially involves condensation of p-maleimidophenylisocyanate (PMPI) with methoxy PEG. The PMPI is generated by thermal Curtius rearrangement of p-maleimidobenzoylazide, which is synthesized from p-maleimidobenzoic acid essentially by procedures described by Yoshitake et al. (25) and Annunziato et al. (26). The acyl azide is stable and may be stored indefinitely with minimal precaution. Work in our laboratories is in agreement with the observation (26) that thermal, Curtius rearrangement of p-maleimidobenzoylazide to the isocyanate is quantitative. Since the resulting isocyanate is difficult to isolate and store without degradation, we have found it convenient to derivatize the hydroxyl containing PEG compounds by generating PMPI in the presence of the PEG. The in situ generation and consumption of PMPI thus obviates the need to isolate and store this sensitive compound. Typically, 500 g of methoxy poly(ethylene glycol) (methoxy PEG5000 of low,
