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(beta) 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 beta-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, (SP-PEG10K)(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 O(2) 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.

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Our recent studies on PEG-Hb [poly(ethylene glycol)-Hb] conjugates generated by thiolation-mediated maleimide-chemistry based PEGylation demonstrated that the vasoactivity of the PEG-Hb conjugates is a function of the configuration of the PEG chains on the surface of the protein and is independent of the PEG/protein-mass ratio [Manjula, A. G. Tsai, Intaglietta, H.-C. Tsai, Ho, Smith, Perumalsamy, Kanika, Friedman and Acharya (2005) Protein J. 24, 133-146]. A Hb conjugated with six PEG5k chains (SP-PEG5k)6-Hb, was vasoinactive. In an attempt to understand whether the chemistry of conjugation of PEG to Hb has any influence on the modulation of its functional and solution properties, we have now generated a new hexaPEGylated-Hb, (propyl-PEG5k)6-Hb, by reductive alkylation chemistry. CD (circular dichroism) spectral measurements indicated that the overall secondary structure of Hb is not adversely influenced upon PEGylation. (Propyl-PEG5k)6-Hb exhibited an increased O2 affinity with decreased co-operativity and decreased modulation by allosteric effectors comparable with that of (SP-PEG5k)6-Hb, although its Cys-93(b) is not derivatized as in the latter. On a molecular mass basis, PEG linked to Hb by reductive alkylation increased its COP (colloidal osmotic pressure) more efficiently than when linked by thiolation-mediated maleimide-chemistry. These results demonstrate that the functional properties of PEG-Hb conjugates may be a direct consequence of surface decoration of Hb with PEG, but are independent of the site (pattern) and/or the chemistry of PEGylation. However the solution properties of PEGylated Hb are influenced by the site (pattern) and/or the chemistry of PEGylation and the presence or absence of an 'extension arm' between the conjugating site of Hb and the PEG chain.

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