Influence of the chemistry of conjugation of poly(ethylene glycol) to Hb on the oxygen-binding and solution properties of the PEG-Hb conjugate

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(β) 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.