Role of extension arm in PEG-Hb conjugates on the stability of the tetramer: non-conservative EAF maleimide thio-PEG mediated PEGylation.

PEGylation induced increase in colloidal osmotic pressure (COP) of Hb, one of the unique molecular properties of PEG-Hb conjugates, facilitates the neutralization of the vasoconstrictive activity of acellular Hb is a function of chemistry of conjugation of PEG-chains. The dependence of COP with the chemistry is a consequence of PEGylation induced weakening of interdimeric interactions. The conservative Extension Arm Facilitated (EAF) PEGylation exerts least influence on lowering the tetramer stability as compared to direct PEGylation. We have now designed a new, non-conservative EAF PEGylation that uses acylation chemistry to introduce the extension arm onto proteins to delineate the role of the extension arm and of the charge at the site of attachment in PEG-Hb conjugate on tetramer stability. The non-conservative EAF PEGylation does not lower the tetramer stability just as the non-conservative direct PEGylation of Hb. The impact of the extension arm in PEGylated Hb in terms of their structure and potential significance of higher tetramer stability of PEG-Hb conjugates generated by EAF-PEGylation in the in vivo toxicity and in the design of oxygen carrying plasma expander has been discussed.