PEGylated Hbs have emerged as a novel class of nohypertensive, oxygen carrying low viscosity nitric oxide producing resuscitation fluids. The limitations of PEGylation of Hb are that it induces a weakening of its interdimeric interactions and increases oxygen affinity. Besides, the influence of oxygen affinity of PEG-Hb on the tissue oxygen is still a unknown zone in the blood substitute field. In this study, we prepared two PEGylated intramolecularly crosslinked Hb and compared their performance in the extreme hemodilution model.


Hb-PEG adducts were prepared by incubation of PEG reagents with Hb to conjugate PEG chains to the amino groups on the surface of Hb molecule. The unreacted PEG was removed by tangential flow filtration against PBS using Minim (PAL Co.) at 4 °C. The oxygen equilibrium curves of Hb-PEG solutions were obtained using a Hemox analyzer (TCS Scientific, New Hope, PA). Extreme hemodilution: By progressive hemodilution, the hamster’s systemic Hct is reduced to 18% with a non- O2 carrying colloid plasma expander (dextran 70). It is at the critical transition of Hb where O2 carrying capacity becomes limited that the study materials are introduced. This final hemodilution step is performed with the study material until an 11% hematocrit is reached, a control being generated using dextran 70. O2 delivery to the microcirculation and O2 extraction to the tissue were determined by measured by the Phosphorescence Decay (PD) Technique.


The extension arm facilitate (EAF) PEGylation as well direct PEGylation using either reductive alkylation chemistry or acylation chemistry based PEGylation of αα fumaryl-Hb generates PEG Hbs with an higher oxygen affinity (∼20 mm Hg), as compared to the oxygen affinity of 30 mm Hg for αα fumaryl-Hb. The thiocarbmoylation chemistry based PEGylation of αα-fumaryl Hb using isothiocyanate phenyl PEG 5K (ITP-PEG5K) has been investigated here. TetraPEGylated αα fumaryl-Hb [(TCP-PEG5K)4 αα-Hb] generated by thiocarbmoylation chemistry generates thiourethane linkage and exhibits an oxygen affinity (34 mm Hg), which is slightly lower than that of parent molecule (30 mm Hg). The tetraPEGylation by ITP PEG exhibits very high site selectivity to the four α-amino groups, which is the same as the reductive PEGylation. The reductive PEGylation results in a significant increase in the oxygen affinity of αα-fumaryl Hb (15 mm Hg). The oxygen affinity of (TCP-PEG 5K)4 ββ-Hb is around 8 mm Hg, which is comparable to that of ββ-Hb. Thus PEG-chains on the amino termini of crosslinked Hb with thiourethane linkage is distinct as it does not increase the oxygen affinity of crosslinjed Hb. Despite the low P50 of (TCP-PEG5K)4 αα-Hb, it neutralized the in vivo hypertensive activity of αα-fumaryl Hb. Besides, the tissue oxygenation by (TCP-PEG5K)4 αα-Hb is 3.5 mmHg, and is lower than that for affinity reductive PEGylated Hb, 5.4 mmHg.


(i) Conjugation chemistry dictates the influence on the oxygen affinity of PEGylated crosslinked Hbs and not the PEG shell, (ii) The number and amount of PEG-chains in the PEG-shell dictates the plasma expander like properties of PEG Hb; (iii) PEGylation mediated attenuation of the hypertensive activity of Hb is not a correlate of its oxygen affinity; and (iv) The tissue oxygenation PEGylated αα Hb is not correlated with oxygen affinity. Accordingly, the choice of the oxygen affinity for PEG-Hb and chemistry of PEGylation for its application as an oxygen therapeutic will be dictated by the overall efficacy of tissue oxygenation expected for a PEG Hb solution in a given clinical setting. We speculate that (TCP-PEG5KK)4 ββ-Hb will achieve a tissue oxygenation better or comparable to PEG5K2 Hb, the molecule that has given the best tissue oxygenation as an oxygen therapeutic in extreme hemodilution model so far.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.