Molecular Aspects of the High Oxygen Afinity of Non-Hypertensive Hexa Pegylated Hemoglobin, [(SP-PEG5K)₆-Hb]

Abstract

The development of hexaPEGylated Hb, (SP-PEG5K)(6)-Hb, using the newly designed thiolation-mediated maleimide chemistry based PEGylation, has validated the concept that engineering 'plasma volume expander' -like properties to Hb neutralizes its vasoactivity. The high O(2) affinity of hexaPEGylated Hb has been attributed to the two PEG-5K chains on its two Cys-93(beta) residues. In an attempt to map the influence of the additional four PEG-5K chains of HexaPEGylated Hb on the O(2) affinity, we have now investigated the influence of PEGylation of the surface amino groups alone on the subunit interface interactions and O(2) affinity of Hb using rHb(betaC93A). The molecular radius of PEGylated rHb(betaC93A) was slightly smaller than that of (SP-PEG5K)(6)-Hb, and the overall site-selectivity of PEGylation in the PEGylated rHb(betaC93A) at Lys-residues was comparable to that of (SP-PEG5K)(6)-Hb. Proton NMR studies have shown that the conjugation of the protein with PEG-5K does not have any significant influence on its subunit interface interactions. Surprisingly, the influence of PEGylation on the O(2) affinity and Bohr effect of HbA and rHb(betaC93A) is also nearly the same. Apparently, conjugation of PEG-chains to Lys residues of Hb by the thiolation mediated PEGylation induces unique changes in the structure of the hydration shell of Hb (layer of tightly bound water molecules), which, in turn, induces constraints in its R to T conformational transition to favor the more hydrated R-state.

Design of Nonhypertensive Conjugated Hemoglobins as Novel Resuscitation Fluids

“…This observation is consistent with the observation that EAF P5K6 rHb generated using the rHb is also an high oxygen affinity species (Li et al 2007) reflecting the high oxygen affinity inducing activity of EAF PEGylation targeted to exclusively to the e-amino groups. At Einstein, Hb has also been EAF PEGylated exclusively at e-amino groups under oxy conditions achieved through the reversible protection of Cys-93(b) during EAF PEGylation and this product is also high oxygen affinity species.…”

“…All three PEGylated derivatives are high oxygen affinity molecular species since all have the Cys-93(b) PEGylated and attenuate the in vivo hypertensive activity of the acellular Hb to a considerable degree (Li et al 2006(Li et al , 2007. The oxygen affinities of these PEGylated species are comparable.…”

Hemoglobin Conjugates with Antioxidant Enzymes (Hemoglobin-Superoxide Dismutase-Catalase) via Poly(Ethylene Glycol) Crosslinker for Protection of Pancreatic Beta RINm5F Cells in Hypoxia

“…It was shown in a recent study that the conjugation of PEG-chains to lysine residues of Hb by the thiolation-mediated PEGylation induced structural changes by increasing the hydration shell, which further helped in the stabilization of favorable relaxed state conformation of Hb. 33 This stabilization of relaxed state by PEG's hydration shell or modifications that help the transition of Hb from T to R conformation may be responsible for the lowering of p50. 13 However, in the current method of conjugation, it is not feasible to know about which amino group perturbation is critical for lowering the p50 of Hb molecules and thus the lowering of the p50 of the final conjugates.…”

Synthesis and characterization of hemoglobin conjugates with antioxidant enzymes via poly(ethylene glycol) cross-linker (Hb–SOD–CAT) for protection from free radical stress

“…It was shown in a recent study that the conjugation of PEG-chains to lysine residues of Hb by the thiolation mediated PEGylation induced structural changes by increasing the hydration shell, which further helped in the stabilization of relaxed high affinity conformation [44]. It was shown that methemoglobin levels needs to be maintained below 10% in PEG-conjugated hemoglobins to efficiently oxygenate tissues [45].…”