PEGylation Promotes Hemoglobin Tetramer Dissociation

Abstract

Hemoglobin conjugated with poly(ethylene glycol) (PEG) acts as an oxygen carrier free in plasma, substituting red blood cells in supplementing oxygen in hypo-oxygenation pathologies. Given the complexity of oxygen delivery controls, subtle structural and functional differences in PEGylated hemoglobins might be associated with distinct physiological responses and, potentially, adverse effects. We have compared hemoglobin PEGylated under anaerobic conditions, called PEG-Hb(deoxy), with hemoglobin PEGylated under aerobic conditions, called PEG-Hb(oxy), a product that mimics Hemospan, produced by Sangart, Inc. SDS PAGE and MALDI-TOF analyses demonstrated that PEG conjugation yields products characterized by a broad distribution of PEG/hemoglobin ratios. The elution profiles in size-exclusion chromatography indicate that both products exhibit a more homogeneous distribution of molecular weight/hydrodynamic volume under deoxy conditions and at higher concentrations. PEG-Hb(oxy) shows high oxygen affinity, low modulation of allosteric effectors, almost no cooperativity, a fast and monophasic CO binding, and a limited dependence of functional properties on concentration, whereas PEG-Hb(deoxy) exhibits oxygen binding curves that significantly depend on protein concentration, and a slow CO binding, similar to native hemoglobin. PEGylated CO-hemoglobins, probed by flash photolysis, exhibited a lower amplitude for the geminate rebinding phase with respect to native hemoglobin and a negligible T state bimolecular CO rebinding phase. These findings are explained by an increased dissociation of PEGylated hemoglobins into dimers and perturbed T and R states with decreased quaternary transition rates. These features are more pronounced for PEG-Hb(oxy) than PEG-Hb(deoxy). The detected heterogeneity might be a source of adverse effects when PEGylated Hbs are used as blood substitutes.

A Trivalent Enzymatic System for Uricolytic Therapy of HPRT Deficiency and Lesch-Nyhan Disease

“…tetramers containing 4 unmodified protein subunits) (Table I). The calculation was carried out by means of a combinatorial procedure that we previously reported for PEGylated hemoglobin (42). The results show that i) Dr Uox PEGylation with MAL-PEG of different molecular weight resulted in similar PEGylation yields, and ii) despite the relative abundance of non-PEGylated monomers, after quaternary assembly only a low percentage of unmodified tetramers occurs (Table I).…”

“…In order to change the target aminoacid for the PEGylation reaction, while using the same PEG derivative (MAL-PEG), we took advantage of the extension arm-facilitated PEGylation, a two-step procedure which has already been used in PEGylation of human hemoglobin to increase the number of reaction sites (33,44) while avoiding the derivatization of cysteines with crucial role in protein activity (42,45). Dr Urad was pretreated with 2-iminothiolane (IMT), a cyclic molecule that reacts with the primary amino groups of lysine and N-terminal residues to form sulfhydryl groups.…”

Enzymatic Synthesis of Poly(α‐ethyl β‐aspartate) by Poly(ethylene glycol) Modified Poly(aspartate) Hydrolase‐1

“…The present conflict between the molecular masses of the PEG-modified enzymes estimated from SDS-PAGE analysis and their individual molecular masses seems to be consistent with the previous results on the SDS-PAGE and MALDI-TOF MS analyses of the PEGmodified proteins. [21,22] They demonstrated that the apparent molecular weights of the PEG-modified proteins on SDS-PAGE were higher than their actual molecular weights on MALDI-TOF MS. Therefore, taking the present and previous findings into consideration, it was concluded that the PEG-modified PAA hydrolases-1 made their molecular weights seem higher on SDS-PAGE than the actual ones and possibly contained 1-5 PEGs per molecule.…”

Design of Nonhypertensive Conjugated Hemoglobins as Novel Resuscitation Fluids

“…The tetramer dimer-tetramer dissociation constant of these PEGylated molecules with Cys-93(b) free is indistinguishable from that of unmodified Hb; the presence of PEGylation on Cys-93(b) has only very small influence on the dissociation constant. On the other hand, a comparison of the stability of quaternary structure of MP4 versus Euro PEG Hb has suggested that in MP4 the quaternary structure of the molecule (Caccia et al 2009) has been weakened leading to the presence of dissociated PEGylated species. This may be the influence of one-step versus two-step EAF PEGylation platform.…”