PEGylation promotes hemoglobin tetramer dissociation.
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.
Caccia
Dario
D
Dipartimento di Scienze e Tecnologie Biomediche, Dipartimento di Chimica, Biochimica e Biotecnologie per la Medicina, Universita degli Studi di Milano, Milano, Italy.
Ronda
Luca
L
Frassi
Raffaella
R
Perrella
Michele
M
Del Favero
Elena
E
Bruno
Stefano
S
Pioselli
Barbara
B
Abbruzzetti
Stefania
S
Viappiani
Cristiano
C
Mozzarelli
Andrea
A
D000494:Allosteric Regulation; D001802:Blood Substitutes / Q000032:analysis* / Q000378:metabolism*; D002248:Carbon Monoxide / Q000378:metabolism; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006454:Hemoglobins / Q000032:analysis* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D010100:Oxygen / Q000378:metabolism; D011092:Polyethylene Glycols / Q000032:analysis* / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization
D001802:Blood Substitutes; D006454:Hemoglobins; C109960:PEG-hemoglobin; D011092:Polyethylene Glycols; D002248:Carbon Monoxide; D010100:Oxygen