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PEGylation promotes hemoglobin tetramer dissociation.

Dario Caccia1, Luca Ronda, Raffaella Frassi

  • 1Dipartimento di Scienze e Tecnologie Biomediche, Dipartimento di Chimica, Biochimica e Biotecnologie per la Medicina, Universita degli Studi di Milano, Milano, Italy.

Bioconjugate Chemistry
|June 19, 2009
PubMed
Summary

Comparing oxygen carriers, poly(ethylene glycol)-hemoglobin (PEG-Hb) produced under aerobic conditions (PEG-Hb(oxy)) showed distinct functional differences from those made anaerobically (PEG-Hb(deoxy)). This heterogeneity in PEG-Hb may cause adverse effects when used as blood substitutes.

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Area of Science:

  • Biochemistry
  • Biomaterials Science
  • Physiology

Background:

  • Poly(ethylene glycol)-hemoglobin (PEG-Hb) serves as an acellular oxygen carrier, potentially replacing red blood cells in treating hypoxemia.
  • Subtle differences in PEG-Hb production can lead to variations in structure and function, impacting physiological responses and safety.

Purpose of the Study:

  • To compare the structural and functional characteristics of PEG-Hb produced under aerobic (PEG-Hb(oxy)) versus anaerobic (PEG-Hb(deoxy)) conditions.
  • To investigate the potential implications of PEG-Hb heterogeneity on its performance as a blood substitute.

Main Methods:

  • PEGylation of hemoglobin under aerobic and anaerobic conditions.
  • Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry for structural analysis.
  • Size-exclusion chromatography (SEC) to assess molecular weight distribution.
  • Functional assays including oxygen binding, allosteric effector modulation, carbon monoxide (CO) binding kinetics, and flash photolysis studies.

Main Results:

  • PEGylation resulted in a heterogeneous distribution of PEG/hemoglobin ratios for both PEG-Hb(oxy) and PEG-Hb(deoxy).
  • PEG-Hb(oxy) exhibited high oxygen affinity, minimal cooperativity, and concentration-independent functional properties, unlike PEG-Hb(deoxy) which showed concentration-dependent oxygen binding and slower CO binding.
  • Flash photolysis revealed altered CO binding kinetics and reduced quaternary transition rates in PEGylated hemoglobins, more pronounced in PEG-Hb(oxy), attributed to increased dimer dissociation and perturbed T and R states.

Conclusions:

  • Significant structural and functional heterogeneity exists in PEGylated hemoglobins, influenced by conjugation conditions (aerobic vs. anaerobic).
  • PEG-Hb(oxy) displays functional properties distinct from native hemoglobin and PEG-Hb(deoxy), potentially due to greater dimer dissociation and altered quaternary dynamics.
  • The inherent heterogeneity of PEG-Hb products may contribute to adverse physiological effects, necessitating careful consideration for their use as blood substitutes.