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Conjugation of Multiple Copies of Polyethylene Glycol to Hemoglobin Facilitated Through Thiolation: Influence on Hemoglobin Structure and Function

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Abstract

PEGylation induced changes in molecular volume and solution properties of HbA have been implicated as potential modulators of its vasoconstrictive activity. However, our recent studies with PEGylated Hbs carrying two PEG chains/Hb, have demonstrated that the modulation of the vasoconstrictive activity of Hb is not a direct correlate of the molecular volume and solution properties of the PEGylated Hb and implicated a role for the surface charge and/or the pattern of surface decoration of Hb with PEG. HbA has now been modified by thiolation mediated maleimide chemistry based PEGylation that does not alter its surface charge and conjugates multiple copies of PEG5K chains. This protocol has been optimized to generate a PEGylated Hb, (SP-PEG5K)6-Hb, that carries ~six PEG5K chains/Hb – HexaPEGylated Hb. PEGylation increased the O2 affinity of Hb and desensitized the molecule for the influence of ionic strength, pH, and allosteric effectors, presumably a consequence of the hydrated PEG-shell generated around the protein. The total PEG mass in (SP-PEG5K)6-Hb, its molecular volume, O2 affinity and solution properties are similar to that of another PEGylated Hb, (SP-PEG20K)2-Hb, that carries two PEG20K chains/Hb. However, (SP-PEG5K)6-Hb exhibited significantly reduced vasoconstriction mediated response than (SP-PEG20K)2-Hb. These results demonstrate that the enhanced molecular size and solution properties achieved through the conjugation of multiple copies of small PEG chains to Hb is more effective in decreasing its vasoconstrictive activity than that achieved through the conjugation of a comparable PEG mass using a small number of large PEG chains.

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Abbreviations

Hb:

hemoglobin

PEG:

poly(ethylene glycol)

PEGylation:

conjugation with PEG

PEGylated:

conjugated with PEG

4-PDS:

4,4-dithiopyridine

SP:

succinimidophenyl

SE:

succinimidoethyl

PBS:

phosphate buffered saline

Tris:

tris(hydroxymethyl) amino methane

SEC:

size exclusion chromatography

COP:

colloidal osmotic pressure

FCD:

functional capillary density

MAP:

mean arterial pressure

hypervolemic infusion:

infusion of a known volume of the test solution without removal of an equal volume of blood

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Authors and Affiliations

  1. Departments of Physiology and Biophysics, Albert Einstein College of Medicine, 10461, Bronx, NY, USA

    Belur N. Manjula, Joel M. Friedman & Seetharama A. Acharya

  2. Department of Bioengineering, University of California-San Diego, 92093, La Jolla, CA, USA

    Amy G. Tsai & Marcos Intaglietta

  3. Department of Biological Sciences, Carnegie Mellon University, 15213, Pittsburgh, PA, USA

    Ching-Hsuan Tsai & Chien Ho

  4. BioAffinity Systems, 61073, Roscoe, IL, USA

    Paul K. Smith

  5. Departments of Medicine, Albert Einstein College of Medicine, 10461, Bronx, NY, USA

    Krishnaveni Perumalsamy, Nirmala Devi Kanika & Seetharama A. Acharya

Authors
  1. Belur N. Manjula
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  2. Amy G. Tsai
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  3. Marcos Intaglietta
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  4. Ching-Hsuan Tsai
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  5. Chien Ho
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  6. Paul K. Smith
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  7. Krishnaveni Perumalsamy
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  8. Nirmala Devi Kanika
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  9. Joel M. Friedman
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  10. Seetharama A. Acharya
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Correspondence to Seetharama A. Acharya.

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Manjula, B.N., Tsai, A.G., Intaglietta, M. et al. Conjugation of Multiple Copies of Polyethylene Glycol to Hemoglobin Facilitated Through Thiolation: Influence on Hemoglobin Structure and Function. Protein J 24, 133–146 (2005). https://doi.org/10.1007/s10930-005-7837-2

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  • DOI: https://doi.org/10.1007/s10930-005-7837-2

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