Enhanced molecular volume of conservatively pegylated Hb: (SP-PEG5K) 6-HbA is non-hypertensive

Seetharama A. Acharya, Joel M. Friedman, Belur N. Manjula, Marcos Intaglietta, Amy G. Tsai, Robert M. Winslow, Ashok Malavalli, Kim Vandegriff, Paul K. Smith

Research output: Contribution to journalArticle

32 Scopus citations

Abstract

Recent studies have suggested that the "pressor effect" of acellular Hb is a consequence of perturbation of the macro-and microcirculatory system in multiple ways, and that PEGylation is an effective approach for controlling the same. In an attempt to confirm this concept, a new and simple thiolation mediated, maleimide chemistry-based conservative PEGylation protocol has been developed to conjugate multiple copies of PEG-chains to Hb. This approach combines the high reactivity of maleimides towards thiols with the propensity of iminothiolane to derivatize the ε-amino groups of proteins into reactive thiol groups, with conservation of their positive charge. One of the PEGylated products, namely (SP-PEGSK)6-HbA, that carries on an average six copies of PEG5000 chains per Hb, is non-hypertensive in hamster top load and in rat 50% exchange transfusion models. This hexa-PEGylated-Hb has (i) a hydrodynamic volume corresponding to that of an oligomerized Hb of 256 kDa, (ii) a molecular radius of ∼6.8 nm, (iii) high oxygen affinity, (iv) lowered Bohr effect, and (v) increased viscosity and colloidal osmotic pressure. These properties of (SP-PEGSK)6-HbA are consistent with the emerging new paradigms for the design of Hb based oxygen carriers and confirm the concept that the "pressor effect" of Hb is a multifactorial event. The thiolation mediated maleimide chemistry-based PEGylation protocol described here for the generation of (SP-PEGSK)6-Hb is simple, highly efficient, and is carried out under oxy conditions. The results demonstrate that a non-hypertensive PEG-Hb can be generated by conjugation of a lower number of PEG chains than previously reported.

Original languageEnglish (US)
Pages (from-to)239-255
Number of pages17
JournalArtificial Cells, Blood Substitutes, and Immobilization Biotechnology
Volume33
Issue number3
DOIs
StatePublished - Sep 1 2005

ASJC Scopus subject areas

  • Biotechnology
  • Biomedical Engineering

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    Acharya, S. A., Friedman, J. M., Manjula, B. N., Intaglietta, M., Tsai, A. G., Winslow, R. M., Malavalli, A., Vandegriff, K., & Smith, P. K. (2005). Enhanced molecular volume of conservatively pegylated Hb: (SP-PEG5K) 6-HbA is non-hypertensive. Artificial Cells, Blood Substitutes, and Immobilization Biotechnology, 33(3), 239-255. https://doi.org/10.1081/BIO-200066365