TY - JOUR
T1 - Conjugation of multiple copies of polyethylene glycol to hemoglobin facilitated through thiolation
T2 - Influence on hemoglobin structure and function
AU - Manjula, Belur N.
AU - Tsai, Amy G.
AU - Intaglietta, Marcos
AU - Tsai, Ching Hsuan
AU - Ho, Chien
AU - Smith, Paul K.
AU - Perumalsamy, Krishnaveni
AU - Kanika, Nirmala Devi
AU - Friedman, Joel M.
AU - Acharya, Seetharama A.
N1 - Funding Information:
The assistance of Dr. Dongxia Li for some of the molecular radius measurements is greatly appreciated. This research was supported by a grant-in-aid from the American Heart Association Heritage Affiliate, the National Institutes of Health grants HL58247, HL71064 and USPHS NIH Bioengineering Partnership grant 1R24 HL 64395, and the US Army grant PR023085.
PY - 2005/4
Y1 - 2005/4
N2 - 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, O 2 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.
AB - 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, O 2 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.
KW - Colligative property
KW - Hydrodynamic volume
KW - PEG shell
KW - PEGylated hemoglobin
KW - PEGylation
KW - Vasoactivity
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U2 - 10.1007/s10930-005-7837-2
DO - 10.1007/s10930-005-7837-2
M3 - Article
C2 - 16096719
AN - SCOPUS:23844456246
SN - 1572-3887
VL - 24
SP - 133
EP - 146
JO - Protein Journal
JF - Protein Journal
IS - 3
ER -