Role of Extension Arm in PEG-Hb Conjugates on the Stability of the Tetramer: Non-conservative EAF Maleimide Thio-PEG Mediated PEGylation

Figures & data

Figure 1.  Schematic representation of three types of linkages in PEGylated Hbs investigated for the correlation of PEGylation induced COP of Hb with the chemistry of conjugation.

Table 1. Influence of PEG-conjugation platform on the molecular radius of hexaPEGylated Hb and of αα-fumaryl intramolecular crosslinked Hb

	Hydrodynamic radius (nm)
PEGylation Platform	Hb	αα-fumaryl Hb	References
None	3.0	3.0
EAF-PEGylation	6.3	6.8	[9]
Reductive alkylation	5.4	6.6	[17]
Acylation	5.9	6.9	[18]

Figure 2.  Schematic representation of non-conservative EAF PEGylation to generate thio succinimido caproyl-PEG5K-Hb [(TSC-PEG5K)-Hb].

Figure 3.  Influence of loss of charge at the site of attachment of extension arm on the hydrodynamic volume (A) and of HPLC patterns (B) of Hb and of intramolecularly crosslinked Hb. Comparison of hexaPEGylated (TSC-PEG5K)₆-HbA and (TSC-PEG5K)₆-ααHbA with (SP-PEG5K)₆-HbA by SEC (A) and HPLC (B). Curve a, Hb; curve b, (TSC-PEG5K)₆-Hb; curve c, (SP-PEG5K)₆-Hb; curve d, ααHb; curve e, (TSC-PEG5K)₆-ααHb.

Table 2. Influence of non-conservative extension arm on the PEGylation induced COP, viscosity and molecular hydrodynamic radius of hexaPEGylated Hb

Sample	COP (mm Hg)	Visco- sity (cP)	Radius (nm)
Hb	14.3	1.0	3.0
(TSC-PEG5K)6-Hb	58.0	2.0	5.4
(TSC-PEG5K)6-ααHb	76.4	2.3	5.7
Propionyl(PEG5K)6-Hb^1	91.0	2.4	5.9
Propionyl(PEG5K)6-ααHb^1	50.0	2.4	6.9
(SP-PEG5K)6-Hb^2	70.4	2.5	6.5

Note: 1. Data shown are at 2.5 % from Li et al., 2008 [18] and the rest all at 4 % 2. Data from Manjula et al., 2005 [9].

Acharya S. A., Friedman J. M., Manjula B. N., Intaglietta M., Tsai A. G., Winslow R. M., Malavalli A., Vandegriff K., Smith P. K. Artif Cells Blood Substit Immobil Biotechnol 2005; 33(3)239–255

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Hu T., Prabhakaran M., Acharya S. A., Manjula B. N. Biochem J 2005; 392(Pt 3)555–564

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Hu T., Manjula B. N., Li D., Brenowitz M., Acharya S. A. Biochem J 2007; 402(1)143–151

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