Influence of the chemistry of conjugation of poly(ethylene glycol) to Hb on the oxygen-binding and solution properties of the PEG-Hb conjugate

Abstract

Our recent studies on PEG-Hb [poly(ethylene glycol)-Hb] conjugates generated by thiolation-mediated maleimide-chemistry based PEGylation demonstrated that the vasoactivity of the PEG-Hb conjugates is a function of the configuration of the PEG chains on the surface of the protein and is independent of the PEG/protein-mass ratio [Manjula, A. G. Tsai, Intaglietta, H.-C. Tsai, Ho, Smith, Perumalsamy, Kanika, Friedman and Acharya (2005) Protein J. 24, 133-146]. A Hb conjugated with six PEG5k chains (SP-PEG5k)6-Hb, was vasoinactive. In an attempt to understand whether the chemistry of conjugation of PEG to Hb has any influence on the modulation of its functional and solution properties, we have now generated a new hexaPEGylated-Hb, (propyl-PEG5k)6-Hb, by reductive alkylation chemistry. CD (circular dichroism) spectral measurements indicated that the overall secondary structure of Hb is not adversely influenced upon PEGylation. (Propyl-PEG5k)6-Hb exhibited an increased O2 affinity with decreased co-operativity and decreased modulation by allosteric effectors comparable with that of (SP-PEG5k)6-Hb, although its Cys-93(b) is not derivatized as in the latter. On a molecular mass basis, PEG linked to Hb by reductive alkylation increased its COP (colloidal osmotic pressure) more efficiently than when linked by thiolation-mediated maleimide-chemistry. These results demonstrate that the functional properties of PEG-Hb conjugates may be a direct consequence of surface decoration of Hb with PEG, but are independent of the site (pattern) and/or the chemistry of PEGylation. However the solution properties of PEGylated Hb are influenced by the site (pattern) and/or the chemistry of PEGylation and the presence or absence of an 'extension arm' between the conjugating site of Hb and the PEG chain.

Preparation of ultrapure bovine and human hemoglobin by anion exchange chromatography

“…Compared to values reported in the literature [29], the P 50 for purified hHb is similar. Hu et al [10] reported a P 50 of 14 torr and a cooperativity of 2.8 for hHb, whereas [29] reported a P 50 of 15.1 torr and a cooperativity of 2.97 for hHb. The difference between these published values and the values in this work are easily explained in terms of the different buffer conditions used for measuring the oxygen binding equilibria.…”

“…The difference between these published values and the values in this work are easily explained in terms of the different buffer conditions used for measuring the oxygen binding equilibria. For example, the oxygen binding equilibria of hHb in this study was measured in Hemox buffer, whereas [10] used PBS (pH = 7.4) as the measurement buffer. These changes in the buffer composition explain the observed variation in P 50 .…”

“…A variety of HBOCs exist and include: recombinant Hbs [4], cross-linked Hbs [5], polymerized Hbs [6][7][8][9], PEGylated Hbs [10], liposome encapsulated Hbs [11][12][13], polymersome encapsulated Hbs [14] and other particle encapsulated Hbs [15][16][17]. In order to synthesize and/ or formulate these HBOCs, a pure source of Hb is needed.…”

Design of Nonhypertensive Conjugated Hemoglobins as Novel Resuscitation Fluids

“…Accordingly, it can be concluded that site selectivity of PEGylation is also not the determinant of the PEGylation induced in vivo neutralization of the vasoconstrictive activity of acellular Hb. Besides, we have hexaPEGylated Hb by direct PEGylation using reductive alkylation chemistry (Hu et al 2005) and active ester chemistry , and all the hexaPEGylation protocols induce the neutralization of the vasoactivity of Hb. Though all hexaPEGylated Hbs are high oxygen affinity Hbs, there were noticeable differences in the oxygen affinity of these molecules and also in the COP, but that does not seem to have much correlation with the attenuation of natural vasoconstriction brought upon by the presence of intravascular acellular Hb.…”

Combining the Influence of Two Low O2 Affinity-Inducing Chemical Modifications of the Central Cavity of Hemoglobin

“…Accordingly, we have been considering the use of low O 2 -affinity Hbs instead of using normal adult human Hb as substrates for the generation of PEGylated Hbs using the same protocols discussed above (19)(20)(21)(22).…”