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ABSTRACT: The propensity of site-specific carboxymethylation and propylation of Val-1(α) of Hb to attenuate the reductive hexaPEGylation-induced dissociation of tetramers has been investigated. Only reductive propylation of Val-1(α), which increases the stability of oxy Hb, attenuates the reductive hexaPEGylation-induced dissociation. Increasing the stability of the oxy conformation of Hb by chemical or genetic approaches is a strategy to generate PEGylated Hbs with native-like tetramer stability using direct PEGylation platforms. This new approach and EAF-PEGylation are the only two alternate PEGylation strategies available to design stable second-generation vasoinactive uncrosslinked PEGylated Hbs with native-like tetramer stability.
Artificial Cells Blood Substitutes and Biotechnology (formerly known as Artificial Cells Blood Substitutes and Immobilization Bi 04/2011; 39(2):69-78. · 0.94 Impact Factor
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ABSTRACT: Cys-93(beta) of hemoglobin (Hb) was reversibly protected as a mixed disulfide with thiopyridine during extension arm facilitated (EAF) PEGylation and its influence on the structural and functional properties of the EAF-PEG-Hb has been investigated. Avoiding PEGylation of Cys-93(beta) in the EAF-PEG-Hb lowers the level of perturbation of heme pocket, alpha1beta2 interface, autoxidation, heme loss, and the O(2) affinity, as compared to the EAF-PEG-Hb with PEGylation of Cys-93(beta).The structural and functional advantages of reversible protection of Cys-93(beta) during EAF PEGylation of oxy-Hb has been compared with Euro PEG-Hb generated by EAF PEGylation of deoxy Hb where Cys-93(beta) is free in the final product. The alphaalpha-fumaryl cross-linking and EAF PEGylation targeted exclusively to Lys residues has been combined together for generation of second-generation EAF-PEG-Hb with lower oxygen affinity. The PEG chains engineered on Lys as well as PEGylation of Cys-93(beta) independently contribute to the stabilization of oxy conformation of Hb and hence increase the oxygen affinity of Hb. However, oxygen affinity of the EAF-PEG-alphaalpha-Hb is more sensitive to the presence of PEGylation on Cys-93(beta) than that of the EAF-PEG-Hb. The present modified EAF PEGylation platform is expected to facilitate the design of novel versions of the EAF-PEG-Hbs that can now integrate the advantages of avoiding PEGylation of Cys-93(beta).
Bioconjugate Chemistry 10/2009; 20(11):2062-70. · 4.93 Impact Factor
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ABSTRACT: A hexaPEGylated hemoglobin (Hb), (Propyl-PEG5K)(6)-Hb, is essentially in alphabeta dimers (Hu et al. (2007) Biochem. J. 402, 143-151). In order to provide a biochemical insight into the tetramer-dimer dissociation of this PEGylated Hb, we prepared and characterized two PEGylated Hbs site-specifically modified at Val-1(alpha) and at Val-1(beta), respectively. PEGylation at Val-1(alpha) and at Val-1(beta) increase the tetramer-dimer dissociation constant (K(d)) of Hb by 2 and 1 order of magnitude, respectively. Accordingly, the sites of PEGylation can determine the tetramer stability of the PEGylated Hb. In order to determine the role of the polyethylene glycol (PEG) chains on the tetramer stability of Hb, we prepared a propylated Hb site-specifically modified at Val-1(alpha). Interestingly, site-specific propylation of Hb at Val-l(alpha) stabilizes the Hb tetramer by 1 order of magnitude. Therefore, conjugation of the PEG chains at Val-1(alpha) can greatly destabilize the tetramer stability of Hb. On the structural aspects, the PEG chains conjugated at Va-1(alpha) unfavorably alter the heme environment and quaternary structure and destabilize the alpha1beta2 interface of Hb. On the functional aspects, the PEG chains conjugated at Val-1(alpha) decrease the Hill coefficient, the Bohr effect of Hb and the sensitization to the presence of the allosteric effectors. In contrast, PEGylation of Hb at Val-1(beta) gives rise to less pronounced structural alteration and different functional change.
Biochemistry 02/2009; 48(3):608-16. · 3.42 Impact Factor
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ABSTRACT: A hexaPEGylated hemoglobin (Hb), (Propyl-PEG5K)6-Hb, is essentially in αβ dimers (Hu et al. (2007) Biochem. J. 402, 143−151). In order to provide a biochemical insight into the tetramer−dimer dissociation of this PEGylated Hb, we prepared and characterized two PEGylated Hbs site-specifically modified at Val-1(α) and at Val-1(β), respectively. PEGylation at Val-1(α) and at Val-1(β) increase the tetramer−dimer dissociation constant (Kd) of Hb by 2 and 1 order of magnitude, respectively. Accordingly, the sites of PEGylation can determine the tetramer stability of the PEGylated Hb. In order to determine the role of the polyethylene glycol (PEG) chains on the tetramer stability of Hb, we prepared a propylated Hb site-specifically modified at Val-1(α). Interestingly, site-specific propylation of Hb at Val-l(α) stabilizes the Hb tetramer by 1 order of magnitude. Therefore, conjugation of the PEG chains at Val-1(α) can greatly destabilize the tetramer stability of Hb. On the structural aspects, the PEG chains conjugated at Va-1(α) unfavorably alter the heme environment and quaternary structure and destabilize the α1β2 interface of Hb. On the functional aspects, the PEG chains conjugated at Val-1(α) decrease the Hill coefficient, the Bohr effect of Hb and the sensitization to the presence of the allosteric effectors. In contrast, PEGylation of Hb at Val-1(β) gives rise to less pronounced structural alteration and different functional change.
01/2009;
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ABSTRACT: The PEGylated hemoglobin (Hb) has been evaluated as a potential blood substitute. In an attempt to understand the autoxidation of the PEGylated Hb, we have studied the autoxidation of the PEGylated Hb site-specifically modified at Cys-93(beta) or at Val-1(beta). PEGylation of Hb at Cys-93(beta) perturbed the heme environment and increased the autoxidation rate of Hb, which is at a higher level than that caused by PEGylation at Val-1(beta). The perturbation of the heme environment of Hb is attributed to the maleimide modification at Cys-93(beta) and not due to conjugation of the PEG chains. However, the PEG chains enhance the autoxidation and the H 2O 2 mediated oxidation of Hb. Accordingly, the PEG chains are assumed to increase the water molecules in the hydration layer of Hb and enhance the autoxidation by promoting the nucleophilic attack of heme. The autoxidation rate of the PEGylated Hb does not show an inverse correlation with the oxygen affinity. The H 2O 2 mediated structural loss and the heme loss of Hb are increased by maleimide modification at Cys-93(beta) and further decreased by conjugation of the PEG chains. The autoxidation of the PEGylated Hbs is attenuated significantly in the plasma, possibly due to the presence of the antioxidant species in the plasma. This result is consistent with the recent suggestion that there is no direct correlation between the in vitro and in vivo autoxidation of the PEGylated Hb. Therefore, the pattern of PEGylation can be manipulated for the design of the PEGylated Hb with minimal autoxidation.
Biochemistry 10/2008; 47(41):10981-90. · 3.42 Impact Factor
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ABSTRACT: The influence of intramolecular cross-links on the molecular, structural and functional properties of PEGylated {PEG [poly(ethylene glycol)]-conjugated} haemoglobin has been investigated. The sites and the extent of PEGylation of haemoglobin by reductive alkylation are not influenced by the presence of an alphaalpha-fumaryl cross-link at Lys-99(alpha). The propylated hexaPEGylated cross-linked haemoglobin, (propyl-PEG5K)(6)-alphaalpha-Hb, exhibits a larger molecular radius and lower colloidal osmotic pressure than propylated hexaPEGylated non-cross-linked haemoglobin, (propyl-PEG5K)(6)-Hb. Perturbation of the haem microenvironment and the alpha1beta2 interface by PEGylation of haemoglobin is reduced by intramolecular cross-linking. Sedimentation velocity analysis established that PEGylation destabilizes the tetrameric structure of haemoglobin. (Propyl-PEG5K)(6)-Hb and (propyl-PEG5K)(6)-alphaalpha-Hb sediment as stable dimeric and tetrameric molecules, respectively. The betabeta-succinimidophenyl PEG-2000 cross-link at Cys-93(beta) outside the central cavity also influences the molecular properties of haemoglobin, comparable to that by the alphaalpha-fumaryl cross-link within the central cavity. However, the influence of the two cross-links on the oxygen affinity of PEGylated haemoglobin are very distinct, indicating that the high oxygen affinity of PEGylated haemoglobin is not a direct consequence of the dissociation of the haemoglobin tetramers into dimers. alphaalpha-Fumaryl cross-linking is preferred to modulate both oxygen affinity and molecular properties of PEGylated haemoglobin, and cross-linking outside the central cavity could only modulate molecular properties of PEGylated haemoglobin. It is suggested that PEGylation induces a hydrodynamic drag on haemoglobin and this plays a role in the microcirculatory properties of PEGylated haemoglobin.
Biochemical Journal 03/2007; 402(1):143-51. · 4.90 Impact Factor
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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.
Biochemical Journal 01/2006; 392(Pt 3):555-64. · 4.90 Impact Factor
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ABSTRACT: High hydrodynamic volume, high viscosity and high colloidal osmotic pressure (COP) of PEGylated hemoglobin (Hb) have been suggested to neutralize the vasoactivity of acellular Hb. Consequences of non-conservative PEGylation (positive charge of the amino groups at the PEGylation sites is neutralized) using succinimidyl-ester of propionic acid PEG5K on the properties of PEGylated Hb have now been investigated. Non-conservative PEGylation of Hb leads to a much higher increase in the COP and viscosity of Hb than conservative extension arm facilitated (EAF) PEGylation of Hb. Introduction of αα-fumaryl crosslinking decreased the COP of non-conservative PEGylated Hb by stabilization of interdimeric interactions. Compared to the EAF-PEGylated αα-fumaryl Hb, non-conservative PEGylated product shows a comparable COP and higher viscosity. Conservative PEGylation of αα-fumaryl Hb by reductive alkylation chemistry does not increase the COP to this level, but enhanced the molecular volume and viscosity comparable to EAF-PEGylated product. Thus, the molecular properties of PEGylated Hb can be fine tuned using different PEGylation platforms and provide a unique opportunity for the design of second generation PEGylated Hbs.
Biochimica et Biophysica Acta (BBA) - Proteins & Proteomics.
