PEGylation is a successful approach to improve potency of a therapeutic protein. The improved therapeutic potency is mainly due to the steric shielding effect of PEG. However, the underlying mechanism of this effect on the protein is not well understood, especially on the protein interaction with its high molecular weight substrate or receptor. Here, experimental study and molecular dynamics simulation were used to provide molecular insight into the interaction between the PEGylated protein and its receptor. Staphylokinase (Sak), a therapeutic protein for coronary thrombolysis, was used as a model protein. Four PEGylated Saks were prepared by site-specific conjugation of 5 kDa/20 kDa PEG to N-terminus and C-terminus of Sak, respectively. Experimental study suggests that the native conformation of Sak is essentially not altered by PEGylation. In contrast, the bioactivity, the hydrodynamic volume and the molecular symmetric shape of the PEGylated Sak are altered and dependent on the PEG chain length and the PEGylation site. Molecular modeling of the PEGylated Saks suggests that the PEG chain remains highly flexible and can form a distinctive hydrated layer, thereby resulting in the steric shielding effect of PEG. Docking analyses indicate that the binding affinity of Sak to its receptor is dependent on the PEG chain length and the PEGylation site. Computational simulation results explain experimental data well. Our present study clarifies molecular details of PEG chain on protein surface and may be essential to the rational design, fabrication and clinical application of PEGylated proteins.
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.
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.
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).
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.
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