Molecular aspects of the high oxygen affinity of non-hypertensive hexa pegylated hemoglobin, [(SP-PEG5K)(6)-Hb].
The development of hexaPEGylated Hb, (SP-PEG5K)(6)-Hb, using the newly designed thiolation-mediated maleimide chemistry based PEGylation, has validated the concept that engineering 'plasma volume expander' -like properties to Hb neutralizes its vasoactivity. The high O(2) affinity of hexaPEGylated Hb has been attributed to the two PEG-5K chains on its two Cys-93(beta) residues. In an attempt to map the influence of the additional four PEG-5K chains of HexaPEGylated Hb on the O(2) affinity, we have now investigated the influence of PEGylation of the surface amino groups alone on the subunit interface interactions and O(2) affinity of Hb using rHb(betaC93A). The molecular radius of PEGylated rHb(betaC93A) was slightly smaller than that of (SP-PEG5K)(6)-Hb, and the overall site-selectivity of PEGylation in the PEGylated rHb(betaC93A) at Lys-residues was comparable to that of (SP-PEG5K)(6)-Hb. Proton NMR studies have shown that the conjugation of the protein with PEG-5K does not have any significant influence on its subunit interface interactions. Surprisingly, the influence of PEGylation on the O(2) affinity and Bohr effect of HbA and rHb(betaC93A) is also nearly the same. Apparently, conjugation of PEG-chains to Lys residues of Hb by the thiolation mediated PEGylation induces unique changes in the structure of the hydration shell of Hb (layer of tightly bound water molecules), which, in turn, induces constraints in its R to T conformational transition to favor the more hydrated R-state.
Li
Dongxia
D
Department of Physiology, Albert Einstein College of Medicine, Bronx, NY, USA.
Manjula
Belur N
BN
Ho
Nancy T
NT
Simplaceanu
Virgil
V
Ho
Chien
C
Acharya
A Seetharama
AS
D001665:Binding Sites; D001794:Blood Pressure; D001802:Blood Substitutes / Q000737:chemistry*; D006441:Hemoglobin A; D006454:Hemoglobins / Q000737:chemistry*; D006801:Humans; D008239:Lysine; D009682:Magnetic Resonance Spectroscopy; D010100:Oxygen / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000737:chemistry*; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry; D014867:Water
D001802:Blood Substitutes; D006454:Hemoglobins; D021122:Protein Subunits; D014867:Water; D011092:Polyethylene Glycols; D006441:Hemoglobin A; D008239:Lysine; D010100:Oxygen