PEGylation of Val-1(alpha) destabilizes the tetrameric structure of hemoglobin.
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.
Hu
Tao
T
Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Li
Dongxia
D
Manjula
Belur N
BN
Brenowitz
Michael
M
Prabhakaran
Muthuchidambaram
M
Acharya
Seetharama A
SA
D000494:Allosteric Regulation; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D006454:Hemoglobins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007700:Kinetics; D008970:Molecular Weight; D010100:Oxygen / Q000378:metabolism; D011092:Polyethylene Glycols / Q000378:metabolism*; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D014461:Ultracentrifugation; D014633:Valine / Q000378:metabolism*
D006454:Hemoglobins; D011092:Polyethylene Glycols; D014633:Valine; D010100:Oxygen