Analysis of Dimeric alpha beta Subunit Exchange between PEGylated and Native Hemoglobins (alpha(2)beta(2) Tetramer) in an Equilibrated State by Intramolecular beta beta-Cross-Linking

摘要

Various chemical modifications of hemoglobin (Hb) including PEGylation have been investigated to produce red blood cell substitutes. Some of those modifications are designed on the premise that the alpha(2)beta(2) tetrameric structure of Hb is fundamentally stable and that it rarely dissociates into two alpha beta dimers in a physiological condition. However, in the present work using the "clipping" method we detected and quantitatively analyzed the considerable degree of exchange reaction of alpha beta subunits between beta 93Cys-bis-PEGylated and native Hbs through dissociation into alpha beta dimers and restructuring to alpha(2)beta(2) tetramer in a physiological condition. The equilibrium constant (K-eq) of subunit exchange reactions increased from 0.82 to 2.86 with increasing molecular weight of PEG from 2 to 40 kDa, indicating that longer PEG chains enhanced such exchange reaction. The results suggest that the exchange might occur for other modified Hbs even at a practically high concentration for use as a red blood cell substitute.