Analysis of Dimeric αβ Subunit Exchange between PEGylated and Native Hemoglobins (α₂β₂ Tetramer) in an Equilibrated State by Intramolecular ββ-Cross-Linking

Abstract

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 αβ tetrameric structure of Hb is fundamentally stable and that it rarely dissociates into two αβ 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 αβ subunits between β93Cys-bis-PEGylated and native Hbs through dissociation into αβ dimers and restructuring to αβ tetramer in a physiological condition. The equilibrium constant ( K) 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.

Ring-Opening Polymerization of Hemoglobin

“…In the reaction mixture, both Hb-rPEG and dimeric Hb-PEG-Hb, and their corresponding oligomers were generated because a longer PEG chain enhances the exchange of αβ subunits between PEGylated and native Hbs. 26 The existence of such oligomers was ascertained by DBBF crosslinking of the mixture of Hb-rPEG and native Hb (Figures 7B, 7C, and 7D). We reported recently that PEGylated Hb is allowed to exchange αβ subunits with native Hb.…”

“…DBBF crosslinked Hb (XLHb) was also prepared as reported in the literature. 26,30 The Hb derivative concentrations were determined using Hemoglobin B-test Wako (Fujifilm Wako Pure Chemical Corp.). Sodium chloride, potassium chloride, disodium hydrogen phosphate (Na 2HPO4), potassium dihydrogen phosphate (KH2PO4), polyethylene glycol 20,000 (20 kDa PEG), ammonium sulfate, Tris(hydroxymethyl) aminomethane (Tris), sucrose, sodium dodecyl sulfate (SDS), 2-mercaptoethanol, bromophenol blue, and Coomassie brilliant blue (Quick CBB) were purchased from Fujifilm Wako Pure Chemical Corp. (Osaka, Japan).…”

“…Free α subunits remained after crosslinking at every concentration, indicating that DBBF crosslinks PEGylated β subunits selectively in the same manner as that of native Hb. 26 The SEC profile of the reaction products of Hb-rPEG and DBBF at Hb concentration of show that the peak of the polymer appeared abruptly at 6.2 min. The polymer size almost reaches the void volume, and it is the detection limit of the column (protein KW804).…”

Entropy-Driven Supramolecular Ring-Opening Polymerization of a Cyclic Hemoglobin Monomer for Constructing a Hemoglobin–PEG Alternating Polymer with Structural Regularity

“…Cellular HBOCs have been produced by encapsulation of Hbs with synthetic polymer membranes or in phospholipid vesicles (liposomes) mimicking the cellular structure of RBCs. ,, Acellular HBOCs have been produced by various chemical modifications of Hbs. For instance, intra-molecular αα-cross-linking or ββ-cross-linking inhibits the dissociation of an α 2 β 2 tetrameric Hb molecule. , Conjugations of polyethylene glycol (PEG) or albumins decrease the antigenicity of a Hb molecule by surface coating. − Moreover, inter-molecular conjugation of multiple Hbs by glutaraldehyde enlarges the particle …”

Prolonged functional life span of artificial red cells in blood circulation by repeated methylene blue injections

“…or unknown pathogens; (iii) possibility of blood type mismatching by human error, and (iv) blood supply-demand unbalance in an aging society because the majority of blood donors are young people while recipients include many elder people. Hemoglobin-vesicle (HbV) is one HBOC encapsulating purified and concentrated Hb solution in phospholipid vesicles (liposomes) [4,5] We believe the importance of the cellular structure of red blood cells (RBCs) for retardation of any gas reactions, absence of colloid osmotic pressure, prevention of dissociation of Hb tetramer to dimers [6], etc. Actually, Hb-V showed sufficient oxygencarrying capacity in the studies of animal experimental models such as hemorrhagic shock resuscitation, hemodilution, oxygenation of ischemic tissues, etc.…”