Modification of human haemoglobin with glucose 6-phosphate enhances tetramer-dimer subunit dissociation

Abstract

Studies using equilibrium gel-permeation chromatography demonstrate that formation of the covalent adduct of D-glucose 6-phosphate (G6P) with human haemoglobin promotes dissociation of the haemoglobin tetramer into its component alpha beta dimer pairs [Kdoxy = 2.57 X 10(-6) versus Kdoxy (G6P) = 11.22 X 10(-6) M-haem]. On the other hand, Kd for glucosylated haemoglobin is identical with those of the O2- and CO-liganded forms of intact haemoglobin A0. These data are consistent with the phosphate moiety alone being responsible for a 4.5-fold increase in the tetramer-to-dimer apparent Kd. This suggests the glucose 6-phosphate moiety does not bind to the same sites on haemoglobin as do the free organic phosphates, as suggested by ligand-binding kinetics data or structural analysis. My study presents a working model for studying changes in protein subunit assembly as altered by protein phosphorylations.

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

“…For example, the native oxy Hb (0.1 mM) maintains a tetrameric structure between pH 7.1 and 7.4 in a phosphate buffer . It dissociates in the presence of 0.5–1.0 M MgCl 2 . , Generally, native Hb maintains tetrameric structures over the concentration of 0.1 mM at pH 7.4 and dissociates at lower concentrations such as 5 μM . Reportedly, PEGylation promotes dissociation of the α 2 β 2 tetramer. ,, An earlier study demonstrated that the dissociation constants of α 2 β 2 tetramer into dimers ( K d ) are 8.5 μM for native Hb, 10.5 μM for 5 kDa bis-PEGylated Hb, and 43.2 μM for 5 kDa multiple-PEGylated Hb with 6 PEG chains in the average (hexa-PEGylated-Hb), although it was not described how the K d values were measured.…”

“…27 It dissociates in the presence of 0.5−1.0 M MgCl 2 . 35,36 Generally, native Hb maintains tetrameric structures over the concentration of 0.1 mM at pH 7.4 37 and dissociates at lower concentrations such as 5 μM. 38 Reportedly, PEGylation promotes dissociation of the α 2 β 2 tetramer.…”

“…Therefore, the tetrameric structure is expected to be favored. Based on the reported dissociation constant of native carbonyl Hb (K d = 1.05 μM), 37 the tetrameric structure is expected to be maintained as greater than 99% at 0.10 mM and as no less than 90% at 0.050 mM at pH 7.4. The Hb concentrations for PEGylation and for subsequent clipping were 0.50 and 0.25 mM.…”

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

“…45 For native Hb, the Ka of αβ dimers to α2β2 tetramer is reportedly 10 5 −10 6 M −1 . 23,46,47 Judging from the Ka values, the non-covalent interaction between αβ dimers has potential for application to construct a supramolecular polymer with DP > 10 at [M]0 > 1 mM. Actually, in the present SEC results of ββ-cross-linking at [M]0 = 1.00 mM (green chromatograms in Figure 4), CM-20 was efficiently ring-opened (r1 = 0.088) and polymerized to form XLSP-20 with a molecular size reaching to the column void volume (DP >10).…”