Increased Inter Dimeric Interaction of Oxy Hemoglobin is Necessary for Attenuation of Redutive Pegylation Promoted Dissociation of Tetramer

Figures & data

Figure 1. Schematic representation of carboxymethylation (a) and propylation (b) of Hb through reductive alkylation chemistry.

Table 1. Tetramer-Dimer Dissociation Constants of Modifed Hbs.

Sample	Kd (μM)
HbA	2.7
Propyl-Hb at Val-1(α)	0.3
Carboxymethylated Hbs	7.0
NEM-Hb	3.7

The dissociation constant (Kd) was measured in PBS buffer, pH 7.4.

Figure 2. Size exclusion chromatography analysis of the hexaPEGylated Hbs. hexaPEGylated αα-Hb (1), hexaPEGylated Hb (2), hexaPEGylated [cm-Val-1(α)]2-Hb (3), hexaPEGylated [cm-Val-1(β)]2-Hb (4), were loaded on two HR10/30 Superose 12 columns and eluted with PBS, pH 7.4 at a flow rate of 0.5 ml/min.

Figure 3. Size exclusion chromatography analysis of the PEGylated proteins. HbA (a), [Propyl-Val-1(α)]₂-Hb (b), αα-fumaryl Hb (c), the hexaPEGylated Hb (d), the hexaPEGylated [Propyl-Val-1(α)]₂-Hb (e), and the hexaPEGylated αα-fumaryl Hb (f) were loaded on two HR10/30 Superose 12 columns (1 × 31 cm²) and eluted with PBS, pH 7.4 at a flow rate of 0.5 ml/min.

Figure 4. RP-HPLC analysis of the PEGylated proteins. HPLC analysis of HbA (a), [Propyl-Val-1(α)]₂-Hb (b), the hexaPEGylated Hb (c), and the hexaPEGylated [Propyl-al-1(α)]₂-Hb (d) was carried out on a Vydac C4 column (4.6 × 250 mm²). The column was eluted with a linear gradient of 35–50% acetonitrile containing 0.1% TFA in 100 min and 50–70% acetonitrile containing 0.1% TFA in 30 min at a flow rate of 1.0 ml/min.

Figure 5. The tetramer-dimer dissociation constant (K_d) of the PEGylated proteins. HbA (a), the hexaPEGylated [Propyl-Val-1(α)]₂-Hb (b), the hexaPEGylated Hb prepared using extension arm facilitated PEGylation (c), and the diPEGylated Hb with PEGylation at Val-1(β) (d) were diluted to a series of Hb concentrations and subjected to two Superose 12 columns (1 × 31 cm²) in series for the K_d measurement.

Table 2. The oxygen affinity of the HexaPEGylated [Propyl-Val-1(α)]₂-Hb Sample.

Sample	P50 (mmHg)/n
HbA	14.0/2.8
[Propyl-Val-1(α)]2-Hb	7.6/2.7
HexaPEGylated Hb	6.3/1.9
EAF-HexaPEGylated Hb [Cys 93(β)-PEGylated]	7.0/2.0
EAF-HexaPEGylated Hb [Cys-93(β)-Free]	8.5/2.1
HexaPEGylated [Propyl-Val-1(α)]2-Hb	4.1/2.1

Oxygen equilibrium curves of the Hb samples were measured at 37°C in PBS (pH 7.4) at Hb tetramer concentration of 0.5 mM. n is the Hill coefficient. P₅₀ is the partial oxygen pressure at 50% saturation and is expressed in mmHg on the left of the Hill coefficient.

Figure 6. Molecular models of [Propyl-PEG5K-Val-1(α)]₂-Hb and [Propyl-PEG5K-Val-1(β)]₂-Hb. The α- and the β-globin chains are shown in red and blue, respectively. The models were generated as described under Experimental Procedures.

Table 3. Loss of Accessible Surface Area on PEGylation of Hb.

	ASA^c (Å^2 )	Loss of ASA (Å^2)
Hb	21976.2	0
PEG5K	3543.2	0
2 PEG5K at αα-end of Hb	7086.4	0
2 PEG5K at ββ-end of Hb	4963.4	2123.0
Hb PEGylated at Val-1(α)^a	20189.8	1786.4
Hb PEGylated at Val-1(β)^a	20425.2	1551.0
Hb-PEG complex at αα-end^b	25401.1	3572.8
Hb-PEG complex at ββ-end^b	23673.3	5225.0

^aLoss of the protein surface area resulting from PEGylation.

^bLoss of protein and PEG-surface area.

^cSolvent accessible surface area.

Figure 7. Schematic representation of the extension arms sandwiched between the PEG-chains and side chains of Hb in the EAF-PEGylated Hb. Note the extension arm between the amino group and succinimidyl phenyl PEG chains, which helps to attenuate the influence of the PEG-chains on the interdimeric interactions (central cavity interactions) of Hb tetramer.