PEGylation of αα-Hb using succinimidyl propionic acid PEG 5K: Conjugation chemistry and PEG shell structure dictate respectively the oxygen affinity and resuscitation fluid like properties of PEG αα-Hbs

Figures & data

Figure 1. Schematic representation of the conjugation chemistry used for PEGylation of αα-fumaryl Hb. Succinimidyl esters are used to generate either isopeptide and of urethane linkage between Hb and PEG: (A) succinimidyl ester of propionic acid PEG (SPA PEG), (B) N-succinimidyl propionate, and (C) succinimidyl carbamate PEG (SC PEG).

Table I. Estimation of PEGylation αα-Hb.

	Number of copies of PEG5K/ αα-Hb molecule^a
	^1HNMR	Tryptic peptide mapping
(Propionyl-PEG5K)6 αα-Hb	6	6
(Propionyl-PEG5K)10-αα-Hb	10	8

^aApproximated to the nearest integer.

Figure 2. SEC analysis of modified Hbs. 1, HbA control; 2, αα-Hb; 3, (Propionyl-PEG5K)₆-αα-Hb; 4, (Propionyl-PEG5K)₁₀-αα-Hb.

Table II. Molecular and solution properties of PEGylated αα-Hb.

Properties	HbA	(Propionyl- PEG5K)6-αα-Hb	(Propionyl- PEG5K)10-αα-Hb
Molecular radius (nm)	3.1	6.0	6.7
Molecular volume (nm^3)	125	904	1259
PEG shell volume (nm^3)	0	779	1134
PEG mass^a (Dal)	0	31000	53000
PEG Density (Dal/nm^3)	0	39.8	46.7
COP^b (mmHg)	7.4	41.4	100.6
Viscosity^b (cP)	0.8	2.0	3.1

^aThe PEG mass are calculated from the extent of PEGylation as determined by ¹H-NMR method and as described in context.

^bHb concentration is 4%. The COP is tested at room temperature. The viscosity is tested at 37°C.

Figure 3. SEC analysis of modified Hbs: 1, HbA; 2, αα-Hb; 3, (Propionyl-PEG5K)₆-αα-Hb; 4, (Propionyl-PEG5K)₁₀-αα-Hb; 5, (Urethane-PEG5K)₆-αα-Hb; 6, (Urethane-PEG5K)₁₀-αα-Hb.

Table III. Influence of conjugation chemistry on the plasma expander like solution properties of PEG-Hb conjugates.

	Hydrodynamic radius (nm)	Viscosity^a (cP)	COP^a (mmHg)
HbA	3.1	0.9	10.3
Αα-Hb	3.1	–	–
(Propionyl-PEG5K)6-Hb	5.8	2.1	55.4
(Propionyl-PEG5K)6-αα-Hb	6.2	2.0	41.4
(Urethane-PEG5K)6-Hb	5.8	2.6	63.8
(Urethane-PEG5K)6-αα-Hb	6.4	2.3	49.5

^aHb concentration is 4%. The COP is tested at room temperature. The viscosity is tested at 37°C.

Table IV. Influence of conjugation chemistry on O₂ affinities of PEG-Hb conjugates.

	P50/mmHg	Hill number
HbA	14.0	2.8
αα-HbA	32.0	2.8
(Propionyl-PEG5K)6-Hb	10.0	1.8
(Propionyl-PEG5K)6-αα-Hb	22.8	1.7
(Urethane-PEG5K)6-Hb	8.0	2.0
(Urethane-PEG5K)6-αα-HA	23.3	2.1

Table V. Influence of PEGylation of the function properties of αα-fumaryl Hb.

	Oxygen affinity^a		Bohr effect^b
	P50 (n) mmHg	Increase fold in P50	−ΔlogP50/ ΔpH	Reduction %
HbA	13.7(3.0)	–	1.76	–
Αα-Hb	32.0(2.8)	2.3	0.73	59
(Propionyl-PEG5K)6- αα-Hb	22.8 (1.8)	1.7	0.62	65
(Propionyl-PEG5K)10- αα-Hb	17.6 (1.6)	1.2	0.44	75
(Propionyl)6-αα-Hb	17.5 (1.7)	1.2	0.42	75
(Propionyl)10-αα-Hb	13.3 (1.6)	1.0	0.36	80

^aThe P₅₀ and n were determined in PBS, pH 7.4.

^bThe oxygen affinity were determined in 100 mM phosphate buffer, pH 6.6, 7.0, 7.4, 8.0 and 8.5.

Figure 4. The oxygen affinity of HbA control, αα-Hb, (Propionyl-PEG5K)₆-αα-Hb, and (Propionyl-PEG5K)₁₀-αα-Hb at different pH of 100 mM phosphate buffer.

Figure 5. Kinetics of the reactions of SH groups of HbA, αα-Hb, Propionyl₆-αα-Hb, Propionyl₁₀-αα-Hb, (Propionyl-PEG5K)₆-αα-Hb, and (Propionyl-PEG5K)₁₀-αα-Hb with 4,4-dipyridine disulfide (4-PDS).

Table VI. Influence of conjugation chemistry and of PEG shell on the reactivity of Cys-93(β).

	Pseudo-first-order rate (min^−1)
HbA	0.165
αα-HbA	0.151
Propionyl6-αα-Hb	0.118
Propionyl10-αα-Hb	0.103
(Propionyl-PEG5K)6-αα-Hb	0.112
(Propionyl-PEG5K)10-αα-Hb	0.098

The rates were calculated from initial slop of the time courses in Figure 4.

Table VII. Site of PEGylation of αα-Hb.

Residue	(Propionyl-PEG5K)6-αα-Hb	(Propionyl-PEG5K)10-αα-Hb
	Modification %	Modification %
Lys-11(α)	13	23
Lys-16(α)	17	31
Lys-40(α)	12	14
Lys-56(α)	16	20
Lys-90(α)	75	93
Lys-139(α)	25	44
Lys-8(β)	17	21
Lys-17(β)	23	29
Lys-82(β)	42	44
Lys-120(β)	52	56

Figure 6. Schematic representation of the PEGylated αα-Hb molecule.