Non-hypertensive tetraPEGylated canine haemoglobin: correlation between PEGylation, O2 affinity and tissue oxygenation

Abstract

TetraPEGylated canine Hb, [SP (succinimidophenyl)-PEG5K]4-canine-Hb, with PEGylation at its four reactive cysteine residues (a111 and b93) has been prepared and characterized. The hydrodynamic volume and the molecular radius of (SP-PEG5K)4-canine-Hb are intermediate to those of di- and hexaPEGylated human Hb as expected. However, the COP (colloidal osmotic pressure) of tetraPEGylated canine Hb is closer to that of hexaPEGylated human Hb than to that of diPEGylated human Hb. The O2 affinity of tetraPEGylated canine Hb is higher than that of canine Hb and comparable with that of hexaPEGylated Hb. The O2 affinity of tetraPEGylated canine Hb is not responsive to the presence of DPG (diphosphoglycerate) or chloride, but it retains almost full response to L-35, an allosteric effector that interacts at the aa-end of the central cavity. The tetraPEGylated canine Hb is vasoinactive in hamster in 10% top load infusion studies. It is also essentially non-hypertensive in an extreme exchange haemodilution protocol in hamster just as di- and hexaPEGylated human Hb. The O2 delivery by tetraPEGylated canine Hb is comparable with that of hexaPEGylated Hb but not as efficient as diPEGylated Hb. These results demonstrate that PEGylation-induced solution properties of PEG [poly(ethylene glycol)]-Hb conjugates are dictated by the level and chemistry of PEGylation and the interplay of these plays a critical role in tissue oxygenation. The studies imply the need to establish the right level (and/or pattern) of PEGylation and O2 affinity of Hb-PEG adducts in designing O2-carrying plasma volume expanders, and this remains the primary challenge in the design of PEGylated Hb as blood substitutes.

Figure 1. SEC analysis of canine Hb PEGylated with maleimide PEG5K as a function of incubation time

The PEGylation of Hb was monitored as reflected by the enhancement in the molecular volume (hydrodynamic volume) of Hb by SEC of the reaction mixture. The canine Hb incubated with maleimido-phenyl PEG5K for different periods of time has been analysed by SEC. FPLC patterns of canine Hb PEGylated with maleimido-phenyl PEG5K at 4 °C for 1.5 h (curve b), 6 h (curve c) and 22 h overnight (curve d) are shown. The pattern of unmodified canine Hb (curve a) is also given for comparison.

Figure 2. Ion-exchange chromatographic purification of the tetraPEGylated canine Hb on a Q-Sepharose high performance column

Purification was carried out as described previously for purification of human Hb diPEGylated at Cys⁹³(β) with maleimide PEG [25]. The major peak corresponds to tetraPEGylated canine, and the region of the chromatogram pooled to isolate this material is indicated.

Figure 3. Structural analysis of PEGylated canine Hb

(A) Comparison of SEC patterns of tetraPEGylated canine Hb (the major component eluting from the Q-Sepharose HP column) with that of HbA, and hexaPEGylated Hb generated using thiolation-mediated maleimide chemistry-based PEGylation maleimide PEG5K [21]. (B) RP-HPLC pattern of canine Hb. (Upper curve) PEGylated canine Hb before chromatographic purification. (Lower curve) PEGylated canine Hb after chromatographic purification on Q-Sepharose HP. (C) SDS gel patterns of tetraPEGylated canine Hb before and after Q-Sepharose chromatography and (SP-PEG5K)₂-Hb. The tetraPEGylated canine Hb carries one major band. The mobility of this band corresponds to that of human β-globin PEGylated with PEG5K at its Cys⁹³(β), present in the sample of diPEGylated Hb. Lane 1, protein markers; lane 2, canine Hb; lane 3, tetraPEGylated canine Hb before Q-Sepharose purification; lane 4, tetraPEGylated Hb after purification on the Q-Sepharose column; and lane 5, diPEGylated human Hb. (D) IEF pattern of PEGylated canine Hb. Lane 1, canine Hb; lane 2, tetraPEGylated canine Hb after purification on a Q-Sepharose column; lane 3, diPEGylated human Hb; lane 4, hexaPEGylated Hb generated by EAF PEGylation; lane 5, control human Hb.

Figure 4. COP and viscosity of tetraPEGylated canine Hb as a function of protein concentration

(A) COP and (B) viscosity data for (SP-PEG5K)₂-Hb compared with (SP-PEG5K)₆-Hb. Open squares, canine Hb; filled circles, (SP-PEG5K)₂-Hb; open triangles, tetraPEGylated canine Hb; filled triangles, (SP-PEG5K)₆-HbA. The COP and viscosity of each sample were measured in triplicate and values presented are the averages.

Figure 5. Systemic and microcirculatory responses to a 10% top load of canine Hb (Dog-Ao), tetraPEGylated canine Hb, di (SP-PEG5K)₂-Hb and hexaPEGylated [SP-PEG5K)₆-Hb] human Hb

As the PEGylation increased from di- to tetra- and hexa-, the molecular volume, the viscosity and COP are all increased. (A) Changes in the MAP, (B) arteriolar diameter and (C) FCD. Studies performed in this current investigation with native canine Ao and tetraPEGylated canine Hb were both performed in four and five animals in which a total of 32 arterioles (average diameter at baseline of 58±12 μm) were followed in both groups. P <0.05: *, relative to baseline; †, Dog-P5K4 compared with Dog-Ao;§, P5K4 compared with P5K2, P5K6. Light grey bar, native canine Ao (the present study); open bar, di-, P5K2-Hb [24]; dark grey bar, tetra-, P5K4-canine Hb (the present study); black bar, hexa-, P5K6-Hb [20].

Figure 6. Microcirculatory and tissue oxygenation studies of tetraPEGylated Hb in extreme haemodilution

Hamster window chamber model haemodiluted to Hct 11% with 70 kDa dextran. TetraPEGylated canine Hb (Tetra) is compared with hexaPEGylated Hb, (SP-PEG5K)₆Hb (Hex) [27], and diPEGylated Hb, (SP-PEG5K)₂Hb (Di) [24]. (A) MAP; (B) microvascular flow in arterioles (A, n=54 vessels with diameter 66±19 μm) and venules (V, n=45 vessels with diameter 83±29 μm); (C) FCD; (D) tissue pO₂. P<0.05: *, relative to baseline; †, Di compared with Tetra and Hex.

Figure 7. Comparison of the chemical reactivity of cysteine residues of animal Hbs with maleimide phenyl PEG5K

The Hbs at a concentration of 0.5 mM were reacted with 12.5 mM maleimide PEG5K at pH 7.4 (PBS) at 4 °C for 18 h. Curve a, control human Hb; curves b–e, animal Hbs PEGylated with maleimide PEG5K; curve b, human Hb; curve c, feline Hb; curve d, chicken Hb; curve e, canine Hb.