Enhanced Molecular Volume of Conservatively Pegylated Hb: (SP-PEG5K)₆-HbA is Non-Hypertensive

Abstract

Recent studies have suggested that the "pressor effect" of acellular Hb is a consequence of perturbation of the macro-and microcirculatory system in multiple ways, and that PEGylation is an effective approach for controlling the same. In an attempt to confirm this concept, a new and simple thiolation mediated, maleimide chemistry-based conservative PEGylation protocol has been developed to conjugate multiple copies of PEG-chains to Hb. This approach combines the high reactivity of maleimides towards thiols with the propensity of iminothiolane to derivatize the epsilon-amino groups of proteins into reactive thiol groups, with conservation of their positive charge. One of the PEGylated products, namely (SP-PEG5K)6-HbA, that carries on an average six copies of PEG5000 chains per Hb, is non-hypertensive in hamster top load and in rat 50% exchange transfusion models. This hexa-PEGylated-Hb has (i) a hydrodynamic volume corresponding to that of an oligomerized Hb of 256kDa, (ii) a molecular radius of approximately 6.8 nm, (iii) high oxygen affinity, (iv) lowered Bohr effect, and (v) increased viscosity and colloidal osmotic pressure. These properties of (SP-PEG5K)6-HbA are consistent with the emerging new paradigms for the design of Hb based oxygen carriers and confirm the concept that the "pressor effect" of Hb is a multifactorial event. The thiolation mediated maleimide chemistry-based PEGylation protocol described here for the generation of (SP-PEG5K)6-Hb is simple, highly efficient, and is carried out under oxy conditions. The results demonstrate that a non-hypertensive PEG-Hb can be generated by conjugation of a lower number of PEG chains than previously reported.

Sanguinate's effect on pial arterioles in healthy rats and cerebral oxygen tension after controlled cortical impact

“…Proximity of cell-free hemoglobin to endothelial cells (Alayash, 2004), extravasation (Urbaitis et al, 1991), and depletion of vascular endothelium derived nitric oxide (NO) by extracellular-hemoglobin (Hb) (Alayash et al, 2007;Doherty et al, 1998;Olson et al, 2004) are the primary hypotheses for the vasoconstriction and increases in systemic and pulmonary blood pressures observed after HBOC infusion. Over the years, structural and chemical modifications have reduced extravasation of the HBOCs, mitigating some of their vasoconstrictive effects (Acharya et al, 2005). Sanguinate (Prolong Pharmaceuticals, South Plainfield, NJ, USA) is composed of a carbon monoxide (CO)-releasing purified bovine Hb conjugated with Microvascular Research 107 (2016) [83][84][85][86][87][88][89][90] Abbreviations: CCI, Controlled cortical impact; HBOC, hemoglobin based oxygen carrier; PbtO 2 , partial pressure of oxygen in brain interstitium; PO 2 , partial pressure of oxygen; PQM, phosphorescence quenching method; TBI, traumatic brain injury.…”

Enhancing Nitrite Reductase Activity of Modified Hemoglobin: Bis-tetramers and Their PEGylated Derivatives

“…In addition, increasing the affinity for oxygen can be achieved by modifying hemoglobin at key residues (e.g. β-Cys93) (9,27). mmHg and an average weight of 150 kDa (range: 64 -400 kDa) (37).…”

Design of Nonhypertensive Conjugated Hemoglobins as Novel Resuscitation Fluids

“…In this new approach for Hb (or other proteins for that matter) oligomerization protocol, the principles of extension arm chemistry (detailed discussion in Sect. 11.3.3), the chemical protocol to engineer new (extrinsic) functional groups on the molecular surface of proteins, has been integrated with the use of short flexible bifunctional bis maleimide PEG reagents (Li et al 2006;Manjula et al 2005;Acharya et al 2005). In this two step oligomerization approach, flexible PEG spacer arm based intermolecular crosslinks are engineered between the newly introduced thiols of a thiolated intramolecularly crosslinked Hbs using Bis Mal PEG.…”