Combining the Influence of Two Low O₂ Affinity-Inducing Chemical Modifications of the Central Cavity of Hemoglobin

Abstract

HexaPEGylated Hb, a non-hypertensive Hb, exhibits high O 2 -affinity which makes it difficult to deliver desired levels of oxygen to tissue. PEGylation of very low O 2 -affinity Hbs is now contemplated as the strategy to generate PEGylated Hbs with intermediate levels of O 2 -affinity. Towards this goal, a doubly modified Hb with very low O 2 -affinity has been generated. The amino terminal of β-chain of HbA is modified by 2-hydroxy, 3-phospho propylation first to generate a low oxygen affinity Hb, HPPr-HbA. The oxygen affinity of this Hb is insensitive to DPG and IHP. Molecular modeling studies indicated potential interactions between the covalently linked phosphate group and Lys-82 of the trans β-chain. To further modulate the oxygen affinity of Hb, the αα-fumaryl crossbridge has been introduced into HPPr-HbA in the mid central cavity. The doubly modified HbA (αα-fumaryl-HPPr-HbA) exhibits an O 2 -affinity lower than that of either of the singly modified Hbs, with a partial additivity of the two modifications. The geminate recombination and the visible resonance Raman spectra of the photoproduct of αα-fumaryl-HPPr-HbA also reflect a degree of additive influence of each of these modifications. The two modifications induced a synergistic influence on the chemical reactivity of Cys-93(β). It is suggested that the doubly modified Hb has accessed the low affinity T-state that is non-responsive to effectors. The doubly modified Hb is considered as a potential candidate for generating PEGylated Hbs with an O 2 -affinity comparable to that of erythrocytes for developing blood substitutes.Developing low O 2 -affinity Hb has been the subject of considerable interest both in terms of understanding the structure-function correlation of Hb and for the development of Hb based oxygen carriers. Central cavity modifications such as crosslinking and affinity labeling of the effector binding domains of Hb has been the prominent approaches to reduce the O 2 -affinity of Hb (1-6). However, interest in such molecules subsides since most of these potential Hb based oxygen carriers turned out to be vasoactive (7-9). The vasoactivity was considered to be a consequence of the NO scavenging activity of acellular Hb (10-12). Design of mutant Hbs with reduced NO binding activity has been one of the approaches advanced to generate nonhypertensive Hb based oxygen carriers (11,(13)(14)(15).

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

“…HbA was intramolecularly cross-linked with bis-(3,5-dibromosalicyl) fumarate (DBBF) as described previously (Chatterjee et la. 1986, Nacharaju et al 2007). Briefly, HbA (1 mM) was incubated overnight with 8 mM sodium tripolyphosphate in 50 mM Bistris acetate buffer (pH 6.5) at 4 ° C to block modifications of the β chains by DBBF.…”