Identification of the sites of deoxyhaemoglobin PEGylation

Abstract

The 2-iminothiolane reaction with protein amino groups adds a spacer arm ending with a thiol group, which can be further treated with molecules carrying a maleimido ring. This approach is currently used for the preparation of a candidate 'blood substitute' in which human Hb (haemoglobin) is conjugated with long chains of PEG [poly(ethylene glycol)]. To identify the thiolation sites by MS, we have carried out the reaction using deoxyHb bound to inositol hexaphosphate to protect some of the residues crucial for function and NEM (N-ethylmaleimide) to block and stabilize the thiol groups prior to enzymatic digestion by trypsin and pepsin. Under the conditions for the attachment of 5-8 PEG chains per tetramer, the thiolated residues were Lys7, Lys11, Lys16, Lys56 and Lys139 and, with lower accessibility, Lys90, Lys99 and Lys60 of the a-chain and Lys8, Lys17, Lys59, Lys61 and Lys66 and, with lower accessibility, Lys65, Lys95 and Lys144 of the b-chain. The a-amino groups of a- and b-chains were not modified and the reaction of the Cysb93 residues with NEM was minor or absent. After the modification with thiolane and NEM of up to five to eight lysine residues per tetramer, the products retained a large proportion of the properties of native Hb, such as low oxygen affinity, co-operativity, effect of the modulators and stability to autoxidation. Under identical anaerobic conditions, the conjugation of the thiolated Hb tetramer with five or six chains of the maleimido derivative of 6 kDa PEG yielded products with diminished co-operativity, Hill coefficient h=1.3-1.5, still retaining a significant proportion of the effects of the modulators of oxygen affinity and stability to autoxidation. Co-operativity was apparently independent of the topological distribution of the PEGylated sites as obtained by treating partly the thiolated protein with NEM prior to PEGylation [poly(ethylene glycol)ation].

Effects of Endogenous Ascorbate on Oxidation, Oxygenation, and Toxicokinetics of Cell-Free Modified Hemoglobin after Exchange Transfusion in Rat and Guinea Pig

“…4Aa), guinea pig Hb (Fig. 4Ba), human HbA 0 , and non cross-linked PEGylated Hb have been shown to readily dissociate in vitro (Iafelice et al, 2007). The overall impact of Hb monomer formation in vivo could result in acute renal failure based on globin chain deposition in the glomeruli of the kidneys (Savitsky et al, 1978).…”

Hemoglobin(βC93A)–Albumin Cluster: Mutation of Cysteine‐β93 to Alanine Allows Moderate Reduction of O2 Affinity by Inositol Hexaphosphate

“…Red blood cell (RBC) substitutes are currently required worldwide because of a shortage of blood supplies. Poly(ethylene glycol) (PEG)‐conjugated human hemoglobins (PEG‐Hbs) are the most widely developed hemoglobin (Hb)‐based O 2 carriers (HBOCs) as RBC substitutes . PEGylation is achieved through the coupling of Hb with monofunctionalized PEG.…”

“…Typically, maleimide‐PEGs react with two sulfhydryl groups of Cys93 in β subunits (Cys‐β93), yielding PEG‐(βC93)Hbs . PEGylation and chemical modification of Cys‐β93, which is located at the α 1 β 2 /α 2 β 1 interface and a neighbor of proximal His‐β92, is known to increase O 2 affinity and decrease O 2 binding cooperativity . Inositol hexaphosphate (IHP), a powerful allosteric effector, binds to the β cleft of the central cavity of native Hb and stabilizes the tense (T) state quaternary structure .…”

CO binding improves the structural, functional, physical and anti-oxidation properties of the PEGylated hemoglobin

“…Typically, PEGylation of Hb was conducted in an air-equilibrated environment and metHb formation may occur in the PEGylation process. PEGylation in anaerobic condition has been carried out in the presence of Cland inositol hexaphosphate (IHP) to inhibit the metHb formation of Hb (Iafelice et al 2007). However, achieving such an oxygen-free environment requires considerable effort to remove O 2 from Hb.…”