Identification of the sites of deoxy haemoglobin PEGylation

Roberto Iafelice , Simone Cristoni , Dario Caccia , Rosaria Russo , Luigi Rossi-Bernardi , Kenneth C. Lowe , Michele Perrella (1)

1 Dipartimento di Scienze e Tecnologie Biomediche

The 2-iminothiolane reaction with protein amino groups adds a spacer arm ending with a thiol group, which can be further reacted with molecules carrying a maleimido ring. This approach is currently used for the preparation of a candidate "blood substitute" in which human haemoglobin is conjugated with long chains of polyethylene glycol. To identify the thiolation sites by mass spectrometry, we have carried out the reaction using deoxy haemoglobin bound to inositol hexaphosphate to protect some of the residues crucial for function and N-ethyl maleimide to block and stabilize the thiol groups prior to enzymatic digestion by trypsin and pepsin. Under the conditions for the attachment of 5-8 polyethylene glycol chains per tetramer, the thiolated residues were K 7}, K 11}, K 16}, K 56}, K 139}, and, with lower accessibility, K 90}, K 99}, K 60} of the {alpha} chain and K 8}, K 17}, K 59}, K 61}, K 66}, and, with lower accessibility, K 65}, K 95}, K 144}, of the {beta} chain. The {alpha}-amino groups of {alpha} and {beta} chains were not modified and the reaction of the Cys{beta}93‘s with N-ethylmaleimide was minor or absent. After the modification with thiolane and N-ethylmaleimide of up to 5-8 lysines per tetramer, the products retained a large proportion of the properties of native haemoglobin, such as low oxygen affinity, cooperativity, effect of the modulators and stability to auto-oxidation. Under identical anaerobic conditions, the conjugation of the thiolated haemoglobin tetramer with 5-6 chains of the maleimido derivative of 6kDa polyethylene glycol yielded products with diminished cooperativity, Hill‘s n=1.3-1.5, still retaining a significant proportion of the effects of the modulators of oxygen affinity and stability to auto-oxidation. Cooperativity was apparently independent of the topological distribution of the pegylated sites as obtained by reacting partly the thiolated protein with N-ethyl maleimide prior to pegylation.

Mots clés: Life Sciences

Roberto Iafelice, Simone Cristoni, Dario Caccia, Rosaria Russo, Luigi Rossi-Bernardi, et al.. Identification of the sites of deoxy haemoglobin PEGylation. Biochemical Journal, 2006, 403 (1), pp.189-196. ⟨10.1042/BJ20061556⟩. ⟨hal-00478685⟩