Identification of the sites of deoxyhaemoglobin PEGylation

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

Research output: Contribution to journalArticlepeer-review


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, Lys 11, Lys16, Lys56 and Lys139 and, with lower accessibility, Lys90, Lys99 and Lys 60 of the α-chain and Lys8, Lys17, Lys59, Lys61 and Lys66 and, with lower accessibility, Lys65, Lys95 and Lys144 of the β-chain. The α-amino groups of α- and β-chains were not modified and the reaction of the Cysβ93 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, cooperativity, 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 cooperativity, 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].

Original languageEnglish
Pages (from-to)189-196
Number of pages8
JournalBiochemical Journal
Issue number1
Publication statusPublished - Apr 1 2007


  • Blood substitute
  • Haemoglobin (Hb)-based oxygen carrier
  • N-ethylmaleimide (NEM)
  • Protein mass spectroscopy (protein MS)
  • Protein poly(ethylene glycol)ation (protein PEGylation)
  • Protein thiolation

ASJC Scopus subject areas

  • Biochemistry


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