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Probing the reactivity of nucleophile residues in human 2,3‐diphosphoglycerate/deoxy‐hemoglobin complex by aspecific chemical modifications
Author(s) -
Scaloni Andrea,
Ferranti Pasquale,
De Simone Giuseppina,
Mamone Gianfranco,
Sannolo Nicola,
Malorni Antonio
Publication year - 1999
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(99)00601-8
Subject(s) - chemistry , nucleophile , methylation , hemoglobin , reactivity (psychology) , globin , edman degradation , peptide , stereochemistry , peptide sequence , biochemistry , gene , medicine , alternative medicine , pathology , catalysis
The use of aspecific methylation reaction in combination with MS procedures has been employed for the characterization of the nucleophilic residues present on the molecular surface of the human 2,3‐diphosphoglycerate/deoxy‐hemoglobin complex. In particular, direct molecular weight determinations by ESMS allowed to control the reaction conditions, limiting the number of methyl groups introduced in the modified globin chains. A combined LCESMS‐Edman degradation approach for the analysis of the tryptic peptide mixtures yielded to the exact identification of methylation sites together with the quantitative estimation of their degree of modification. The reactivities observed were directly correlated with the p K a and the relative surface accessibility of the nucleophilic residues, calculated from the X‐ray crystallographic structure of the protein. The results here described indicate that this methodology can be efficiently used in aspecific modification experiments directed to the molecular characterization of the surface topology in proteins and protein complexes.