Probing the reactivity of nucleophile residues in human 2,3-diphosphoglycerate/deoxy-hemoglobin complex by aspecific chemical modifications

The use of aspecific methylation reaction in combination with MS procedures has been employed for the characterization of the nucleophilic residues pr esent on the molecular surface of the human 2,3-diphosphoglycerate/deoxy-he moglobin complex, In particular, direct molecular weight determinations by ESMS allowed to control the reaction conditions, limiting the number of met hyl groups introduced in the modified globin chains. A combined LCESMS-Edma n degradation approach for the analysis of the tryptic peptide mixtures yie lded to the exact identification of methylation sites together with the qua ntitative estimation of their degree of modification, The reactivities obse rved were directly correlated with the pK(a) and the relative surface acces sibility of the nucleophilic residues, calculated from the X-ray crystallog raphic structure of the protein. The results here described indicate that t his methodology can be efficiently used in aspecific modification experimen ts directed to the molecular characterization of the surface topology in pr oteins and protein complexes. (C) 1999 Federation of European Biochemical S ocieties.