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

FEBS Letters

De Simone, Giuseppina²  Ferranti, Pasquale¹  Scaloni, Andrea¹  Mamone, Gianfranco¹  Malorni, Antonio¹  Sannolo, Nicola¹ 

[1] Centro Internazionale Servizi di Spettrometria di Massa, CNR, 80131 Napoli, Italy;Centro di Studio di Biocristallografia, CNR, 80134 Napoli, Italy

关键词: Aspecific chemical modification;    Mass spectrometry;    Surface topology;    Methyl bromide;    Hemoglobin adduct;    DPG;    2;    3-diphosphoglycerate;    ES;    electrospray;    GCEIMS;    gas chromatography electron ionization mass spectrometry;    h-deoxy-Hb;    human deoxy-hemoglobin;    LC;    liquid chromatography;    MeBr;    methyl bromide;    MS;    mass spectrometry;    PTH;    phenylthiohydantoin;    RP-HPLC;    reversed phase high performance liquid chromatography;    TBDMS;    tert-butyl-dimethyl-silyl;   

DOI  :  10.1016/S0014-5793(99)00601-8

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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 pK _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.

【 授权许可】

Unknown