Disulphide bond assignment in human tissue inhibitor of metalloproteinases (TIMP)

作者：Ritchie Williamson, Fiona A. O. Marston, Sarojani Angal, PETER A. KOKLITIS, Maria Antonietta Panico, Howard R. Morris, Alan Carne, Bryan John Smith, Tess J Harris, Robert B. Freedman · 发表于：Biochemical Journal · 年份：1990 · DOI：10.1042/bj2680267 · 被引用次数：178 · 研究领域：Protease and Inhibitor Mechanisms、Peptidase Inhibition and Analysis、Monoclonal and Polyclonal Antibodies Research

Disulphide bonds in human recombinant tissue inhibitor of metalloproteinases (TIMP) were assigned by resolving proteolytic digests of TIMP on reverse-phase h.p.l.c. and sequencing those peaks judged to contain disulphide bonds by virtue of a change in retention time on reduction. This procedure allowed the direct assignment of Cys-145-Cys-166 and the isolation of two other peptides containing two disulphide bonds each. Further peptide cleavage in conjunction with fast-atom-bombardment m.s. analysis permitted the assignments Cys-1-Cys-70, Cys-3-Cys-99, Cys-13-Cys-124 and Cys-127-Cys-174 from these peptides. The sixth bond Cys-132-Cys-137 was assigned by inference, as the native protein has no detectable free thiol groups.