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

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 allo...

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Bibliographic Details
Published in:Biochemical journal 1990-06, Vol.268 (2), p.267-274
Main Authors: Williamson, R A, Marston, F A, Angal, S, Koklitis, P, Panico, M, Morris, H R, Carne, A F, Smith, B J, Harris, T J, Freedman, R B
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Cell Line
Chromatography, High Pressure Liquid
disulfide bonds
Disulfides - analysis
DNA - analysis
Glycoproteins
Humans
Hydrolysis
Metalloendopeptidases
Mice
Microbial Collagenase - antagonists & inhibitors
Molecular Sequence Data
Pepsin A
Recombinant Proteins - genetics
Tissue Inhibitor of Metalloproteinases
Trypsin
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Summary: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.
ISSN:0264-6021
1470-8728
DOI:10.1042/bj2680267