Disulphide bond assignment in human tissue inhibitor of metalloproteinases (TIMP). |
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| Authors: | R A Williamson F A Marston S Angal P Koklitis M Panico H R Morris A F Carne B J Smith T J Harris and R B Freedman |
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| Institution: | Biological Laboratory, University of Kent, Canterbury, U.K. |
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| Abstract: | 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. |
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