Disulphide bond assignment in human tissue inhibitor of metalloproteinases (TIMP).
Disulphide bond assignment in human tissue inhibitor of metalloproteinases (TIMP).
复制标题
人体组织金属蛋白酶抑制剂 (TIMP) 中的二硫键分配。
作者：
R. Williamson;F. Marston;S. Angal;P. Koklitis;M. Panico;H. Morris;A. Carne;Bryan J. Smith;T. Harris;R. Freedman
利用反相高效液相色谱法测定重组组织金属蛋白酶抑制剂（TIMP）的蛋白水解酶切产物，并根据还原时保留时间的变化对其进行测序，确定其二硫键。该程序允许直接指定Cys-145-Cys-166，并分离另外两个肽各含有两个二硫键。进一步的肽切割结合快速原子轰击质谱分析，从这些肽中鉴定出Cys-1-Cys-70、Cys-3-Cys-99、Cys-13-Cys-124和Cys-127-Cys-174。第六键Cys-132-Cys-137是通过推断确定的，因为天然蛋白没有可检测到的游离巯基。
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.