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
Biological Laboratory, University of Kent, Canterbury, U.K.
Biochem J. 1990 Jun 1;268(2):267-74. doi: 10.1042/bj2680267.
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.
通过在反相高效液相色谱上解析金属蛋白酶组织抑制剂（TIMP）的蛋白水解消化产物，并对那些因还原后保留时间变化而被判定含有二硫键的峰进行测序，确定了人重组金属蛋白酶组织抑制剂（TIMP）中的二硫键。该方法直接确定了Cys-145-Cys-166，并分离出另外两个各自含有两个二硫键的肽段。结合快原子轰击质谱分析的进一步肽段裂解，确定了这些肽段中的Cys-1-Cys-70、Cys-3-Cys-99、Cys-13-Cys-124和Cys-127-Cys-174。第六个二硫键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。
