天花粉蛋白C-端顺序的测定

C-Terminal Sequencing of Trichosanthin

采用肼解法、乙内酰硫脲法和羧肽酶法检测了天花粉蛋白的C-端氨基酸有二,分别为Met和Ala借计算机辅助的羧肽酶法,测定了天花粉蛋白C-端顺序,其结果显示C-端是非均一的,有两个末端,分别为—ArgAsnAsnMetOH和—ArgAsnAsnMetAlaOH.这些结论已经从天花粉蛋白的溴化氰降解物中获得游离的Ala.

The identification of C-terminus amino acid of trichosanthin(T)had been firsttried by means of chemical and enzymic methods such as hydrazinolysis,thiohydantoinreaction and carboxypeptidase hydrolysis.All findings indicated that the results werecomplicated by the possible presence of more than one terminus,i.e.Met and Ala and,moreover,even complicated by side reactions with the chemical reagents.A computer-assisted carboxypeptidase method was first used by the authors to determine C-terminal sequence of T.The results showed that its C-terminal sequence is heterogeneousand has been determined to be-ArgAsnAsnMetOH and-ArgAsnAsnMetAlaOHrespectively.These two sequences have been later confirmed by the results from otherexperiments,in one of which,some free Ala was always found in the CNBr degradationproducts of T,and anothers,two fragments of trypsin digest of T had been isolated from itsfinger prints as well as from HPLC fractons and their sequences determined by DABITC/PITC manual method were the same as those determined by computer simulation.