C-Terminal sequencing of trichosanthin

Abstract

Preliminary identification of C-terminus of trichosanthin by chemical and enzymic methods, such as hydrazinolysis, thiohydantoin reaction and carboxypeptidase hydrolysis, showed that there may be the possible presence of more than one terminus, i.e., Met and Ala but complicated by side reactions. A computer-assisted carboxypeptidase method was first introduced by the authors to determine the C-terminal sequence of trichosanthin, and showed that trichosanthin is heterogeneous at its C-terminus and has two C-terminal sequences determined as -Arg-Asn-Asn-Met-OH and -Arg-Asn-Asn-Met-Ala-OH respectively. These results have been later unambiguously confirmed by the results from other experiments through the identification of the free alanine always present in the CNBr degradation products of trichosanthin, and the actual separation of two fragments from the finger prints as well as from the HPLC fractions of the trypsin digest of this protein. All shows that their amino acid sequences, determined by manual DABITC/PITC technique, agree well with those of the two C-terminal sequences determined by the computer-carboxy peptidase method.