Isolation and characterization of new isoforms of trichosanthin from Trichosanthes kirilowii

Three ribosome-inactivating proteins (RIPs) were isolated from the previously characterized 'trichosanthin' fraction prepared from the root tuber of Trichosanthes kirilowii. They were designated as α-, β-, and γ-trichosanthin (TCS). All three trichosanthin isoforms exhibited similar biochemical activities, namely in vitro inhibition of protein synthesis, N-glycosidase activity on 28S rRNA, and conversion of supercoiled DNA to open-circular and linear DNA. SDS-PAGE analysis indicated that their molecular weights were around 26 kDa. Amino acid composition analysis revealed that α-TCS, the major protein, corresponded closely to the previously reported trichosanthin. Protein fingerprinting analysis indicated that γ-TCS is structurally distinct from α-, and β-TCS while the latter two are very similar. The identity of γ-TCS was confirmed by N-terminal and internal protein sequencing. Cytotoxicity assay indicated that α-TCS is distinctively more toxic towards lymphocytes than β- and γ-TCS, while for fibroblast, β-TCS is the least toxic. Their difference in cytotoxicity in spite of their structural similar warrant further investigation into their structure and function relationship.