Aim: To localize the active site of ribosome inactivation of trichosanthin (Tri), a Chinese herb protein.
Methods: Hydroxylamine was used to specifically cleave the unique Asn-Gly peptide bond of Tri. Preparative SDS-polyacrylamide gel electrophoresis was applied to get 2 cleaved fragments, HATf1 and HATf2. Western blotting was used to determine the different epitopes of Tri and screen the antibodies. A cell-free system, rabbit reticulocyte lysate, was introduced to quantitate the inhibitory activity of Tri and its fragments on protein biosynthesis.
Results: HATf1 and HATf2 were separated with the purity of 96.9% and 80.5% respectively. HATf1, like intact Tri, retained the inhibitory activity on protein biosynthesis. The mAb No 14 and No 16 against Tri showed different immunoreactivities with 2 fragments and were selected as representatives in further blocking tests. The mAb No 14 hindered the activities of Tri and HATf1, whereas the mAb No 16 did not.
Conclusion: The active site of Tri responsible for inhibitory activity on protein biosynthesis was on the HATf1 side near the junction of two portions.