ACTIVE-SITE OF TRICHOSANTHIN ACTING AS A RIBOSOME-INACTIVATING PROTEIN

AIM: To localize the active site of ribosome inactivation of trichosan thin (Tri), a Chinese herb protein. METHODS: Hydroxylamine was used to specifically cleave the unique Asn-Gly peptide bond of Tri. Preparati ve SDS-polyacrylamide gel electrophoresis was applied to get 2 cleaved fragments, HATf1 and HATf2. Western blotting was used to determine th e different epitopes of Tri and screen the antibodies. A cell-free sys tem, rabbit reticulocyte lysate, was introduced to quantitate the inhi bitory activity of Tri and its fragments on protein biosynthesis. RESU LTS: HATf1 and HATf2 were separated with the purity of 96.9 % and 80.5 % respectively. HATf1, like intact Tri, retained the inhibitory activ ity on protein biosynthesis. The mAb No 14 and No 16 against Tri showe d different immunoreactivities with 2 fragments and were selected as r epresentatives in further blocking tests. The mAb No 14 hindered the a ctivities 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.