The N-terminal amino group essential for the biological activity of notexin from Notechis scutatus scutatus venom

Abstract

Notexin from Notechis scutatus scutatus snake venom was modified with trinitrobenzenesulfonic acid, and the major trinitrophenylated (TNP) derivative was separated by high-performance liquid chromatography. Modification resulted in the incorporation of only one TNP group on the N-terminal α-amino group. The TNP derivative showed a precipitous decrease in enzymatic activity and lethal toxicity, whereas the antigenicity remained unchanged. However, trinitrophenylation did not significantly affect the secondary structure of the toxin molecule as revealed by the CD spectra. The results, that the modification reaction was accelerated by the Ca 2+ and that the TNP derivative retains its affinity for Ca 2+, indicate that the N-terminal α-amino group did not participate in the Ca 2+-binding. The TNP derivative could be regenerated with hydrazine hydrochloride. The biological activities of the regenerated notexin are almost the same as those of native notexin. These results suggest that the N-terminal α-amino group is essential for the phospholipase A 2 activity and lethal toxicity of notexin, and that incorporation of the TNP group on the N-terminal α-amino group might give rise to a distortion of the active conformation of notexin.