Studies on the status of free amino and carboxyl groups in cobrotoxin

Abstract

The status of free amino groups in cobrotoxin was studied by stepwise modification with trinitrobenzene sulfonate. Lys-27 was selectively modified without altering the activity of cobrotoxin. However, complete loss of activity was observed when Lys-27 and Lys-47 were trinitrophenylated, suggesting that the ε-amino group of Lys-47 is essential for the activity of cobrotoxin. The α-amino group of N-terminal leucine had no correlation with activity, demonstrated by the guanidination of the lysine residues with O- methylisourea followed by trinitrophenylation of the α-amino group. The carboxyl groups in cobrotoxin were modified with glycine methyl ester after activation with water-soluble carbodiimide. Six out of seven free carboxyls reacted in the absence of guanidine · HCl without altering the biological activity. When the remaining carboxyl was modified in the presence of 5 M guanidine · HCl, the resulting toxin was devoid of activity. This “buried” carboxyl is essential for activity and was identified as the γ-carboxyl group of Glu-21.