Studies on the status of free amino and carboxyl groups in cobrotoxin.
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 epsilon-amino group of Lys-47 is essential for the activity of cobrotoxin. The alpha-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 alpha-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 gamma-carboxyl group of Glu-21.
Chang
C C
CC
Department of Biochemistry, Kaohsiung Medical College, Kaohsiung, Taiwan, Republic of China.
Yang
C C
CC
Nakai
K
K
Hayashi
K
K
D000595:Amino Acid Sequence; D000818:Animals; D002250:Carbon Radioisotopes; D003039:Cobra Neurotoxin Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000633:toxicity; D004220:Disulfides / Q000737:chemistry; D004546:Elapid Venoms / Q000737:chemistry; D017815:Elapidae; D007202:Indicators and Reagents; D008239:Lysine; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D013053:Spectrophotometry; D014302:Trinitrobenzenesulfonic Acid
D002250:Carbon Radioisotopes; D003039:Cobra Neurotoxin Proteins; D004220:Disulfides; D004546:Elapid Venoms; D007202:Indicators and Reagents; D010446:Peptide Fragments; D014302:Trinitrobenzenesulfonic Acid; D008239:Lysine