Structure and Function of Cobrotoxin.
NATIONAL TSING HUA UNIV HSINCHU (TAIWAN) INST OF MOLECULAR BIOLOGY
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The two-dimensional structure of cobrotoxin has been established which permits a study of structure-function relationships. Previous studies on the chemical modification of cobrotoxin suggested that either the intact disulfide bonds or Tyr-25 are structurally important for maintenance of the active conformation of the toxin and at least two cationic groups held at certain distances in the molecule are functionally important for its neuromuscular blocking activity. The reaction of cobrotoxin with 4,4-difluoro-3,3-dinitrodiphenylsulfone FNPS was studied in an attempt to introduce new tertiary bonds into toxin. At pH 10.5, cobrotoxin was reacted with a 10-fold molar excess of FNPS at 25 C for 8 h. Dimers and higher molecular weight derivatives arising from intermolecular reactions were separated from intramolecularly cross-linked monomeric derivatives by gel filtration on a Sephadex G-50 column. The monomeric fraction F III was further purified by chromatography on CM-cellulose column. Six peaks were obtained, of which three fractions, IIIa, IIIb and IIIc were proved to be NPS-containing cobrotoxin derivatives and were all bifunctionally cross-linked with FNPS. The results of amino acid analyses of these derivatives revealed that the modification resulted in the cross-linkage between phenolic OH group of Tyr-residue and N-terminal alpha-NH2 group IIIa, and between Tyr-residue and epsilon-NH2 group of Lys-residue IIIb and IIIc. The lethality of IIIa, IIIb and IIIc decreased to 4, 4 and 13 with a concomitant increase in antigenic activities to 106.1, 123.9 and 131.3, respectively.