Unfolding/folding studies on cobrotoxin from Taiwan cobra venom: pH and GSH/GSSG govern disulfide isomerization at the C-terminus.
Refolding of cobrotoxin was assessed by the exposure degree of its single Trp determined by an acrylamide quenching study. The change in the accessibility of Trp for acrylamide quantitatively reflected the formation of folded cobrotoxin, and the data were confirmed by HPLC and gel electrophoresis analyses. However, the site-specific information provided by quenching Trp fluorescence revealed that the ordered structure in the neighborhood of Trp was attained prior to the complete formation of the tertiary structure of cobrotoxin. HPLC analyses showed that, in addition to refolded cobrotoxin, two novel species (cobrotoxin II and cobrotoxin III) with isomerization of disulfide bonds at the C-terminus of the toxin molecule were produced along the folding reaction. The disulfide pairings in cobrotoxin II and cobrotoxin III were Cys43-Cys55 and Cys54-Cys60 and Cys43-Cys60 and Cys54-Cys55, respectively. Among the three possible two-disulfide species at the C-terminus, the disulfide linkages Cys43-Cys60 and Cys54-Cys55 of cobrotoxin III caused a marked decrease in lethality and resulted in a conformation which was notably different from that observed with the native toxin molecule as evidenced by CD spectra. The refolding reaction was accelerated by the addition of GSH/GSSG, and the resulting products were mostly folded cobrotoxin. However, if GSH/GSSG was not added into the initial folding materials, the yields of cobrotoxin II and cobrotoxin III greatly increased. The conversion of cobrotoxin to its isomers was to be irreversible and pH-dependent: the higher the pH, the faster the rate of conversion. However, this conversion could be partly inhibited by GSH/GSSG. Cobrotoxin II and cobrotoxin III were purified from Taiwan cobra venom as well, and their yields in comparison to that of cobrotoxin in venom were similar to that noted with the folded products in the presence of GSH/GSSG. Moreover, the rate of disulfide isomerization was expected to be slow in venom fluid in which the pH was approximately pH 6.2. Thus, the finding that cobrotoxin represents the predominant neurotoxin species in Taiwan cobra venom is probably associated with the synergistic effects of GSH/GSSG and pH.
Chang
L S
LS
Department of Biochemistry, Kaohsiung Medical College, Taiwan, Republic of China. lschang@mail.nsysu.edu.tw
Lin
S R
SR
Chang
C C
CC
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003039:Cobra Neurotoxin Proteins / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D017815:Elapidae; D004591:Electrophoresis, Polyacrylamide Gel; D005978:Glutathione / Q000737:chemistry*; D019803:Glutathione Disulfide / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007536:Isomerism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013624:Taiwan; D014364:Tryptophan / Q000737:chemistry
D003039:Cobra Neurotoxin Proteins; D004220:Disulfides; D014364:Tryptophan; D005978:Glutathione; D019803:Glutathione Disulfide