Disulfide isomerization and thiol-disulfide exchange of long neurotoxins from the venom of Ophiophagus hannah.
Selective reduction on the Cys28-Cys32 disulfide of Ophiophagus hannah neurotoxins, Oh-4 and Oh-5, revealed that isomerization of this disulfide linkage caused the two toxins to have distinct conformation and different retention time on a reversed-phase column. The Cys28-Cys32 disulfide of Oh-4 and Oh-5 was prone to form mixed disulfides with glutathione following pseudo-first-order kinetics. In addition to glutathionylated proteins, Oh-4 could be promoted to convert into Oh-5 by thiol compounds. Isomerization of Oh-5 into Oh-4 was not observed in the presence of thiol compounds. Dethiolation of glutathionylated proteins produced Oh-4 and Oh-5. Oxidation of the partially reduced toxin with reduced Cys28 and Cys32 was exclusively converted into Oh-5 regardless of the absence or presence of GSH/GSSG. Acrylamide quenching studies revealed difference in degree of exposure of the single Trp27 between Oh-4 and Oh-5. Synthesized peptides with substitution of Trp27 or Phe31 with Gly abolished entirely the formation of disulfide-linked dimeric product noted with the peptide of wild-type sequence. These results suggest that disulfide formation and isomerization of Cys28-Cys32 could be regulated by thiolation, and that the bulky aromatic residues Trp27 and Phe31 facilitate favorably the occurrence of disulfide isomerization of Cys28-Cys32.
Chang
Long-Sen
LS
Institute of Biomedical Sciences, National Sun Yat-Sen University-Kaohsiung Medical University Joint Center, National Sun Yat-Sen University, Kaohsiung 804, Taiwan. lschang@mail.nsysu.edu.tw
Lin
Shinne-Ren
SR
Huang
Hsien-Bin
HB
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D003039:Cobra Neurotoxin Proteins / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D005978:Glutathione / Q000737:chemistry; D007536:Isomerism; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D013438:Sulfhydryl Compounds / Q000737:chemistry*
D003039:Cobra Neurotoxin Proteins; D004220:Disulfides; D013438:Sulfhydryl Compounds; D005978:Glutathione