Reduction and reoxidation of the neurotoxin II from the scorpion Androctonus australis Hector.
Reoxidation of the totally reduced scorpion neurotoxin II from Androctonus australis Hector (four disulfide bridges) has been investigated. The totally reduced toxin was highly insoluble in neutral and alkaline conditions, which prevented the use of the usual air oxidation process for renaturation. We tested a new method in which the reduced molecules were first solubilized in 10% (v/v) acetic acid and then oxidized by air through dialysis against a series of buffers with a slow pH gradient from 2.2 to 7.0 or 8.0. In this system, up to 95% of the protein was recovered in solution. Addition of reduced and oxidized glutathione accelerated refolding and also permitted a better recovery of fully active peptide as measured by both toxicity to mice and ability to displace 125I radiolabeled toxin II from its binding site on rat brain synaptosomal fractions. The reoxidation reaction could also be monitored directly by high pressure liquid chromatography. A strong effect of guanidine hydrochloride concentration as well as the temperature was observed both on the solubility of the reoxidation intermediates and on the refolding pathway. Finally, the method used, i.e. dialysis reoxidation with a pH gradient from 2.2 to 8.0 in 0.1 M sodium phosphate, 0.1 M sodium chloride, 20 mM guanidine hydrochloride, 1 mM oxidized and reduced glutathione allowed regeneration in high yield (70%) of a reoxidized toxin form indistinguishable from the native toxin. A minor stable and inactive molecular species (about 30%) showing a difference in mobility by electrophoresis was also detected.
Sabatier
J M
JM
U.D.C.- C.N.R.S. UA 1179, Faculty of Medicine North, Marseilles, France.
Darbon
H
H
Fourquet
P
P
Rochat
H
H
Van Rietschoten
J
J
D000818:Animals; D001921:Brain / Q000378:metabolism; D007700:Kinetics; D009498:Neurotoxins / Q000378:metabolism; D010084:Oxidation-Reduction; D051381:Rats; D011919:Rats, Inbred Strains; D011950:Receptors, Cholinergic / Q000378:metabolism*; D030162:Reptilian Proteins; D012604:Scorpion Venoms / Q000378:metabolism; D015222:Sodium Channels; D013574:Synaptosomes / Q000378:metabolism
D009498:Neurotoxins; D011950:Receptors, Cholinergic; D030162:Reptilian Proteins; D012604:Scorpion Venoms; D015222:Sodium Channels; C029574:scorpion toxin receptor; C053969:scorpion toxin II, Androctonus