Oxidative folding of reduced and denatured huwentoxin-I.
Huwentoxin-I, a neurotoxic peptide with 33 amino acid residues and three disulfide bonds, was used to investigate the pathway of reduction/denaturation and of oxidative folding in small proteins with multiple disulfide bonds. Titration of thiol groups, reversed-phase HPLC, 1D NMR spectroscopy, and biological activity assays were used to monitor the extent of reduction/ denaturation and renaturation of the toxin. The reduction and denaturation of huwentoxin-I resulted in a 100% loss of bioactivity as measured in a mouse phrenic nerve-diaphragm preparation. About 90% of full biological activity could be restored under optimized conditions of oxidative refolding of the reduced peptide. Several reaction conditions employing air oxidation, oxidized and reduced glutathione (GSSG and GSH), and cystine/cysteine were investigated in order to find optimal conditions for renaturation of huwentoxin-I. The best renaturation yield was achieved in 0.1 mM GSSG and 1 mM GSH at pH 8.5 and 4 degrees C over 24 hr. High concentrations of glutathione and high temperatures reduced renaturation yields. Oxidative refolding of huwentoxin-I in air requires about 6 days for maximal yields and is inhibited by EDTA.
Liang
S
S
College of Life Sciences, Hunan Normal University, Changsha, China.
Shu
Q
Q
Wang
X
X
Zong
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X
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003964:Diaphragm / Q000294:innervation; D005978:Glutathione / Q000737:chemistry; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008969:Molecular Sequence Data; D009498:Neurotoxins / Q000737:chemistry* / Q000494:pharmacology; D010084:Oxidation-Reduction; D010791:Phrenic Nerve / Q000187:drug effects / Q000502:physiology; D011489:Protein Denaturation; D017510:Protein Folding; D030162:Reptilian Proteins; D013111:Spider Venoms / Q000737:chemistry* / Q000494:pharmacology
D009498:Neurotoxins; D030162:Reptilian Proteins; D013111:Spider Venoms; C083624:huwentoxin I; D005978:Glutathione