Oxidative folding of hepcidin at acidic pH.
Hepcidin is a four disulfide 25-residue peptide hormone which has a central role in the regulation of iron homeostasis. To support studies on hepcidin we have sought to establish reliable and robust synthetic methods for the preparation of correctly folded materials. While correctly-folded hepcidin has good aqueous solubility, we have found that its direct synthetic precursor, linear (reduced) hepcidin peptide, is resistant to solubilization, prone to precipitation at pH > or = 6, and thus difficult to fold efficiently. Attempts to directly fold either the crude or purified linear hepcidin peptide by air or DMSO oxidation methods under basic conditions were ineffective. However, addition of a glutathione redox pair system improved folding of purified linear hepcidin at mild basic pH (pH 7.5). Under acidic conditions, it was possible to oxidatively fold both crude and purified hepcidin using a polymer-supported oxidizing strategy. Peptide precipitation was also avoided under acidic conditions. Isolated folding yields of human hepcidin under acidic polymer-assisted conditions were superior to yields under basic folding conditions. These studies enabled identification of a reliable synthetic route for correctly-folded hepcidin.
Chemistry Research and Discovery, Amgen, Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA.
Jingwen
J
Zhang
Stephanie
S
Diamond
Tara
T
Arvedson
Barbra J
BJ
Sasu
Les P
LP
Miranda
D023181:Antimicrobial Cationic Peptides / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D020650:Combinatorial Chemistry Techniques; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D064451:Hepcidins; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010084:Oxidation-Reduction; D017510:Protein Folding
D023181:Antimicrobial Cationic Peptides; C578702:HAMP protein, human; D064451:Hepcidins; D004121:Dimethyl Sulfoxide