Oxidative folding of hepcidin at acidic pH

Abstract

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.

Synthetic hepcidin from fish: Uptake and protection against Vibrio anguillarum in sea bass (Dicentrarchus labrax)

“…Synthesis of cysteine rich peptides like hepcidin are more biologically active molecules than the peptides produced by the recombinant system [45]. In addition, oxidative folding is a key step for the four disulfide bonds of hepcidin to generate a tightly folded peptide [46]. In chemical synthesis these disulfide bonds are usually performed in a one-step oxidative procedure that is sufficient for the correct peptide folding.…”

Scorpion toxins for the reversal of BoNT-induced paralysis

“…37 This resin has demonstrated improved yields over air oxidation techniques for the formation of disulfide bonds in peptides such as the conotoxins 38 and hepcidin. 39 However, in our hands the yield of the oxidative folding of ChTX was approximately 7-fold greater under air oxidation conditions (pH 8, 5% yield) 33 than in the presence of CLEAR-OX (0.75% yield). Furthermore, the oxidation in the presence of the resin was sluggish, as a reaction time of 24 h was required, as opposed to 8 h under the slightly basic conditions.…”

Optimal Cleavage and Oxidative Folding of α-Conotoxin TxIB as a Therapeutic Candidate Peptide

“…Higher pH may facilitate efficient oxidative folding by regulating the degree of ionization of thiols to thiolates; disulfide bond reshuffling is also more significant at higher pH [38,39,40]. In this study, we concluded that pH 8 was optimal for oxidative folding of TxIB in NH 4 HCO 3 buffer, while pH 9 also exhibited a higher proportion of globular isomer.…”