Oxidative folding of hepcidin at acidic pH

Invited ReviewOxidative Folding of Hepcidin at Acidic pH

Jingwen Zhang,1 Stephanie Diamond,1 Tara Arvedson,2 Barbra J. Sasu,2 Les P. Miranda11Chemistry Research and Discovery, Amgen, Inc., One Amgen Center Drive, Thousand Oaks, CA 91320

2Hematology and Oncology Research, Amgen, Inc., One Amgen Center Drive, Thousand Oaks, CA 91320

Received 16 September 2009; revised 10 December 2009; accepted 22 December 2009

Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bip.21383

INTRODUCTION

Hepcidin is a peptide hormone secreted primarily by

the liver, that plays a central role in the regulation of

iron homeostasis through its interactions with the iron

transporter ferroportin.1–4 Genetic studies have dem-

onstrated that the hepcidin pathway is a critical com-

ponent in the control of iron metabolism.5–7 Hepcidin excess

leads to iron deficiency anemia,8 whereas hepcidin deficiency

results in hereditary hemochromatosis, a disease where iron

accumulates in vital organs.9 Inappropriate iron distribution is

implicated in multiple diseases, such as anemia of inflammation

(AI),10 atherosclerosis,11 and neurodegenerative disorders.12

The human hepcidin gene encodes an 84-residue pre-pro-

hepcidin polypeptide. This peptide is processed to produce a

mature 25-residue hepcidin peptide. Hepcidin circulates in

the serum and is cleared through the kidney, leading to its

identification in human urine and blood ultrafiltrate.13

N-terminally truncated forms, including hepcidin 20 and

hepcidin 22, have also been detected in urine.13 Hepcidin

contains eight cysteine residues (32% of its total amino

acids), and all are paired in four disulfide bonds generating a

tightly folded peptide (see Figure 1).

Previous structural NMR studies of human and bass hep-

cidin reported a C7��C23, C11��C19, C13��C14, and

C10��C22 (C1��C8, C3��C6, C4��C5, C2��C7) disulfide

connectivity, which included a rare vicinal disulfide bond.14

Using several structural techniques including variable tem-

perature NMR and X-ray crystallography, we have recently

determined a different disulfide connectivity for hepcidin:

C7��C23, C10��C13, C11��C19, and C14��C22 (C1��C8,

C2��C4, C3��C6, C5��C7) (see Figure 1).15 These orthogo-

nal techniques found no data to support the presence of the

previously proposed vicinal disulfide bond. Both the NMR-

derived aqueous structure and the crystal structure demon-

strate that hepcidin consists of two b-sheet structural motifs

and a b-hairpin loop.

Invited ReviewOxidative Folding of Hepcidin at Acidic pH

Correspondence to: Les P. Miranda; e-mail: [email protected]

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 � 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. # 2010 Wiley Periodicals, Inc.

Biopolymers (Pept Sci) 94: 257–264, 2010.

Keywords: hepcidin; iron homeostasis; ferroportin

VVC 2010 Wiley Periodicals, Inc.

PeptideScience Volume 94 / Number 2 257

To provide adequate quantities of hepcidin and related

derivatives for structural and biological studies, we pursued

several methods for its preparation. While the solid-phase as-

sembly of the linear hepcidin peptide was straightforward,

initial attempts to solubilize and fold the linear (reduced)

peptide according to standard methods were not effective for

the production of multi-milligram quantities. Here, we

report the results of several methods which were examined

for the oxidative folding of human hepcidin under both basic

and acidic pH conditions (see Figure 2).

MATERIALS AND METHODS

Solid-Phase Assembly of Human HepcidinHuman hepcidin and Abu ((L)-2-aminoisobutyric acid)

[Abu7,10,11,13,14,19,22,23] hepcidin peptide chains were chemically syn-

thesized using an ABI433 synthesizer (Applied Biosystems, Foster

City, CA) employing an Na-Fmoc/side-chain tBu orthogonal protec-

tion strategy with 1.0M N,N0-dicyclohexylcarbodiimide (DCC)/1-

hydroxybenzotriazole hydrate (HOBT) (1:1) coupling chemistry in

N-methyl-pyrrolidone (NMP) and 20% (v/v) piperidine/NMP

deprotection chemistry.16 The synthesis was carried out on Fmoc-

Thr(tBu)-Wang resin (0.125 mmol equiv scale, Novabiochem). The

following side-chain protection strategy was used with Na-Fmoc-

protected amino acids: Asp(tBu), Asn(Trt), Thr(tBu), His(Trt),

Cys(Trt), Arg(Pbf), Ser(tBu), and Lys(Boc). Single amino acid cou-

pling cycles at 1 mmol scale were used for the synthesis, and con-

sisted of 58-min coupling times and 3 + 15 min Fmoc-deprotection

times.

Side-Chain Deprotection and Resin-CleavageSide-chain deprotection and cleavage from the solid-support was

accomplished by treatment with trifluoroacetic acid (TFA)/H2O/

triisopropylsilane (TIS)/3,6-dioxa-1,8-octane-dithiol (DODT)

(92.5:2.5:2.5:2.5 v/v) for 2 h, the solution was filtered and then

evaporated in vacuo. The residue was treated with ice-cold diethyl

ether (250 mL) and the precipitated peptide was collected by cen-

trifugation (5 min at 3800 rpm). The ether solution was decanted

and the peptide was dried in vacuo.

Purification of Reduced Human HepcidinThe dried crude linear hepcidin peptide was reconstituted in neat

TFA (2 mL) and then diluted drop-wise with stirring into a fresh

6M guanidine/0.25M Tris/10 mM EDTA pH 5 buffered solution

(100 mL) containing Tris(2-carboxyethyl)phosphine hydrochloride

FIGURE 1 Primary structure of human hepcidin using single let-

ter amino acid code. Disulfide connectivity and residue numbering

are indicated.

FIGURE 2 General scheme outlining the oxidative folding routes evaluated for human hepcidin.

Routes A–C involved folding at basic pH, whereas routes D and E involved folding at acidic pH.

Routes A and E were performed directly on crude linear hepcidin. The backbone structure of hepci-

din with disulfide connectivity used in the figure was derived from its crystallographic structure.15

258 Zhang et al.

Biopolymers (Peptide Science)

(TCEP, 1 mmol) and stirred for 2 h. The reduced human hepcidin-

containing solution was then loaded onto a Phenomenex Jupiter 10

lm 300 A C18 250 3 21.2 mm column for preparative purification

and fractions containing the expected molecular mass of reduced

human hepcidin were pooled (C113H178N34O31S9, Calc. mass:

2795.09 Da). Isolated purification yield with >95% purity was typi-

cally 21 mg (6%).

Air Oxidation of HepcidinAir oxidation was carried out by dissolving crude hepcidin cleavage

material in 30% aqueous acetonitrile at a concentration of 0.2 mg

peptide/mL. The pH was adjusted to 7.0 or 8.5 with NH4OH, and

stirred in an open atmosphere at room temperature for 24–48 h.

Prior to analysis by RP-HPLC, the solution was acidified to pH 3

with TFA and filtered through a 0.2-lm filter.

DMSO Oxidation of HepcidinDMSO oxidation was carried out by dissolving crude hepcidin

cleavage material in 2M Guanidine/DMSO/isopropanol (8:1:1, v/v)

at a concentration of 0.2 mg peptide/mL. The pH was adjusted to

5.8 with NH4OH, and stirred at room temperature for 24 to 48 h.

Prior to analysis by RP-HPLC, the solution was acidified to pH 3

with TFA and filtered through a 0.2 lm filter.

Oxidized and Reduced Glutathione-Assisted

Oxidation of Purified Linear Human HepcidinPurified linear human hepcidin (15 mg) was diluted to 94 mL with

water and acetonitrile to give an approximate final acetonitrile com-

position of 30% (v/v) and peptide concentration of 0.16 mg mL�1.

Disulfide bond formation was carried out for *16 h in the presence

of a 1:1 glutathione/glutathione disulfide (GSH/GSSG) redox sys-

tem (13.8 mg GSSG (0.023 mmol) and 7.05 mg GSH (0.023 mmol))

at pH 7.5 (solution adjusted with 28–30% NH4OH, Baker) with

stirring at 70 rpm. After folding for *16 h, the human hepcidin

containing solution was then adjusted to pH 2 with neat TFA and

the acetonitrile solvent component was evaporated. The crude fold-

ing solution containing human hepcidin was then loaded onto a

Phenomenex Jupiter 10 lm, 300 A, C18, 100 3 7.8 mm column for

preparative purification. The elution linear gradient method was

10–25% buffer B (0.9% TFA in acetonitrile) in 10 min followed by

25–35% buffer B in 40 min at a flow rate 3.5 mL min�1. Fractions

were analyzed by LC/MS and fractions containing >95% hepcidin

were pooled and lyophilized. Yield ¼ 0.95 mg (6.4%). The disulfide

connectivity of the product was determined by reductive-alkylation

techniques and was found to be C7��C23, C10��C13, C11��C19, and

C14��C22.15 C113H170N34O31S9 Calc. mass: 2787.03 Da; Exp.

Observed mass: 2787.23 Da. Amino acid analysis (AAA): Asx 1.0,

Serine 0.9, Glycine 2.0, Histidine 2.0, Arginine 1.0, Threonine 1.7,

Proline 1.0, Cysteine 8.0, Methionine 1.0, Lysine 2.1, Isoleucine 2.0,

and Phenylalanine 1.9.

CLEAR-OX2

Oxidation of Human HepcidinCLEAR-OX

2

resin (0.2 mmol equiv/g; Peptides International; 10

molar excess to peptide) was placed into a fritted peptide synthesis

reaction vessel, swollen in DCM for 30 min, and then washed

successively with 20 mL of DCM, DMF, MeOH, and 50% aqueous

acetonitrile. Reduced hepcidin peptide (14 mg) was added to the

resin and 30% aqueous acetonitrile was added to give a peptide con-

centration of 6 mg mL�1. The pH was adjusted via the addition of

1M Tris buffer to pH 5.5 and the vessel was gently agitated at 218C.Analytical samples were taken periodically, acidified with acetic

acid, filtered through a 0.45-lm filter, and then analyzed by LC/MS.

Upon completion of folding, the bulk reaction was collected and the

resin washed three times with 50% aqueous acetonitrile. The filtrate

was acidified as above, filtered through a 0.45-lm filter, lyophilized,

and then loaded onto a Phenomenex Jupiter 10 lm, 300 A,

C18, 100 3 7.8 mm column for purification. The elution linear gra-

dient method was 10–25% buffer B in 10 min followed by 25–35%

buffer B in 40 min at a flow rate 3.5 mL min�1. Fractions were

analyzed by LC/MS and fractions containing >95% hepcidin were

pooled and lyophilized. C113H170N34O31S9 Calc. mass: 2787.03 Da;

Exp. observed mass by ES-MS: 2787.23 Da. Isolated purified yield

starting with purified reduced hepcidin (14 mg) at pH 5.5 Yield ¼1.3 mg (12%). Isolated purified yield starting with crude reduced

hepcidin (38 mg) at pH 4.0 ¼ 4.7 mg (12%).

RESULTS AND DISCUSSION

Solid-Phase Synthesis of Linear Human Hepcidin

The aim of this work was to identify a reliable route for the

preparation of human hepcidin. The starting linear hepcidin

peptide was assembled by solid-phase peptide synthesis

(SPPS) using an Na-Fmoc/tert-butyl strategy on Fmoc-

Thr(tBu)-Wang resin.16 Peptide-chain assembly was carried

out using N,N0-dicyclohexylcarbodiimide (DCC)/1-hydroxy-

benzotriazole hydrate (HOBT) (1:1) coupling chemistry in

N-methyl-pyrrolidone (NMP) and 20% (v/v) piperidine/

NMP deprotection chemistry. We found that the on-resin

chain assembly of the hepcidin peptide proceeded well with-

out any difficult couplings. Following TFA-mediated side-

chain deprotection and cleavage from the resin, the major

product in the crude material, as determined by LC/MS anal-

ysis was the expected reduced linear peptide with a molecular

weight of 2797 Da (see Figure 3). The later eluting peaks cor-

respond in mass to modified linear hepcidin compounds,

such as +56 tBu adducts.

Handling of Crude Linear Human Hepcidin

While the quality of the crude cleavage material was accepta-

ble, we found that the bulk peptide was poorly soluble in a

variety of different solvents, including aqueous acetonitrile

mixtures, DMSO, guanidine-based buffer, aqueous acetic

acid, aqueous isopropanol, and trifluoroethanol (TFE). In

some cases, gelatinous suspensions formed, and heating and

sonication did not entirely obviate the problem. Repeated

diethyl ether precipitation and lyophilization steps after TFA

cleavage did improve peptide solubility properties in aqueous

acetonitrile, although purification yields were found to be

Oxidative Folding of Hepcidin at Acidic pH 259


more consistent after TFA solubilization. Attempts to directly

oxidize crude linear hepcidin with solvent mixtures, includ-

ing 2M Guanidine/DMSO/isopropanol (8:1:1, v/v) at pH 5.8

also led to poor results. Similarly, direct oxidation of crude

linear hepcidin with 30% acetonitrile in water at pH 7–8.5

also resulted in significant peptide precipitation. Little or

no correctly folded peptide was detected using these fold-

ing conditions. In general, we found the handling of

reduced linear hepcidin in solution near pH 6 or higher

resulted in significant peptide precipitation in either an

immediate or gradual manner. Although no reduced linear

hepcidin starting peptide remained after the folding pro-

cess, the oxidized material was a complex product mixture

containing only a minute amount of peptide with the

expected mass (2787 Da) and LC/MS retention time of

correctly folded hepcidin.

Air and DMSO Oxidation of Purified Linear Human

Hepcidin

Given that the direct folding of the crude material was

unsuccessful, we sought to purify linear (reduced) human

hepcidin and further investigate folding. The most reliable sol-

ubilization procedure for bulk crude linear hepcidin was disso-

lution in a few milliliters of neat trifluoroacetic acid (TFA),

followed by 50-fold dilution with 6M guanidine/0.25M Tris/10

FIGURE 3 Reversed-phase HPLC analysis of crude linear

(reduced) human hepcidin. The arrow indicates the peak corre-

sponding to linear hepcidin (reduced), with a monoisotopic molec-

ular mass of 2795 Da.

FIGURE 4 Reversed-phase HPLC analysis of the product after

folding of purified linear (reduced) human hepcidin by oxidation

with 30% acetonitrile in water at pH 8 at room temperature for (A)

1 h and (B) 4 days.

FIGURE 5 Reversed-phase HPLC analysis after folding of purified linear (reduced) human hepci-

din oxidized with 30% aqueous acetonitrile, 1:1 GSH/GSSG at pH 7.5 at room temperature. The RP-

HPLC chromatograms of the folding reaction at t ¼ 0 and t ¼ 16 are shown in panels A and B,

respectively. The corresponding mass spectra (avg.) for the major components are shown on the

right.

260 Zhang et al.


mM EDTA, pH 5. After loading onto a preparative RP-HPLC

column, the eluted fractions containing the expected molecu-

lar mass of reduced human hepcidin (2795 Da) were pooled

and lyophilized in*6% yield (>95% purity). The purified lin-

ear human hepcidin peptide showed significantly improved

solubility in >20% acetonitrile/water mixtures. However, puri-

fied linear hepcidin still precipitated from aqueous solution

when subjected to pH > 6 in a manner similar to that of crude

linear hepcidin. Purified linear hepcidin was also susceptible to

spontaneous but partial oxidation and aggregation, both while

standing in HPLC elution mixtures after HPLC purification,

and during lyophilization processes.

Oxidation of purified linear human hepcidin with 30%

acetonitrile in water, pH 8 (Figure 2, Route B), at room tem-

perature was more successful than oxidation of crude mate-

rial (Figure 2, Route A) but overall unsatisfactory. After over-

night oxidation, LC-MS analysis revealed a complex product

mixture and a small peak at the expected retention time for

folded hepcidin (see Figure 4). We also observed peptide pre-

cipitation during this folding reaction. Oxidation of purified

linear human hepcidin with DMSO solution mixtures gave

similar results (data not shown). Lowering of the peptide

concentration during folding did not noticeably improve the

quality of the crude folding product or avoid precipitation.

Oxidized and Reduced Glutathione-Assisted

Oxidation of Purified Linear Human Hepcidin

The effect of an oxidized and reduced glutathione redox

buffer17 on purified linear human hepcidin oxidation was

examined. Purified linear human hepcidin in its HPLC elu-

tion buffer or 30% aqueous acetonitrile, was treated with

glutathione/glutathione disulfide (GSH/GSSG) mixtures at

pH 7.5 and stirred at 70 rpm for 16 h (Figure 2, Route C).

Several ratios and molar excesses of GSH/GSSG were eval-

uated, and it was found that a 1:1 GSH/GSSG ratio in seven-

fold molar excess over the peptide gave the most satisfactory

results (see Figure 5). Under these conditions, we observed

relatively efficient transformation of the linear (reduced)

hepcidin starting material into an earlier eluting peak as

determined by HPLC analysis. The mass of this product

peak, as determined by electrospray-time of flight mass spec-

trometry, was 2787 Da. The loss of 8 Da is consistent with

the formation of four disulfide bonds. The material also coe-

luted with urinary hepcidin, a reference sample. Importantly,

the extent of peptide precipitation was reduced but not elim-

inated entirely under these conditions. Using this process,

human hepcidin was isolated by RP-HPLC in 6% yield from

the purified linear starting peptide. A previously reported di-

sulfide folding method utilizing cysteine (Cys)/cystine

(Cys2), the most abundant, low-molecular-weight thiol/di-

sulfide redox couple found in the plasma, can also be used.18

CLEAR-OX2

Oxidation of Purified Linear Human

Hepcidin

In an attempt to bypass solubility, purification, and precipi-

tation issues, we decided to explore the folding of hepcidin at

acidic pH rather than under conventional basic or near neu-

tral pH conditions.17 It had been previously shown that solu-

tion phase intramolecular disulfide bond formation could be

facilitated at pH 2–5 with dithiopyridine compounds.19,20

FIGURE 6 Time-course of the CLEAR-OX2

-assisted folding of

purified linear (reduced) human hepcidin as determined by

reversed-phase HPLC analysis. An asterisk indicates the HPLC peak

corresponding to correctly-folded human hepcidin.



Furthermore, capitalizing on the pseudo-dilution effect, a

cross-linked ethoxylate acrylate resin with attached 5,50-dithiobis(2-nitrobenzoic acid), CLEAR-OX

2

, has been

reported as a fast and efficient reagent for disulfide bridge

formation at pH 4.6–8.21 This polymer-supported oxidant

had been previously applied to the oxidative folding of one-,

two-, and three-disulfide peptides,21,22 but to our knowledge

is yet to be attempted for a tightly folded four-disulfide pep-

tide such as hepcidin. Although it was unclear how the poly-

mer-supported folding of a complicated four-disulfide pep-

tide would proceed, we investigated the CLEAR-OX2

-assisted

folding of purified linear hepcidin at pH 3.3, 4.0, 5.5, and

7.5. In agreement with the above solution-phase folding

processes, we found that CLEAR-OX2

-assisted folding under

basic conditions (pH 7.5) resulted in the precipitation of

linear hepcidin from solution, leaving a negligible amount

of reduced hepcidin in solution after overnight folding

(t ¼ 17 h). No folded hepcidin could be detected or recov-

ered using CLEAR-OX2

at pH 7.5. In contrast, the CLEAR-

OX2

-assisted folding of hepcidin at pH 3.3, 4.0, and 5.5 had

no noticeable peptide precipitation and the folding reaction

proceeded with good conversion yield to correctly folded

hepcidin. At pH 3.3, the CLEAR-OX2

-assisted folding was

monitored over a time-course of *90 h (see Figure 6). Fold-

ing at pH 3.3 resulted in a higher purity level of the folding

product than at pH 5.5. In comparison to the optimized so-

lution-phase folding of hepcidin which utilizes glutathione/

glutathione disulfide (GSH/GSSG) and is usually complete

within 20 h, the CLEAR-OX2

-assisted folding of hepcidin

proceeded at a relatively slow but steady rate. LC/MS analysis

of samples taken during the folding time-course showed that

the intermediate peaks, with elution times between the

reduced and fully oxidized hepcidin peaks, corresponded in

molecular mass to partially folded hepcidin compounds.

This indicated that partially folded hepcidin compounds

with only one to three disulfide bonds gradually proceed to

the correct and fully oxidized hepcidin peptide containing

four disulfide bonds. Folding at pH 4.0 with CLEAR-OX2

(Figure 2, Route D) also led to a high quality folding product

but had the advantage of faster folding kinetics over the oxi-

dation at pH 3.3. Importantly, we found that correctly folded

hepcidin could be obtained from directly folding of crude

(unpurified) linear hepcidin using CLEAR-OX2

-assisted

folding at pH < 5.5 (Figure 2, Route E). In addition, this

procedure avoided peptide precipitation. The folding of puri-

fied linear and crude linear hepcidin with CLEAR-OX2

at pH

FIGURE 7 Reversed-phase HPLC analysis time-course of the CLEAR-OX2

-assisted folding at pH 4

with: (A) purified linear hepcidin; (B) crude linear hepcidin.

262 Zhang et al.


4.0 both resulted in a 12% isolated yield of correctly folded

hepcidin from the respective starting peptides (see Figure 7).

However, the overall isolated yield from the crude cleavage

material was significantly higher for the CLEAR-OX2

-

assisted folding of crude hepcidin (12%) because this

approach obviated the losses associated with (1) purification

of the linear peptide, and (2) peptide precipitation. On this

basis, the isolated yield of hepcidin from crude peptide for

the glutathione-assisted and CLEAR-OX2

(pH 4) folding via

purified linear hepcidin peptides were both less than 1%.

Assessment of Human Hepcidin Disulfide

Connectivity and In Vitro Activity

Chemically synthesized, correctly-folded hepcidin peptide

was found to coelute with human hepcidin purified from

urine. Urinary hepcidin was used throughout these studies as

the hepcidin reference sample. As recently reported, chemi-

cally synthesized hepcidin had a disulfide connectivity of

C7��C23, C10��C13, C11��C19, and C14��C22 (C1��C8,

C2��C4, C3��C6, C5��C7).15 This connectivity was identi-

cal to that determined for urinary hepcidin. The chemically

synthesized hepcidin material was also tested in a previously

reported intracellular iron retention assay.15 The activity of

both the synthesized and urinary material were comparable

(EC50 ¼ 45 nM and *20 nM for the synthetic and urinary

material, respectively) (see Figure 8). No activity was

observed with [Abu7,10,11,13,14,19,22,23] hepcidin, a cysteine-

free linear analog of hepcidin.

CONCLUSIONSIn this work we have examined several methods for the fold-

ing of human hepcidin, a four-disulfide peptide, after prepa-

ration of the linear peptide precursor by solid-phase peptide

synthesis. To increase final yields and reduce the difficulty of

hepcidin preparation, we evaluated several oxidation meth-

ods, and identified suitable folding conditions in both

basic and acidic environments. At basic pH, we found a solu-

tion-phase glutathione redox pair system at pH 7.5 that

resulted in fast folding kinetics and good transformation effi-

ciency. At acidic pH, we found the difficulties with low pep-

tide solubility and precipitation encountered at basic pH

could be avoided by folding hepcidin at pH 3.3–5.5 using the

polymer-supported oxidant CLEAR-OX2

. Importantly, the

folding of crude linear human hepcidin at acidic pH with

CLEAR-OX2

bypassed the losses associated with additional

purification steps and peptide precipitation, and thus led

to significantly higher overall yields of biologically active

hepcidin. These improved methods will be useful for the

efficient preparation of hepcidin, and its homologs and

derivatives, for biological and structural studies.

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Oxidative folding of hepcidin at acidic pH

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