International Journal of Peptide Research and Therapeutics

, 14:341

Optimization of the Oxidative Folding Reaction and Disulfide Structure Determination of Human α-Defensin 1, 2, 3 and 5

Authors

  • Shigeru Kubo
    • SAITO Research Center, Peptide Institute, Inc.
  • Kyoko Tanimura
    • SAITO Research Center, Peptide Institute, Inc.
  • Hideki Nishio
    • SAITO Research Center, Peptide Institute, Inc.
  • Naoyoshi Chino
    • SAITO Research Center, Peptide Institute, Inc.
    • Department of Chemistry, Graduate School of ScienceOsaka University
  • Tadashi Teshima
    • SAITO Research Center, Peptide Institute, Inc.
    • Department of Chemistry, Graduate School of ScienceOsaka University
  • Terutoshi Kimura
    • SAITO Research Center, Peptide Institute, Inc.
    • SAITO Research Center, Peptide Institute, Inc.
    • Department of Chemistry, Graduate School of ScienceOsaka University
Article

DOI: 10.1007/s10989-008-9146-z

Cite this article as:
Kubo, S., Tanimura, K., Nishio, H. et al. Int J Pept Res Ther (2008) 14: 341. doi:10.1007/s10989-008-9146-z

Abstract

The optimal conditions were determined for oxidative folding of the reduced human α-defensins, HNP1, HNP2, HNP3 and HD5, preferentially into their native disulfide structures. Since the human α-defensin-molecule in both reduced and oxidized forms raised a solubility problem arising from its basic and hydrophobic compositions, buffer concentration had to be lowered and cosolvent, such as CH3CN, had to be added to the folding medium in the presence of reduced and oxidized gluthathione (GSH/GSSG) to prevent aggregation and also to realize predominant formation of the native conformer. The four synthetic human α-defensins of high homogeneity were confirmed to exhibit the same antimicrobial potencies against E. coli as those reported for the natural products. All these peptides were shown to possess the native disulfide structure by sequence analyses and mass measurements with cystine segments obtained by enzymatic digestion. Edman degradation allowed for disulfide assignment of cystine segments involving adjacent Cys residues composed of three peptide chains, for which two possible disulfide modes could be considered, with the guidance of the cycles detecting diPTH cystine. As for HNP1, HNP2 and HNP3, however, diPTH cystine was expected at the same cycles in both structures, which would have resulted in not being able to distinguish between the two alternative modes. To avoid this, it was necessary to provide an acetyl tag for the specific peptide chain originating from the N-terminus. Edman degradation of cystine segments tagged with the acetyl group would be a practical procedure for analyzing disulfide structures involving adjacent Cys residues.

Keywords

Antimicrobial peptideChemical synthesisDefensinDisulfide structureEdman degradationOxidative folding reaction

Abbreviations

Ac

Acetyl

AcOH

Acetic acid

Boc

t-Butyloxycarbonyl

BrZ

2-Bromobenzyloxycarbonyl

BuOH

1-Butanol

But

t-Butyl

Bzl

Benzyl

cHx

Cyclohexyl

6-Cl-HOBt

6-Chloro-1-hydroxybenzotriazole

ClZ

2-Chlorobenzyloxycarbonyl

CZE

Capillary zone electrophoresis

DCM

Dichloromethane

DIEA

Diisopropylethylamine

DMSO

Dimethyl sulfoxide

DTT

Dithiothreitol

ESI MS

Electrospray ionization mass spectrometry

Fmoc

9-Fluorenylmethoxycarbonyl

For

Formyl

GnHCl

Guanidinium hydrochloride

GSH

Glutathione

GSSG

Oxidized glutathione

hBD

Human β-defensin

HCTU

1-[bis(dimethylamino)methylene]-5-chloro-1H-benzotriazolium 3-oxide hexafluorophosphate

HD

Human α-defensin

HNP

Human neutrophil peptide

HOBt

1-Hydroxybenzotriazole

Hoc

Cyclohexyloxycarbonyl

HPLC

High performance liquid chromatography

IEX

Ion exchange

4-MeBzl

4-Methylbenzyl

NMP

1-Methyl-2-pyrrolidinone

OSu

Succinimide ester

Pbf

2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl

PTH

3-Phenyl-2-thiohydantoin

(PTH-Cys)2

diPTH-cystine

RP

Reversed phase

SPPS

Solid-phase peptide synthesis

TFE

2,2,2-Trifluoroethanol

TIS

Triisopropylsilane

TFA

Trifluoroacetic acid

Tos

p-Toluenesulfonyl

Trt

Trityl

Copyright information

© Springer Science+Business Media, LLC 2008