Abstract

Peptide toxins are usually highly bridged proteins with multipairs of intrachain disulfide bonds. Analysis of disulfide connectivity is an important facet of protein structure determination. In this paper, we successfully assigned the disulfide linkage of two novel peptide toxins, called HNTX-III and HNTX-IV, isolated from the venom of Ornithoctonus hainana spider. Both peptides are useful inhibitors of TTX-sensitive voltage-gated sodium channels and are composed of six cysteine residues that form three disulfide bonds, respectively. Firstly, the peptides were partially reduced by tris(2-carboxyethyl)-phosphine (TCEP) in 0.1 M citrate buffer containing 6 M guanidine-HCl at 40° C for ten minutes. Subsequently, the partially reduced intermediates containing free thiols were separated by reversed-phase high-performance liquid chromatography (RP-HPLC) and alkylated by rapid carboxamidomethylation. Then, the disulfide bonds of the intermediates were analyzed by Edman degradation. By using the strategy above, disulfide linkages of HNTX-III and HNTX-IV were determined as I-IV, II-V and III-VI pattern. In addition, this study also showed that this method may have a great potential for determining the disulfide bonds of spider peptide toxins.

Highlights

  • Venom peptides have been attracting attention from pharmacologists and biochemists

  • Among the advantages of TCEP are that it can selectively reduce the disulfide bonds depending on their accessibility and, more importantly, the process can be performed at acid pH to suppress the scrambling among the disulfide bonds [8, 9]

  • The peptide was partially reduced by TCEP at pH 3.0 and separated by reversed-phase high-performance liquid chromatography (RP-HPLC), which yielded intermediates with intact disulfide bond/bonds and free thiols

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Summary

Introduction

Venom peptides have been attracting attention from pharmacologists and biochemists. We successfully assigned the disulfide linkage of two novel peptide toxins, called HNTX-III and HNTX-IV, isolated from the venom of Ornithoctonus hainana spider. The peptides were partially reduced by tris(2-carboxyethyl)-phosphine (TCEP) in 0.1 M citrate buffer containing 6 M guanidine-HCl at 40°C for ten minutes.

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