Received: July 14, 2008.

Accepted: February 12, 2009.

Abstract published online: March 5, 2009.

Full paper published online: May 30, 2009

DETERMINATION OF DISULFIDE BRIDGES OF TWO SPIDER TOXINS: HAINANTOXIN-III AND HAINANTOXIN-IV

Wang W (1), Liu Z (2), Qian W (3), Fang Y (1), Liang S (2)

(1) College of Pharmaceutical Sciences, Central South University, Changsha, Hunan, China; (2) College of Life Sciences, Hunan Normal University, Changsha, Hunan, China; (3) Administrative Center for Basic Research, Ministry of Science and Technology, China.

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.

KEY WORDS: disulfide bonds, TCEP, partial reduction, HNTX-III, HNTX-IV.

CONFLICTS OF INTEREST: There is no conflict.

FINANCIAL SOURCE: National Science Foundation.

CORRESPONDENCE TO:

SONGPING LIANG, College of Life Sciences, Hunan Normal University, Changsha, Hunan, 410081, China. Phone: +86 731 8872556. Fax: +86 731 8861304. Email: liangsp@hunnu.edu.cn.