[Determination of Jingzhaotoxin-I phospholipid membrane-binding activities by high performance liquid chromatography with fluorescence detection].
Jingzhaotoxin-I (JZTX-I), a 33-residue polypeptide with three disulfide bonds, was a novel spider neurotoxin preferentially inhibiting cardiac sodium channel inactivation. Its activities of phospholipid membrane-binding were studied by a combination of reversed-phase high performance liquid chromatography (HPLC) and fluorescence spectroscopy. Small unilamellar vesicles binding assays showed that the partitioning of JZTX-I into lipid bilayer did not require negatively charged phospholipids. Further, JZTX-I also exhibited a blue shift of 6.4 nm or 4.7 nm as well as red-edge excitation shift of 7.4 nm or 8.0 nm in the presence of 75% 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE )/25% 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (POPG) or 100% 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) vesicles respectively, suggesting that some tryptophan residues on the hydrophobic surface of the toxin were located within a motion restricted membrane interfacial region. Fluorescence quenching experiments suggested that some tryptophan residues of JZTX-I were positioned within the membrane and protected from aqueous quenching agents. These findings should provide further insight into the molecular mechanism of the channel gating of JZTX-I.
Zeng
Xiongzhi
X
The Key Laboratory of Protein Chemistry and Developmental Biology of Ministry of Education, Hunan Normal University, Changsha 410081, China. xiongzhizeng@yahoo.com.cn
Pi
Jianhui
J
Liang
Songping
S
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D008051:Lipid Bilayers / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D010743:Phospholipids / Q000737:chemistry*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013111:Spider Venoms / Q000737:chemistry*
D008051:Lipid Bilayers; D010455:Peptides; D010743:Phospholipids; D013111:Spider Venoms; C500354:jingzhaotoxin-I, Chilobrachys jingzhao