Purification and Partial Characterization of a Novel Neurotoxic Protein from Eggs of Black Widow Spiders (Latrodectus tredecimguttatus)

Abstract

Up to now, there have been a few reports on the toxic components purified from black widow spider (Latrodectus tredecimguttatus) eggs. In the present study, a novel neurotoxic protein was purified from the eggs by gel filtration combined with ion-exchange chromatography. Its molecular weight was 23.752 kDa determined by electrospray mass spectrometry. The protein could block the neuromuscular transmission in mouse-isolated phrenic nerve-hemidiaphragm preparations completely in a reversible manner and activate tetrodotoxin-sensitive sodium current in rat dorsal root ganglion cells. The N-terminal sequence of the protein was identified by the Edman degradation to be N-S-I-A-D-D-R-Y-R-W-P-G-Y-P-G-A-G-L-I-P-Y-I-I-D-S-. When the sequence was used to search against protein database with a sequence query in Mascot engine there was no matched sequence or protein whereas the Basic Local Alignment Search Tool (BLAST) analysis indicated that no significant similarity was found. These results demonstrated that the protein (named Latroeggtoxin-I) is a novel neurotoxic protein purified from the eggs of black widow spiders.

Isolation and identification of a sodium channel-inhibiting protein from eggs of black widow spiders

“…SDS-PAGE showed that the peak 1, the penetrating peak, was primarily composed of a protein of slightly greater than 26 kDa whereas peak 3 consisted of a protein with a molecular weight of between 17 kDa and 26 kDa (Fig. 3), which was confirmed to be Latroeggtoxin-I that had been reported previously [11]. Therefore, further purification was focused on the separation of proteins in the penetrating peak.…”

“…Although the proteomic analysis of tryptic digest of Latroeggtoxin-II indicated that this protein has a certain homology with other several proteins particularly a predicted protein (gi|115929936) in S. purpuratus (PREDICTED: similar to Mgc2744-prov protein in S. purpuratus), there was not any protein that matched the Latroeggtoxin-II completely. In addition, comparison of the N-terminal sequence of Latroeggtoxin-II with that of Latroeggtoxin-I purified and characterized in our previous work [11] showed that there were few similarities, demonstrating that they were two different proteins. Taken together, it can be concluded that Latroeggtoxin-II is a novel active protein purified from the eggs of black widow spiders.…”

“…In the present study, by using a combination of gel filtration, ion exchange chromatography and RP-HPLC, which separate the proteins based on the differences in their size, charge property and hydrophobicity, respectively, we have purified Latroeggtoxin-II, a biologically active protein with a molecular weight of 28.69 kDa, from the eggs of black widow spiders. Different from the situation for Latroeggtoxin-I that had been purified from the eggs by gel filtration combined with anion exchange chromatography [11], Latroeggtoxin-II was finally purified by RP-HPLC after the partial purification with gel filtration and anion exchange chromatography. Generally speaking, the resolution of RP-HPLC is higher than those of molecular sieve and ion exchange chromatography.…”

“…The soluble components in the black widow spider eggs about 1-2 weeks before hatching of newborns were extracted according to the methods previously described [4,11]. Briefly, the eggs were homogenized in a neutral PBS buffer of weak ionic strength or ddH 2 O with a mortar and pestle.…”

“…Moreover, a neurotoxic protein (named Latroeggtoxin-I) with a molecular weight of 23.752 kDa was purified from the eggs by gel filtration combined with ion exchange chromatography. Utilizing the determined N-terminal sequence of the protein, protein database searching with the sequence query method of Mascot engine indicated that there was no matched sequence from any known proteins, and BLAST analysis showed that no significant similarity was found between the purified protein and the existing proteins, demonstrating that the protein was a novel protein purified from the eggs of black widow spiders [11]. In the present work, another active protein was isolated from the black widow spider eggs using a combinative purification strategy.…”