Organizing co mmittee

 
 

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Poster presentation 
194 
OPTIMISATION OF ISOLATION OF PEPTIDES FROM 
Latrodectus mactans VENOM 
 
Z.Zh. Mirakhmetova, N.R. Mukhamedov, M.B. Kayumov, Sh.Ya. Mirzaakhmedov 
 
Institute of Bioorganic Chemistry, 100125, Tashkent, Mirzo Ulugbek str. 83. 
e mail: [e-mail] 
Latrodectus mactans
, known as a black widow is a venomous species of spider in the 
genus 
Latrodectus
. Its venom contains small molecular mass compounds, peptides, and 
proteins. The venom arsenal can be used as a source of potential biologically active 
compounds which promise therapeutic application. Proteins of black widow venom are 
studied well. However, peptides of the venom are less studied so far. 
Therefore, it is the subject of interest in our studies related to isolation, identification, 
and biological activity study of the peptides from venoms of spiders, scorpions, snakes, 
and reptiles. The aim of the research is the optimization of separation and isolation 
conditions of low molecular weight peptides from black widow venom. The following 
tasks were conducted: 1) studying of solubility of the venom; 2) depositing of proteins 
and peptides mixture; 3) ultrafiltration and/or gel-filtration of the venom in parallel 
manner and its precipitate; 4) comparative analysis of the fractions by HPLC. 
The venom was dissolved in water, acidic (0.1 and 0.2 M ammonium acetate pH 4), 
and basic (0.1 and 0.2 M ammonium bicarbonate pH 8) buffers to check its solubility. 
The solubility was almost the same in the case of all three types of solvents. We 
selected acetate buffer as a solvent for further steps. Because low pH prevents 
hydrolysis of the components of venom by proteolytic enzymes. The deposition of 
proteins and peptides was conducted by adding ethanol to the solution of the venom. 
60% of ethanol against water (v:v) was enough to precipitate. 
Ultrafiltration was carried out using ultrafiltration membranes 10 kDa and 3 kDa 
sizes. >10 kDa, <10->3 kDa, and <3 kDa fractions of the venom and its ethanol 
precipitate were obtained. The fractions were lyophilized and stored at -20 
o
C. 
Sephadex G-25 was used for gel filtration and five fractions were obtained in the 
result. The first fraction was of very high intensity which corresponds to proteins with 
large molecular masses, and the rest fractions of low intensity are considered to be 
peptides and low molecular weight compounds. 
HPLC analysis of the venom, its ethanol precipitate and ethanol part and the 
fractions obtained by parallel ultra- and gel-filtrations was carried out using the C-18 
column in the gradient mode of MeCN against 0.1 % TFA. HPLC profiles of all 
samples were analyzed using Origin software. The results indicated that peaks of high 
intensity with retention times 2.3, 13.7, 14.4, and 16.2 minutes belong to the low 
molecular weight compounds in the ethanol part. Therefore, it was found that working 
with ethanol precipitate which is free of low molecular weight compounds is preferred. 
HPLC profile of the ultra-filtration fractions of the precipitate did not demonstrate a 
significant difference from the precipitate itself or each other. In the case of gel-
filtration fractions of 2, 3 and 4 had an increase of some peaks on their HPLC profile. 
It can be concluded that working with peptides of black widow venom precipitated 
by ethanol lets us get rid of low molecular weight organic compounds and the 
separation of peptides by gel-filtration is more efficient compared to using ultra-
filtration membranes. 

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Poster presentation 
195