Organizing co mmittee

PRIMARY STRUCTURE OF THE INSECTOTOXIN FROM THE 
SCORPION VENOM Mesobuthus eupeus 
Sh.J. Fazliddinov
1,2
, J.F. Ziyavitdinov
1
, N.J. Sagdiev
1
, U.J. Ishimov
1,2
, Sh.Ya. 
Mirzaakhmedov
1
 
 
1) Institute of Bioorganic Chemistry, 100125, Tashkent, Mirzo Ulugbek street 83. 
2) Tashkent chemical technological institute, Tashkent. 
e-mail: [e-mail]
 
 
Recently, special attention has been paid to the identification of insect-selective 
toxins that can be used to create recombinant biopesticides as an alternative to highly 
toxic chemicals for pest control. The aim of our work was to isolate and establish the 
structure of insectotoxin from the venom of the scorpion 
Mesobuthus eupeus
subspecies. 
According to RP-HPLC analysis, the whole venom of scorpion 
M. eupeus
is a 
multicomponent mixture containing at least 65-70 different protein-peptide components. 
Non-toxic mucoproteins (<10% by weight of the total venom) were removed from the 
whole venom solution by centrifugation. The supernatant was separated by size 
exclusion chromatography on a TSK HW-55f gel column and cation exchange 
chromatography on a CM-TSK 650M. As a result of chromatographic separation, 17 
fractions were obtained. Each fraction was preliminarily desalted and then tested for 
biological activity. Fraction ME-4-2 had the highest activity against mealworm larvae. 
In next step, the ME-4-2 fraction was separated by RP-HPLC on an Agilent 
Technologies 1200 instrument (USA) using a Nucleosil 5C18 column (4.6 mm x 250 
mm) in a linear gradient of 0.1% TFA solution and acetonitrile. Detection was 
performed at 226 nm. As a result, 14 individual peptides were obtained, numbered as 
ME-4-2-1, ME-4-2-14. Fraction ME-4-2-7 at a dose of 5 mkg/g caused instantaneous 
paralysis of cockroaches, mealworms and bollworms. Therefore, the insectotoxin from 
the venom of the 
M. eupeus
scorpion was given the designation BucalT. 
The molecular mass of the toxin was determined on an Agilent Technologies 6530B 
Chip-HPLC Q-TOF MS/MS mass spectrometer and was 3830.16 Da. The molecular 
mass after reduction and carboxymethylation made 4232.75 Da. The difference of 
402.59 Da led to the conclusion that BucalT contains eight cysteine residues, which 
confirms the results of the amino acid analysis. As a result of amino acid analysis, the 
following ratio PTC derivatives were found in the insectotoxin BucalT: Gly - 4, Ala - 3, 
Asx - 5, Glx - 1, Ser - 1, Met - 3, Thr - 2, Pro - 2, Cys - 8, Leu - 1, Phe - 2, Arg - 2, Lys 
- 2. 
When determining the primary structure of BucaIT using automatic amino acid 
sequencing using the Edman method, the following sequence was determined: 
MCMPCFTTDANMARKCSDCCGGNGKCFGPQCLCNRA 
The C-terminal residue, Ala, was determined from the difference in the molecular 
mass of the sum of amino acids 1-35 and the total molecular weight of BucaIT 
insectotoxin. 
Research to determine the disulfide bonds in the BucaIT insectotoxin molecule is 
going on.