Ministry of Education and Traning Vietnam Academy

MINISTRY OF EDUCATION AND VIETNAM ACADEMY OF SCIENCE TRANING AND TECHNOLOGY

GRADUTAE UNIVERSITY OF SCIENCE AND TECHNOLOGY

VO DO MINH HOANG

ISOLATION AND CHARACTERIZATION OF TOXINS FROM THE HETEROMETRUS LAOTICUS SCORPION VENOM

Major: Organic Chemistry Name code: 62.44.27.01

SUMMARY OF DOCTORAL THESIS

HO CHI MINH CITY– 2017

MINISTRY OF EDUCATION AND VIETNAM ACADEMY OF SCIENCE TRANING AND TECHNOLOGY

GRADUTAE UNIVERSITY OF SCIENCE AND TECHNOLOGY

VO DO MINH HOANG

ISOLATION AND CHARACTERIZATION OF TOXINS FROM THE HETEROMETRUS LAOTICUS SCORPION VENOM

Major: Organic Chemistry Name code: 62.44.27.01

SUMMARY OF DOCTORAL THESIS

SUPERVISOR

1. Dr.Sc. Hoàng Ngọc Anh 2. Prof. Dr.Sc Utkin Yuri Nikolaevich

HO CHI MINH CITY – 2017

1. Introduction Along with the social and economic development of the world, advanced researches and discoveries of modern therapies and treatments on various ailments are increasing issues of society and science. Alongside with synthesized medications rooted from plants, animal venoms are today’s valuable crude materials supplementing for new synthesis of medications. For long, venomous animals such as snakes, bees, scorpions and many other species have been used as the main ingredients for healing. In folks, scorpions are often used in whole or tail (Metasoma) to treat seizures in children, tetanus, paralysis, mumps, stroke ... under powder form or pills. With scientific advancement, there were many of the world's first studies on scorpions more than 100 years ago and have successfully identified neuroactive components in scorpion venom. However, the study of scorpion venom in Vietnam is only in its infancy. There are rather not many researches have been published on the use of scorpions as medicines on humans, but folk remedies which are lack of scientific proof. Scorpion venom contains many polypeptides that are capable of affecting the receptors and ion channels of the cell membrane, these polypeptides have been investigated by scientists to create new therapeutic drugs treating Parkinson, high blood pressure, cancer, Alzheimer ... In order to contribute a new source of raw materials and new scientific research into the pharmaceutical industry, aiming to make medicines and provide the basis for researches. Bearing that goal, our research team conducted a study on venom of Heterometrus laoticus. Through the thesis"Isolation and characterization of toxins from the Heterometrus laoticus scropion venom", a new research direction of the scorpion Heterometrus laoticus, we aimed to provide the basis understandings for further studies regarding pharmaceutical applications of these toxins.

2. Research methods Black scorpions Heterometrus laoticus were purchased in An Giang province in two rounds, the first one in May and the second one in August with the total of 850 counts. The scorpions were then brought in the laboratory to collect venom for further research’s purposes. To collect the scorpion tails containing the venom pocket, the scorpion is directly stimulated into the tail (fifth or sixth metasoma) using a direct electric current with an excitation frequency of 2.63kHz and is maintaining throughout the scorpion's venom ejaculation (5 - 7 seconds). Scorpions are kept fixed on a sheet metal, which is connected to negative electrode. The positive electrode is used to stimulate directly into the tail to completely remove venom in the venom bulb.Crude venom is collected on thin glass slides in the form of colorless or milky white liquids. The collected venom is later dried in anhydrous contained CaCl2for 4 hours. Dewatered venom is collected in a small glass vial and stored at -20 ° C until use. Scorpions after buying were to be kept stabilized one day and then proceeded to collect venom, period between two consecutive harvests is two weeks. Crude venoms are purified into smaller fractions using the gel filtration chromatography on Sephadex G-50 gel and high performance liquid chromatography(HPLC) techniques. The collected Fractions arelater evaluated for toxicity on rats, cricketsand also studied fortheir molecular structures using nuclear magnetic resonance spectra (NMR) and mass spectra(MS).

3. Results and Discussions 3.1. Raise scorpions in the laboratory and harvest venoms H. laoticus collected in An Giang (in two batches, 850 counts) were stocked in bins for laboratory testing, each with an average of 65-75 counts, raised and observed for 6 months, results as shown in Figure 3.1

Round 2 1 0,93 0.9 0.8 0,7594 0,6628 0.7 0,6106 0,5737 0.6 0,5652 0,4864 0.5 0,3942 0,3758 0.4 0,3529 0.3 0,2361 0,21680,1996 0.2 0,489 0.1

Harvested amount of crude venom(mg) ofcrude amount Harvested 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Frequency

Figure 3.1 Amounts of scorpion venom collected over time. The amount of venom obtained from the scorpion raised in the laboratory decreases through time. In the laboratory, scorpions were raised in a rather large density compared to nature, with little space for activity, adequate food supply, no competition for food resources as well as resistance against enemies in natural habitat, therefore the amount of venoms are decrease overtime due to lack of threats.

3.2. Observation, evaluation and analysis of crude venom extracted from H. laoticus scorpion 3.2.1. Analysis of total protein content in crude venom The amount of protein presents in crude venoms were determined by the biuret- colorimetric method with the standard curve constructed using different concentrations of albumin solution. From the result of constructing the baseline and regression equation of the calibration curve, the concentration of protein in H. laoticus collected was 1.293 mg / mL and hence the content of water soluble protein of solution crude venoms obtained from scorpions are 64.65%. 3.2.2. Analysis of crude venom using gel chromatography technique on Sephadex G50 gel and molecular weightdetermination of resulted Fractions. Perform gelchromatography on Sephadex G-50 gel with 4.500 mg of crude venom at the Biomaterials Division of the Institute of Tropical Biology, which collects five fractions of venom with mass ratio of each fraction shown in table 3.1. Table 3.1 Mass of Fractions in scorpion venom Amount Percentage Fraction (mg) (%) 1 102,7 5,42 2 174,2 9,20 3 253,2 13,37 4 688,9 36,39 5 674,2 35,61 Total 1.893,2 Amount By gel chromatography, crude venoms from H.laoticus scorpions were separated into 5 different venom fractions corresponding to different molecular weights. Molecular weights of these 5 fractions were preliminarily determined by SDS-PAGE technique and compared with standard mass samples (Figure 3.2).

kDa M 1 Sg 1 Sg 2 Sg 3 Sg 4 Sg 5

170 130 100 70 55 40

Figure 3.2 Electrolysis result of 5 fractions compared to standard sample

3.2.3. Observation for toxicity of the fractions of H. laoticus scorpion’s venom on mice Table 3.2. Result of toxicity observation from scorpion’s venom on mice Fraction Reactions of mice after injection Remark The mouse scratches the muzzle, Non toxic 1 lying still,thereafter working normally. Mice reduce activity, ruffled hair, Light toxic 2 shortless of breath but recovery after 30 minutes. Mice lying still,secreting saliva, 3 paralysis, shortness of breath, but Toxic recovery after 2 hours. Mice lying down, eyes closed, 4 convulsions, paralysis, secreting Lethal saliva, mice died after 3 hours. Mice shaking, lying quietly, Non toxic 5 functioning normally after 60 minutes. Purified Mice shaking, lying quietly, Non toxic water functioning normally after

Section 4 was isolated from H.laoticus scorpion rattans by gel filtration chromatography, toxicity assay on mice clearly indicated that lethal fraction 4 was lethal. Thus, fraction 4 was identified as acute toxicity to mice and achieved the acute toxicity level of fragment 4 extracted from the scorpion Heterometrus laoticus as LD50 = 24.7 ± 1.09 mg / kg. After confirmation of strong toxicity, fraction 4 was further separated by HPLC technique to obtain secondary Fractions (Figure 3.3).

Figure 3.3 Chromatogram of fraction 4 using HPLC The secondary fractions of fraction 4 continue to be evaluated for toxicity in mice and for the summary results in table 3.3. The secondary toxic phases (fractions 4.6, 4.7, 4.11 and 4.15) were further refined and determined the molecular mass of the peptide components. Table 3.3 Result of toxicity assay of secondary Fractions Fraction Toxicity Fraction Toxicity 4.1 Non toxic 4.14 Non toxic 4.2 Non toxic 4.15 Toxic 4.3 Toxic 4.16 Non toxic 4.4 Non toxic 4.17 Non toxic 4.5 Toxic 4.18 Non toxic 4.6 Lethal 4.19 Non toxic 4.7 Toxic 4.20 Non toxic 4.8 Non toxic 4.21 Non toxic 4.9 Non toxic 4.22 Non toxic 4.10 Non toxic 4.23 Non toxic 4.11 Toxic 4.24 Non toxic 4.12 Non toxic 4.25 Non toxic 4.13 Non toxic Saline Non toxic

3.3. Purification and determining the molecular weight of secondary Fractions Fraction 4.6 is separated into three fractions, 4.6.1, 4.6.2, 4.6.3. fraction 4.6.1 obtained relatively small sample sizes, whereas in the 4.6.2 and 4.6.3 divisions the bulk

7 of the collected samples should be further refined to determine the molecular weight. The results show that the secondary fraction 4.6.2 has a molecular weight of 3,669,155 Da, while the secondary fraction 4.6.3 has a molecular weight of 2,915,040 Da. Fraction 4.7 wasseparated into 4 fractions. Of those, peptide 4.7.2 was purified and examined for molecular weights, resulting in peptide 4.7.1 having a molecular mass of 3,700.2 Da and 4.7.2 peptide having a molecular mass of 3.846.872 D. Fraction 4.11 was separated, resulting in purified 4.11.1 peptide and later was determined molecular weight of 3,695,907 Da. Fraction 4.15 was separated into eight peptide fractions. Among them, we purified and determined the molecular weights of the three peptides of the secondary fraction 4.15.8 to be 2,461,105 Da; 3,285,285 Da and 5,021,4 Da. 3.4. Survey the first-order structure of the polypeptide fractions 4.6 and the effect on the K + channel 3.4.1. Survey the first-order structure of fraction 4.6 Table 3.4 Molecular masses of H.laoticus determined by MALDI mass spectrometry Fraction Molecular weight (Da) 4.6.2 (Hetlaxin) 3.670,8 4.6.3 2.915,4 4.7.1 3.700,2 4.7.2 3.846,2 4.11 3.700,3 2.461,1 4.15 3.285,3 5.021,4

The molecular masses of isolated polypeptides were determined by MALDI mass spectrometry (Table 3.4). Practically all the masses determined are in the range from 3 to 4 kDa. This allowed us to suggest that they belong to the potassium channel inhibitor family of the short scorpion toxin superfamily. To determine the number of disulfide bonds, the polypeptides from fractions 4.6.2 (hetlaxin) and 4.6.3 were reduced, pyridylethylated and analyzed by mass- spectrometry. For each fraction only one signal corresponding to the molecular mass of

4498 Da (4.6.2) and 3538 (4.6.3) Da was observed in the MALDI mass spectrum of the derivative obtained. The increase in mass corresponded to the incorporation of 8 pyridylethyl groups in hetlaxin molecule and 6 groups in polypeptide from fraction 4.6.3. This means that hetlaxin contains 4 disulphide bridges and polypeptide from fraction 4.6.3 – 3 disulphides. N-terminal sequence of hetlaxin was determined by Edman degradation - ISXTGSXQ. Hetlaxin sequence was also analyzed by MALDI mass spectrometry (Fig. 3.4).

Figure 3.4: De novo analysis of the MS/MS spectrum of the hetlaxin. Both MS/MS fragmentation of the complete polypeptide chain and peptide mass fingerprinting with trypsin were performed. Basing on the data of Edman degradation and mass spectrometry summarized in Fig. 3.5 we have deduced the amino acid sequence of hetlaxin. Mass spectrometry data on MS/MS fragmentation of tryptic peptides indicated that C-terminal cysteine residue is amidated. The lysine residue in position 30 was found by MS/MS sequencing of tryptic fragments. The determined amino acid sequence is homologous to those of scorpion alpha-toxins interacting with potassium channels (Fig. 3.6). The highest similarity was found with the toxin (AFB73769, Fig 3.6)which sequence was deduced from cDNA prepared from the venom gland of H. laoticus from Thailand.

KCYDPCKKKTGC

KKTGCPNAKCMNK Mass Spectra IS QCYD KKTGCPNAKCMNKSC CYGC ISCTGSKQCYDPCKKKTGCPNAKCMN

ISXTGSXQ Edman degradation

ISCTGSKQCYDPCKKKTGCPNAKCMNKSCKCYGC Hetlaxin sequence Figure 3.5: Sequences of hetlaxin fragments determined by MALDI mass spectrometry and Edman degradation However no data about biological activity of this toxin are available. Other homologous toxins (HsTX1-P59867 andToxin Vm24-P0DJ31, FIG. 3.20) manifest high affinity to Kv1.1 and Kv1.3 potassium channels (Lebrun et al., 1997; Gurrola et al., 2012). Basing on these data, we have checked the ability of hetlaxin to interact with potassium channels.

1 10 20 30 ISCTGSKQCY DPCKKKTGCP NAKCMNKSCK CYGC* HETLAXIN ISCTGSKQCY DPCKRKTGCP NAKCMNKSCK CYGCG AFB73769 IRCSGSRDCY SPCMKQTGCP NAKCINKSCK CYGC* P84094 (KAX6D_HETSP) ASCRTPKDCA DPCRKETGCP YGKCMNRKCK CNRC* P59867 (KAX63_HETSP) AAAISCVGSPECP PKCRAQ-GCK NGKCMNRKCK CYYC* P0DJ31 (KA211_VAEMS) AAAISCVGSKECL PKCKAQ-GCK SGKCMNKKCK CY-C P0DJ32 (KA212_VAEMS)

Figure 3.6Comparison of hetlaxin amino acicd sequence whith those of know scorpion. Cysteine residues are underlined; the identicalresidues are shaded in gray. *-C-terminal amide. 3.4.2. Hetlaxin interaction whit potassium channels The interaction of Hetlaxin with K+ channels has been investigated in competitive-binding experiments. Chimeric proteins KcsA-Kv1.3 and KcsA-Kv1.1, which exhibit different species in the E. coli membrane, have been used as K + and R- AgTx2 channels to be used as a ligand. The linkage of R-AgTx2 was recorded using a laser-guided scanning microscope. It can be seen that Hetlaxin competes effectively with R-AgTx2 in association with both chimeric channels (Figure 3.7). The IC50 values were determined to be 0.48 ± 0.01 μM and 6.7 ± 0.4 μM respectively for Kv1.3 and Kv1.1, while the Ki values were calculated using the Cheng- Prusoff calculatoris 59 ± 6 nM and 0.8 ± 0.3 mM. These figures show that Hetlaxin interacts efficiently with the Kv1.3 potassium channel, while the interaction with the Kv1.1 channel is about ten times lower. [53,87]

1200 A 4000 B

900 3000

600 2000

relative units relative

relative units relative 300 1000

Signal intensity Signal

0 0 -9 -8 -7 -6 -5 -4 -3 -2 -9 -8 -7 -6 -5 -4 -3 -2 lg [C] lg [C] Figure 3.7.Competition of hetlaxin with R-AgTX2 for the binding to KcsA-Kv1.1 (A) and KcsA- Kv1.3 (B)potassium chanels Up until now, only two toxins have been isolated from H. laoticus in Thailand and identified as Hetroscorpin-1 (HS-1) and HelaTx1. HS-1 has a molecular weight of 8,243 Da, contains 6 cysteine and in the same family with scorpine, toxic against insects, antimicrobial but there are none known information regarding HS-1 interaction with potassium channel. HelaTx1 has a molecular weight of 2,763 Da and contains four cysteine, which are capable of interacting with different potassium channels and interacting most strongly with the Kv1.1 channel (EC50 = 9.9 ± 1.6 μM). HelaTx1 is also linked to Kv1.3 channel, but affiliate affinity is much lower than Kv1.1 channel. At concentration of 30 μM, toxin HelaTx1 interacts only 20% with potassium channels. Based on the results of testing the potential for potassium channel linkage from Hetlaxin isolates from H.laoticus species distributed in Vietnam, results show the potential for binding and inhibition of potassium channel activity of Kv1.3 and Kv1. For the Kv1.3 channel, the new protein was found to be inhibiting at relatively low concentrations, 0.48 ± 0.01 μM and 6.7 ± 0.4 μM respectively for Kv1.3 and Kv1.1. These studies have shown that Hetlaxin has the potential to inhibit Kv1.3 channel activity more efficiently than HelaTx1 has been reported and is the most potent Kv1.3 channel blocker to be isolated from H.laoticus to date (0.48 μM). [90]. In the past, most reports have shown that the toxin of the H.laoticus scorpion affects all Kv1.1 neural channels and but none clear indication proving linkage between these proteins with Kv1.3. Hetlaxin was the first toxin, at the time written this article, extracted from H.laoticus scorpion which had a strong effect on Kv1.3 channel. 3.5. To investigate the toxicity of the fractions of H.laoticus scorpion on crickets

3.5.1. The results of the toxicity assay of the fractions of the collected venoms on crickets The fractions extracted from H.laoticus scorpion’s venoms were tested for insecticidal insecticides by injection, each containing 6 crickets and control against distilled water with the results shown in Table 3.5.

Table 3.5 Toxicity assay of separated fractions on crickets Fraction Cricket’s reactions Remark cricket laid still, then resumed to 1 Non toxic normal cricket laid still, then resumed to 2 Non toxic normal cricket laid still, then resumed to 3 Non toxic normal cricket laid still, then resumed to 4 Non toxic normal The cricket was active, jumping continuously in the sink, then 5 lying still, 26 minutes after the Lethal crickets stopped moving, the limbs fell out, died. Nước cất Cricket shivered, laid still then Non toxic resumed normal

For fraction 5, after being injected, in the first five minutes, the cricket is active and runs continuously. By the 10th minute, the crickets began to rest and by the 26th minute, the crickets were dying, the limbs falling out. fraction 5 was identified as a toxic fraction for insects. 3.5.2. Determine the toxicity level on insect through injection at abdomen. After the injections at abdomen site on crickets of the test plots and the control plots, we identified the followings:

 Determine the maximum dose that produced no mortality LD0 = 20,3 µg/g

 Determine the minimum dose that produced mortality of 100% LD100 = 393,75 µg/g

 Determine the median lethal doseLD50usingBehrens-Karber method in association with data from Table 3.7. We have: ∑ab =1.600,52 n = 60/8 = 7,50 ∑ 푎푏 1600,52 퐿퐷 = 퐿퐷 − = 393,75 − = 180,35 50 100 푛 7,50

Therefore, LD50 = 180,35 µg/g

Table 3.6 Mortality rates in crickets vary between LD0 and LD100 after 24 hours. 20,3 73,6 127,0 180,3 233,7 287,0 340,4 393,7 Dosage 0 5 0 5 0 5 0 5 crikets/lo 6 8 8 8 8 8 8 6 t Dead 0 3 3 4 4 6 7 6 counts Live 6 5 5 4 4 2 1 0 counts Percenta 37,5 100,0 0,00 37,50 50,00 50,00 75,00 87,50 ge (%) 0 0

Table 3.7Behrens-Karber worksheet a 1,50 3,00 3,50 4,00 5,00 6,50 6,50 b 53,35 53,35 53,35 53,35 53,35 53,35 53,35 a.b 80.03 160,05 186,73 213,40 266,75 346,78 346,78 a: Average crickets die at two consecutive doses b: Constant between two consecutive doses .

100 90 80 70 60 50 40 30 Dosage(µg/g) 20 10 0 0 100 200 300 400 % Death

Figure 3.8 Percentage of crickets killed by dosage Based on the percentage of mice that died accordingly with used doses, we determined LD16 and LD84 as follows: LD16 = 43.85 μg / g and LD84 = 330.65 μg / g.

From this, the standard deviation was calculated s = 143.40, with dosage leap d = 53.40. Behrens-Karber constant k = 0.564.

The standard error of the LD50 dose is calculated by the formula:

푘 × 푠 × 푑 퐸 = √ 푠 푛

th Es is 23.99. So, the acute toxicity of the 5 fraction separated from H. laoticus scorpion’s crude venom is LD50 = 180.35 ± 23.99 μg / g. Thus, by the Behrens-Karber method, we determined the insecticidal toxicity level of LD50 = 180.35 ± 23.99 μg / g. 3.5.3. Separation of 5thfraction by high performance liquid chromatography(HPLC) 5thfractionafter being determined to be lethal on insects, continues to be separated to obtain toxins to determine molecular structure and to determine interactivity with Potassium channel. Fraction 5 was isolated using high performance liquid chromatography, column Eclipse XDB C18, chromatogram obtained as shown in figure 3.9. SeparatedFractions were numbered from 5.1 to 5.25 and then used to assess toxicity on crickets. 3.5.4. Toxicity observation on crickets of fragmented fractions Based on the results of the toxicity study on secondary insect pests as shown in Table 3.13, 13 fragments were determined to be toxic to test specimens 5.3; 5.7; 5.8; 5.9; 5.10; 5.12; 5.13; 5.15; 5.17; 5.18; 5.21; 5.24. The mild toxicity was 50% (3/6) of death in each plot after 4 hours, with strong poisonous fractions having a mortality rate of 5% for each plot after 4 hours. The 4 fractions identified as having strong toxicity (lethal toxin, 5/6) were the following: 5.7, 5.12, 5.15, 5.21.

Figure 3.9 Spectrum of fraction 5

Table 3.8 Results of toxicity assay of the Fractions after separation by HPLC on insects.

Fraction Result Fraction Result Fraction Result 5.1 Non 5.9 Light 5.17 Light toxic toxic toxic (0/6) (3/6) (3/6) 5.2 Non 5.10 Light 5.18 Light toxic toxic toxic (0/6) (3/6) (3/6) 5.3 Light 5.11 Non 5.19 Non toxic toxic toxic (3/6) (0/6) (0/6) 5.4 Non 5.12 Lethal 5.20 Non toxic (5/6) toxic (0/6) (0/6) 5.5 Non 5.13 Light 5.21 Lethal toxic toxic (5/6) (0/6) (3/6) 5.6 Non 5.14 Non 5.22 Light toxic toxic toxic (0/6) (0/6) (3/6) 5.7 Lethal 5.15 Lethal 5.23 Non (5/6) (5/6) toxic (0/6) 5.8 Light 5.16 Non 5.24 Light toxic toxic toxic (3/6) (0/6) (3/6) From the preliminary results, insect-repellent fractions were purified to obtain a clean toxin sample to determine molecular weights of these toxins. 3.6. Isolate and determine the molecular mass of secondary toxic fractions 3.6.1. Isolate polypeptides from secondary fractions

Secondary Fractions were determined to be toxic by high performance liquid chromatography in order to obtain clean polypeptide chains that would be able to analyze the structural and potassium channel interaction. The summary results of the isolation process are shown in Table 3.8. 3.6.2. Determine polypeptide chain structure of fraction number 5.21 Fraction 5.21 was chosen to continue the chromatography to isolate the two polypeptide sequences contained in the component and from there on to characterize these two polypeptide chains. The molecular weights of the two polypeptide chains were determined by MALDI-TOF mass spectrometry, resulting in 317,974 Da (fracion 5.21.1) and 832,600 Da (fraction 5.21.2). These are short toxins, unlike the toxins that have been identified as being toxic to insects of the scorpion H. laoticus distributed in Thailand which are long toxins with a molecular weight of 8,293.07 Da. [90] Structure of fraction 5.21.1 were defined using nuclear magnetic resonance(NMR) spectra. The temperature and pH of the sample varies between 10 - 45 ° C and 5 - 7.5. First determine the peptide spin of the DQF-COSY spectrum, followed by the NOESY spectrum to determine the amino acid sequence in the peptide chain. Obtained NMR spectrum is shown in figure 3.10. [12,29,30,46,52,75].

Figure 3.10 NMR spectrum of toxin 5.21.1 In figure 3.10, it is shown that the Hs of the terminal amine have been ionized. Based on the results of the NMR spectra and the MS spectra, the predicted structure of toxin 5.21.1 is the Leu-Trp dipeptide (Figure 3.11). Thus, toxin 5.21.1, which was removed and purified in Fraction number 5 of H.laoticus scorpion’s venom with toxicity against insect, is Leu-Trp, a short-toxin with a molecular weight of 317,974 Da.

A B

C Figure 3.11 Structure of toxin 5.21.1 ( Leu-Trp) A. Flat structure of Leu-Trp B. Position if H groups on NMR spectrum C .3D structure of Leu- Trp The number dipeptides with biological activity discovered from nature at of the time of this article are very limited. Some dipeptides with biological activity, thatwere earlier detected including carnosine (β-alanyl-L-histidine) and anserine (β-alanyl-N- Methyl-L-histidine), are found in the muscle and brain tissue of animals. Alongside with homocarnosine and ophidine, these dipeptides have been or are being studied for their antioxidant potentials and applications in pharmaceutical industry. There is a publication on the activity of carnosine and anserine in the treatment of certain human diseases such as diabetes, atherosclerosis, Alzheimer by inhibiting the formation of glycation products (glycosylic products of protein or lipid without the involvement of regulatory enzymes). Recently, from the muscle tissue of Chum salmon (Oncorhynchus keta), various dipeptides as Phe-Leu, Ala-Trp, Val-Trp, Met-Trp, Ile-Trp, Leu-Trp have been found and experimented which yielded result of inhibition on the angiotensin- converting enzyme (ACE), which has the potential to be used as a medication treating hypertension. [42,50,68,79,95,100,105].

4. Summary Through research on crude venom extracted from scorpion Heterometrus laoticus distributed in An Giang, the following results have been recorded and summarized as follows: Due to the impact of the surrounding environment, competition for food resources, against the enemy, crude venoms extracted from the scorpion Heterometrus laoticus in natural habitat are relatively abundant more. Under laboratory conditions, the scorpion is very inactive,adequate amount of foods and water are sufficiently supplied, therefor the scorpion’s weight increases significantly, but the amount of venom obtained is significantly reduced over time. The test conditions in the laboratory are as follows:  The average number of scorpions is less than 50 heads per cage  Feed once a week, on average 2.00 g crickets / scorpions.  The amount of venom collected from 850 scorpions in 6 incubated months was 6.9653 g of venom, averaging 8.19 mg / head. 1. By gel filtration chromatography on Sephadex G-50 gel with solvent ammonium acetate buffer (pH = 4.7), the crude venoms of H.laoticus scorpion were separated into 5 fractions, of which there are 3fractions that have toxicity were fraction 2th, 3th, and 4th. Out of these three toxic Fractions, only the 4thFraction was the most lethalagainst mice, while the second and third fractions were slightly toxic but the mice later recovered. When examining the toxicity of refracted Fractions against insects, 5thfractionexhibited lethal toxicity on crickets. Comparison of electrophoresis shows that 4th fractioncontains proteins with molecular weights ranging from 3 to 10 kDa and within 65 kDa.5thfraction contains proteins with molecular weights in the range of 3 kDa and 9 kDa. 2. Study lethal toxicity of 4thFraction on mice.  Acute toxicity of 4thFraction through IV injection on mice was 24.07 ± 1.09 mg / kg, classified as type II (highly toxic), but when compared with acute toxicity of crude venom,4thFraction toxicity is weaker than that of crude venom, which clearly indicated that in the crude venom, in addition to the peptides of low molecular weights that are toxic against animals, peptides withhigh molecular weight such as the enzyme phospholipase also have toxic effects on animals. They interact with each other making the crude venom has more severe toxicity than that of 4thfraction

 By HPLC, the 4thfraction was split into 25 different secondary fractions. There is a fraction with lethal toxicity to animals that is 4.6 and 5 other fractions that are toxic but not lethal which are secondary fractions number 4.3, 4.5, 4.7, 4.11, 4.15.  Section 4.6 was later separated and identified the molecular weights of the polypeptides contained in Section 4.6: Fraction 4.6.2 weights 3.670.8Da; fraction 4.6.3 weights 2.915.4 Da; fraction 4.7.1 weights 3,700.2 Da; 4.7.2 weights 3.846,2 Da; 4,11.1 weights 3,700,3Da; fraction 4.15 weights 2,461,1Da; 3,285,3 Da; 5,021,4 Da.  In the separated polypeptides, polypeptides separated from 4.6.2 is identified as new toxin and has a strong effect on Kv1.3 channel. This toxin is named Hetlaxin and is classified in the alpha-toxin family. Hetlaxin is the first toxin to be extracted from H.laoticus which has a strong affinity for potassium channel Kv1.3 with the following structure: ISCTGSKQCYDPCKKKTGCPNAKCMNKSCKCYGC 3. Observe toxicity on crickets  Determination of acute toxicity on crickets (through injection at intravenous line) was LD50 = 180.35 ± 23.99 μg / g.  The insect-specific lethal – toxicity Fraction was later separated into24 secondary fractions by HPLC technique, in which the strongestpoisonous fractions (100% dead rate on crickets) are thefractions 5.3; 5.7; 5.8; 5.9; 5.10; 5.12; 5.13; 5.15; 5.17; 5.18; 5.21; 5.24 and mild toxicity (50% mortality)Fractions are 5.7; 5.12; 5.15; 5.21.  Determine molecular weight of the secondary fraction 5.21.1 were 317,974Da and the secondary Fraction 5.21.2 was 832,600Da.  Using Nuclear Magnetic Resonance spectrum, define structure of toxin 5.21.1 as Leu-Trp