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Journal of Venomous Animals and Toxins

Print version ISSN 0104-7930On-line version ISSN 1678-4936

J. Venom. Anim. Toxins vol.6 n.1 Botucatu  2000 

Original paper



Small synthetic peptides inhibit, in mice, the lethalithy of toxins derived from animal, plant and bacteria




1 Ophidia Products, Inc. - 11320 South Post Oak, Suite 203 - Houston, Texas 77035, USA.



ABSTRACT: Lethal Toxin Neutralizing Factor (LTNF), isolated from opossum with having molecular weight 63 kDa, is a potent antidote for animal, plant, and bacterial toxins. This communication deals with the identification of a small fragment of LTNF eliciting the anti-lethal activity of animal, plant, and bacterial toxins when tested in mice.
Purified LTNF was treated with trypsin to cause fragmentation at the arginine and lysine sites. The fragments were separated by high-performance liquid chromatography (HPLC) and were tested against anti-LTNF for binding affinity by enzyme-linked immunosorbent test (ELISA). The fragment showing the most binding to anti-LTNF was sequenced. Synthetic peptides consisting of 15 and 10 amino acids from the N-terminal were constructed and designated as LT-15, with amino acid sequence Leu-Lys-Ala-Met-Asp-Pro-Thr-Pro-Pro-Leu-Trp-Ile-Lys-Thr-Glu, and LT-10, with sequence Leu-Lys-Ala-Met-Asp-Pro-Thr-Pro-Pro-Leu.
Death due to intramuscular (IM) injection of predetermined lethal doses of toxins derived from animal, plant, and bacteria was prevented treating the mice with synthetic peptides LT-15 and LT-10. The lethality was inhibited when the treatment was given before or after the toxin injection. Synthetic LTNF can be made in abundance and should become a universal therapy against intoxication caused by animal, plant, and bacteria.
KEY WORDS: Lethal Toxin Neutralizing Factor (LTNF), opossum, Crotalus atrox, Naja kaouthia, Daboia russelli, Oxyuranus s. scutellatus, Androcetonus australis, Apis mellifera, ricin, Ricinus communis, botulinum, Clostridium botulinum.




The commonly held view that small synthetic peptides cannot mimic effects of large polypeptide ligands is now considerably out of date (16). Bioactive peptides are small peptides that elicit a biological activity. To date, over 500 of these peptides have been isolated, averaging 20 amino acids in size. They have been found in systems ranging from bacteria to human and have been shown to possess a wide range of biological activities. Recent research indicates that many of these peptides function by binding to and inhibiting selected protein targets with incredible specificity.

Several investigators have made synthetic nerve growth factor (NGF) peptide derivatives, which prevent neuronal death and show neurite on PC12 cells. Longo et al. (9) made cyclized peptides corresponding to the beta-loop region of NGF from mouse submaxillary glands and found the highest activity corresponding to a loop region 29-35, which is capable of interacting with p75 receptor. To date, according to these authors, no agents known to promote neurotrophic effects by acting via NGF receptor have been described. Lesauteur et al. (4) designed and synthesized small monomeric cyclic analogs that mimic the beta-turn regions of NGF.

Wrighton et al. (17) reported small peptides as potent mimetics of protein hormone erythropoitein. Likewise, it was reported that a 12-amino acid peptide elicited migratory, cytoprotective, growth stimulatory, growth inhibitory, and angiogenic response, paralleling these of the native epidermal growth factor (EGF) (11).

The synthetic peptide corresponding to KKYRYYLKPLCKK, the region comprising amino acid residues 115-129 of myotoxin II from Bothrops asper venom, was identified. The antibodies against the synthetic peptide 115-129 of myotoxin inhibited myotoxic activity in mice (1) by determining the plasma creatine kinase activity.



VENOMS AND TOXINS. Crotalus atrox (western diamondback rattlesnake), Naja kaouthia (Thailand cobra), Daboia russelli (Asian viper), Oxyuranus s. scutellatus (Australian taipan), Androcetonus australis (scorpion), Apis mellifera (honeybee), cobra toxin from Naja kaouthia, plant toxin ricin Ricinus communis from castor seeds, and botulinum toxin from Clostridium botulinum were purchased from Sigma Co.

TRYPSIN DIGESTION OF NATURAL LTNF. Purified homogenous preparation of LTNF from opossum serum was treated with trypsin dissolved in 0.1 M ammonium bicarbonate buffer, pH 8.0. The protein and trypsin were mixed in 40:1 ratio and 20 mg of LTNF was mixed with 0.5 mg trypsin. The mixture was incubated at 37°C for 16 to 18 h for trypsin to cause fragmentation at arginine and lysine sites. After the incubation period, the reaction was stopped by cooling the mixture to 4°C.

SEPARATION OF FRAGMENTS FROM TRYPSIN DIGEST. The trypsin digested fragments were separated on HPLC. The separation was performed in two runs by loading half the mixture each time. Trypsin-digested LTNF resolved into eight different fractions. The fractions were collected individually and dialyzed against water using 500 molecular weight cutoff tubing (Spectrum USA). The protein concentration of each fraction was measured by using a Bio-Rad USA protein kit, and the concentration for each fraction was adjusted to 100 µg/ml with 0.05 M phosphate buffered saline (PBS).

Balb/C mice were used for the production of polyclonal antibodies against natural LTNF from opossum serum. The US Public Health Service regulations on Humane Care and Use of Animals were abided by. Mice were injected intramuscularly (IM). The first injection consisted of 0.2 ml/mouse containing 10 µg of LTNF mixed with an equal volume of Freund's complete adjuvant. The subsequent three injections were given two weeks apart, consisting of LTNF at the same concentration mixed with Freund's incomplete adjuvant. At the end of the immunization period, the mice were bled through the ophthalmic vein and serum was separated.

The fractions were tested for binding affinity to anti-LTNF by ELISA. In short, 96-well microtiter plate was coated with 10 µg/ml concentration of each fraction. Threefold diluted mouse anti-LTNF was reacted with the fractions in usual ELISA test (5,6). Mouse anti-IgG made in goat horseradish peroxidase was reacted, after which OPD was added for color reaction (the reagents for ELISA were from Sigma Co.). The fragment showing the most binding or highest ELISA titer for the anti-LTNF was sequenced.

Sequencing and synthesis was contracted out to the Protein Core Laboratory of Baylor College of Medicine, Houston, Texas. The fraction showing the highest binding affinity or titer was sequenced and found to consist of 19 amino acids. Initially, a peptide consisting of 15 amino acids from the N-terminal was constructed, and designated as LT-15, with sequence Leu-Lys-Ala-Met-Asp-Pro-Thr-Pro-Pro-Leu-Trp-Ile-Lys-Thr-Glu. On testing it turned out to be active, therefore a peptide consisting of 10 amino acids, excluding the last 5 of the 15 amino acids, was constructed and designated as LT-10, with Leu-Lys-Ala-Met-Asp-Pro-Thr-Pro-Pro-Leu.

ICR mice were injected with venom or toxin at various concentrations to obtain lethal dose (7). Six mice for each venom or toxin were injected IM, giving a lethal dose in 0.1 ml volume. The injected mice were divided into two groups. The control mice received 0.5 ml PBS at the site of the injection, and the mice in the other group, 500 µg of LT-15 in 0.5 ml. The injected mice were observed for a week. All mice in the control group must be dead for the test to be valid. The results are shown in Table 1.


TABLE 1. Inhibition by synthetic LTNF (LT-15) of venom and toxin lethality.

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The results in Table 1 clearly show that the synthetic LT-15, consisting of 15 amino acids of natural LTNF from opossum serum, was capable of inhibiting venom and toxin lethality.

Nine mice were used for each venom or toxin experiment. The mice were injected IM with a predetermined lethal dose in 0.1 ml. The mice were divided into three groups, three in each group. The mice in group II were given 500 µg LT-10 two hours prior to venom or toxin injection. The control mice in group I received PBS two hours after venom or toxin injection. Likewise, the mice in group III received 500 µg LT-10 two hours after venom or toxin injection. The injected mice were observed for a week. All mice in the control group must die to make the test valid. The results are shown in Table 2.


TABLE 2. Inhibition by synthetic LTNF (LT-10) of venom and toxin lethality.

image308.gif (5279 bytes)


The results in Table 2 clearly prove that synthetic LT-10, consisting of 10 amino acids of natural LTNF from opossum, serum was effective in inhibiting the lethality of C. atrox snake, scorpion, and honeybee venoms. LT-10 was also effective to neutralize the lethality of cobratoxin, plant-derived ricin, and botulinum toxin. In addition, LT-10 inhibited the lethality when given 2 h before venom or toxin injection. Most importantly, the lethality was inhibited even when LT-10 was administered 2 h after venom or toxin injection.



Antihemorrhagic protein, with a molecular weight 68,000 daltons and serum metalloproteinase inhibitor homologous to human alpha 1 B glycoprotein were isolated from opossum (Didelphis virginiana) serum by Menchaga and Perez (10); Catanes and Kress (2), respectively. Perales et al. (13) isolated anti-bothropic fraction (ABF) from the sera of South American Didelphidae and found that ABF has anti-Bothrops jararaca venom activity when tested in mice. Antihemorrhagic factors have also been isolated from the sera of mongoose and Trimeresurus flavoviridis (14), Vipera palestinae (12), and Crotalus atrox (15) venomous snakes. Lipps and Lipps (8) isolated lethal toxin neutralizing factor from opossum serum, which is similar to antihemorrhagic factor.

Although naturally occurring bioactive peptides have been utilized as therapeutic agents in medicine for several years, there has recently been an increasing interest in synthetically derived bioactive peptides used as novel pharmaceutical agents due to the inherent ability of these to bind and inhibit specific protein targets. Small synthetic peptides can be delivered by oral route for treatment, as it is known that peptides having less than 2.0 kDa molecular weight are absorbed into the circulation from the gut. Furthermore, synthetic peptides can be made in abundance, with batch to batch reproducibility.

Currently, the use of antivenoms is the only available treatment for envenomation caused by venomous animals namely, snakes, scorpions, spiders, ticks, and jellyfish. Antivenoms are produced in large animals, generally horses. A large percentage of the population is allergic to horse proteins. It has been estimated that an incidence of allergic reactions to polyvalent Crotalidae antivenom used in the US occurs in about 75% of the cases, which is understandable since a patient with moderate envenoming may receive up to 30 vials of anti venom, each containing 1-2 g horse protein including many non-antibody proteins (3).

Synthetic LT-10 inhibits the lethality of venoms and toxins even when administered 2 h later, therefore it has a potential as a universal treatment for intoxication by animal, plant, and bacterial toxins. Furthermore, synthetic LTNF can be made in abundance without depending upon the natural source, opossum serum.

So far, experiments were performed using 500 µg of LT-10 to inhibit the lethality of various venoms and toxins in mice. Research in this laboratory is ongoing to determine the concentration of LT-10 required to inhibit the lethality in mouse from which a human dose can be calculated on weight basis. Research on lethality inhibition of venoms or toxin by orally delivered synthetic LT-10 is also in progress. As it stands now, 500 µg is required for a mouse weighing 20 g converts to a human dose of 2500 x 500µg =1.2g, this would be only 1/50 of the protein given as serotherapy for snakebites. In the future, LT-10 should become a treatment for intoxication caused by animal, plant, and bacterial toxins.



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Received 10 May 1999
Accepted 1 July 1999

B. V. LIPPS - Ophidia Products, Inc. - 11320 South Post Oak, Suite 203 - Houston, Texas 77035, USA.

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