Asiatic Herpetological Research

December 1993

Vol. 5, pp. 143-146

A Study on the Purification and Pharmacological Properties of Two
Neurotoxins from the Venom of the King Cobra (Ophiophagus Hannah)
JIANX1NG SONG, YULIANG XlONG, WANYU WANG, AND XlAOCHUN PU
Toxinologica! Laboratory,Kunming Institute of Zoology. Academia Sinica, Kunming, Yunnan, China

Abstract- Using Sephadex G-50, CM-Sephadex C-25 and CM-cellulose 52 columns, two neurotoxins ofOphiophagus hannah were purified to be homogeneous on acidic PAGE which contain 53 and 73 amino

The two neurotoxins were used as a substitute for morphine. Using a morphinewithdrawal jumping model, the results demonstrated that the effects of the two neurotoxins whenadministered by injecting are very significant (P<0.01), and when administered orally are significantacid residues respectively.

(P<0.05).

Key words: King Cobra, Ophiophagus hannah, neurotoxins, morphine addiction, naloxone jumping model.

Introduction

this paper, the research concentrated on theThe King Cobra (Ophiophagus hannah)
the largest poisonous snake in the world(Tu, 1977). It is extensively distributed inis

southern China, India, Thailand and other
Asian countries. Joubert (1973) reported
on the purification and sequence
determination of two toxins from thevenom of King Cobras grown in Thailand.
Shun (1981) reported on the purification offour postsynaptically acting toxins from thevenom of the King Cobra in Guangxi,China. Our research showed that amongfour toxins, there is a postsynapticallyacting toxin containing only 63 amino acidWe

determined the complete
residues.
amino acid sequence of neurotoxin, whichThis
contain 73 amino acid residues.
neurotoxin's sequence is analogous to thatof the neurotoxins determined by Joubert(1973), but its C-terminal four amino acidresidues are very similar to that ofbungartoxininhydrophobicity (Lin andNow, the sequence of theWang,neurotoxin containing 53 amino acid1984).

residues isdetermined.

intheprocessof being

Xiong and Wang (1987) reported theclinical observation results of using theneurotoxin from cobra venom to achieve
better analgesic effect on moiphine addicted
The possibility to use thepatients.
neurotoxins from snake venom as amorphine was also firstreported by Xiong (1990). The mechanism
substitute forhas been discussed in the other papers.

1993 by Asiatic Herpetological ResearchInpurifying of two neurotoxins from thevenom of King Cobras from Guangxi,
China and using them as a substitute formorphine during tests on the mice-jumping
model.

Methods

Venom of King Cobras was purchasedfrom Guangxi Province. Male and female
mice were provided by the feed lot in ourNaloxone and morphine wereinstitute.
fromQinghai Medical Factory.
purchased
CM-Sephadex C-25, Sephadex G-50, andCM-Cellulose 52 were provided by ourpharmacy. Other chemicals of A. R. gradewere produced in China.
1.
The purification of two neurotoxinsfrom the venom of King Cobras: The dryvenom powder was dissolved in the buffer
solution (pH 5.8, 0.05 M HAc-NaAcbuffer), then the solution was centrifuged
Theto discard the insoluble material.
wasloadedonaGSephadex
supernatant
50 column (25 x 200 cm). The same bufferThe
was used to elute the column.
fractions which contained only low
molecular weight components werecollected (The process was directed byacidic PAGE).
The collected material was desalted andconcentrated. It was then loaded on a CMSephadex C-25 column (4x80 cm). The
column was eluted first by the equilibrium
buffer, then was eluted by the bufferAsiatic Herpetological ResearchVol. 5 p. 144

December 1993

{0<f27b5"432Li

FIG. 1. The Acidic PAGE of the different fractions
after separation with a Sephadex G-50 column (2.5x 200 cm).

1:

Crude venom; 2-11:Different

FIG. 2.

The acidic PAGE of the neurotoxins fromfraction after separation with a Sephadex G- 50column. Fractions 7-11 were collected together forCM-Sephadex C-25 CM-Cellulose columns. 1.
The fraction containing
Crude venom; 2.

the further isolation.

neurotoxins from Sephadex G-50; 3. Neurotoxin
Neurotoxin 2 from1; 4.
Sephadex C-25; 5.
cellulose 52.
Neurotoxin 2 further purified onCMcontaining NaCl gradient (00.4) and finally
was eluted by the buffer containing NaCIgradient (0.4-0.8). The two neurotoxins
were eluted during the first gradient

The neurotoxins from CM- Sephadex C25 still showed two other minor bands on
acidic PAGE. So each of them was further
purified on the CM- Cellulose 52 Column.

The equilibrium buffer is 0.05M, pH 5.8
HAc-NaAc buffer. The NaCI gradient is0-0.5M in the same buffer.
Determination of purities:Acidic

PAGE was used.

The separating gel was
15 %, and the spacal gel was 2.5 %. The
gel was stained by R- 250 dissolved in 375
ml ethanol, 125 ml water and 5 ml
methanol.
3.

5. Determination of LD 50: Mice wereused as the material. The method wasaccording to Gu (1965).

process.

2.

CM

Determinationofamino acidcomposition: The samples were analyzed
with a Hitachi Model 835-50 High Speed
Amino Acid Analyzer.

Determination of amino acidsequence of the neurotoxin: The method
was in accordance with the method of Lin
4.

and Wang (1984).

6.

model:Morphine withdrawal jumping
Mice were divided into random

groups, with each group consisting of 10
mice, half male and half female, with body
The groups wereweights of 20±2g.
treated by injecting S. V. with morphine,for four days, three times at a dose of 10mg/kg, three times at a dose of 20 mg/kg
and 12 times at a dose of 30 mg/kg. Aftermorphine was given the last time, theneurotoxins were administered either orallyor by injecting. The control group wastreated with physiological saline of 0.2 ml
per mouse. Three hours later, naloxone
was administered S. V. to different groups
at a dose of 15 ml/kg. Then the jumping
numbers of the mice during a 30 minute
The data wereperiod were recorded.
analyzed with statistical methods.

Results

The lyophilized venom powder wasdissolved in the buffer (0.05M,

pH 5.8.

December 1993

Asiatic Herpetological Research

TABLE 1. The amino acid compositions of neurotoxins 1 and 2.

Vol. 5 p. 145Vol. 5 p. 146

Asiatic Herpetological Research

TABLE 2. The effects of the two neurotoxins on mice jumping

December 1993