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Fractionation of egyptian cobra venom
Toxtcon, 1974, Vol. 12, pp . 493 99 . Per~amon Prru. Printed in Grcrt Britain
FRACTIONATION OF EGYPTIAN COBRA VENOM A. KAMEL* Physiology Department, Faculty of Medicine, Ain-Shams University, Cairo, Egypt (.lccepted for publirnlion
17 Febru~y 1974)
Abstract-Five toxic fractions were isolated from Egyptian Ngja hgje venom by filtration using Sephadex G-75. Applying immunotüffusion techniques, fractions four and five were found to contain y(cardiotoxic} and a(neurotoxic}like antigenic activity, respectively. The maximum phospholipase A activity was recorded in fraction three. Quomatographic puriScation of fractions four and five, using Biorex 70 and various buffers, showed that the best resolution was obtained with ammonium atxtate, at pH 63 . The Snal concentrated fractions were submitted to immtmoekctrophorais as a test of their purity . INTRODUCTION
isolated only 2 major fractions from crude and dialysed Egyptian cobra venom. One had neurotoxic properties while the other was hemolytic. The present study was carried out to further fractionate this venom using various techniques. Lethality, and some immunological and enrymatic properties of the different fractions were also studied. KtiALIFAt
MATERIALS AND METHODS
A pool of freeze-dried Naja haje venom was used. Filtration on Sephadex G-75
Two glass columns measuring 2~5 x 100 cm were prepared for gel filtration (PORATH, 1959, 1960) using Sephadex G-75 (40-120 nm, Pharmacia Uppsala-Sweden) prepared with 0~2 M ammonium acetate buffer, pH 6~5. One g of venom was dissolved immediately before use in 6 ml of the buffer and then filtered through MilliporeO filters (04700, SS 3 ftm) . The rate of the effluent fluid was adjusted to 20 ml per hr (4°C). Chromatography on Biorex 70
Fractions IV and V obtained from venom filtration on Sephadex G-75 were fractionated on analytic grade cation exchange resin Biorex 70 (200-4U0 mesh, sodium type, Uppsala Sweden). The Biorex was prepared for chromatography in a glass column of 1 ~2 x 50 cm (KAttissoN et al., 1971) using in one run phosphate buffer and in the other acetate buffer, both at pH 6~5. The chromatography began with a low concentration (0~1 M) of buffer and increased gradually. All experiments were performed at 4°C and the rate of efliuent fluid was adjusted to 8 ml per hr. " This work was performed at the Pasteur Institute (Larches-France) in the Service des Recherches sur les Venins . t M.D. Thesis, Ain~hams University, Faculty of Medicine, Cairo, Egypt. 495 TOXICON r97~ Yo1. r7
496
A. KAMEL
The protein concentration in the eluate was determined spectrophotometrically at 280 nm . The fractions were lyophilized, weighed and stored at 4°G. Lethality, immunochemical and phospholipase studies ~so of the Sephadex fractions was assessed by injecting different concentrations i.v . to 20 g white mice (20 animals per dose). The mortality rate was recorded during 24 hr following the injection, and the i,nso calculated according to BoQusr et al. (1967) . The phospholipase A activity of these fractions was also tested (BoQusr et al., 1967). The method which is specific for phospholipase A activity depends upon hydrolysis of ovolecithin by the venom phospholipase A enzyme into lysolecithin, which is responsible for hemolysis of a suspension of horse red cells. Double diffusion in gel was carried out by the method of OUCHTERLONY (1948) as previously described (BoQusr et al., 1966, 1973 ; JouAnxgr, 1968). In the first series the crude venom was allowed to react with different types of antisera in order to choose the one most suitable for precipitation with the different fractions. The fractions were compared with proteins of different types. (a) Gamma and alpha proteins of Ethiopian N. nigricollis and N. haje venoms, respectively, extracted according to the method of IZARD et al. (unpublished observations) by ion exchange chromatography of the venoms (one run on Biorex 70, followed by filtration on Sephadex G-75). The y is a cardiotoxin, while a is a neurotoxin . (b) T3 of N. naja (siamenesis) extracted according to the method of KAxLt ssoN et al . (1971) by ion exchange of the venom (one run on Biorex 70 followed by filtration on Sephadex G-50), it is a minor neurotoxin with a curariform-like activity. (c) Lot X of Ethiopian N. nigricollis extracted according to the method of Dumarey et al . (unpublished observations) by ion exchange chromatography of the venom (one run on Biorex 70 followed by filtration on Sephadex G-50). It is a toxic basic phospholipase. Immuncelectrophoresis was performed on the final concentrated fractions according to the method Of GRABAR and WILLIAMS (1955), as modified by BoQusr et al. (1966) . RESULTS AND DISCUSSION Filtration on Sephadex G-75
The venom was separated into 5 fractions (Fig . 1) . The weights (mg) obtained after gel filtration of 1 g of venom were 225, 133, 155, 110 and 335 for fractions one through five, respectively. The corresponding intravenous Lnso values in mice (mg/kg) were 035, 0~4, 0~5, 015 and 0~2. All the fractions were soluble in distilled water and physiological saline solution, while fraction 1V was the most potent and fraction III the least potent. Fraction III was the most powerful hemolytic (phospholipase A) producing 100 per cent hemolysis at a dose of 1 hg/~ . Complete hemolysis was produced by fractions 1V and II at doses of 30 and 100 Elg/ml, respectively. Only 20 per cent hemolysis was caused by fraction V even at a dose of 100 fig/ml. Fraction I was devoid of hemolytic activity up to a dose of 100 pg/ml. By double diffusion according to Ouchterlony the total venom reacted maximally with Ethiopian anti Naja nigricollis serum, so this antiserum was used in the subsequent studies. There was an identity reaction pattern between fraction IV and y antigen of Ethiopian N. nigricollis venom and between fraction V and a antigen of Ethiopian N. haje venom, indicating the presence of y-like antigen in fraction IV and a-like antigen in fraction V (Fig. 2). The immunological relationship between fraction lv and y antigen of N. nigricollis T10XICON J97~ Yob !1
Egyptian Cobra Venom
497
8'0 TS
E c 0 m N
"N
T0 6~ 6 6'0 5'S 5' 0 4~ 5
m
O U t
a O
3'~ 2tf 2'0 l' S l' 0 0"5 0
luiL_ L
fla . 1 . Get Fn.rw~rlox ox
S
t
10 I S 20 2S 30 3S 40 40 SO SS 60 6S 70 7S 80 No. of tubes
~8
serzunex G-75
COLUMN OF veNOM.
1 g tYOrztn.>rz® eaYrnwN N.
hgje
Elution was with 0~2 M ammonium acetate buffer pH 6"S at a rate 20 ml per hr.
venom indicated that this fraction belongs to the group of cardiotoxic substances of 60 amino acids, containing 8 half cystines (BoQusr et al., 1973), while the same relation between fraction V and a antigen of N. haje venom showed that it belongs to a curarelike proteins of 61-62 amino acids carrying 4 S.S. bridges (HoQvEr et al., 1973) . There was no reaction between the five fractioas and T3 or Lot X antigens of N. naja and N. nigricollis venoms . Chromatography on Biorex 70
One hundred mg of fraction IV was separated into two sûbfractions a and b. Subfraction a (65 mg) was obtained at 0"2 M and b (29 mg) at 0"5 M phosphate buffer pH 6"5 (Fig. 3). Using ammonium acetate buffer, 100 mg of fraction IV was separated into five subfractions; cl (43 mg) and c2 (10 mg) were obtained at a buffer concentration 0"2 M, while c3 (15 mg), c4 (8 mg) and cs (22 mg) were obtained at a concentration of 0~5 M (Fig. 4). Better separations could thus be achieved with ammonium acetate buffer at pH 6"5. By chromatography of fraction V (200 mg) on Biorex 70 using ammonium acetate buffer at pH 6"5, six subfractions were obtained ; al (55 mg) and aZ (22 mg) were extracted at 0"1 M, a3 (14 mg), and a4 (12 mg) at 0~2 M, as (8 mg) and a6 (85 mg) at 035 M concentrations of buffer (Fig. ~. By double diffusion according to Ouchterlony, the subfractions (a,b) and (ct -cs) obtained by chromatography of fraction IV on Hiorex 70 with phosphate and ammonium acetate buffers, respectively, were reacted with y antigen of Ethiopian N. nigricollis venom. Only b and c s gave an identity reaction pattern with the y antigen denoting that they alone TOXICON 197~ Yol. 11
49B
A. KAMEL
l'S
E c O m N c m v ô u
o
s
~o u zo zs so 3s ~o so eo ~o so ao ioo ia izo po i~o iso No . of tubea
iO COLUMN OF I00 mg LYOPHiI1Z® FRACTION of N. lrgje vENOM. Elution was with 02-0"S M phosphate buffer at pH 6"S at a rate 8 ml per hr .
FIG. 3 . CHROMATOGRAPHY ON THE BIOREX
IV
No . of tubas
FIG. 4. CHROMATnGRAPHY ON THE BIOREX 7O OOLUMN OF of N. llgje vENOM.
100 mg
LYOPHIIJZHD FRACTION IV
Elution was with 02-03 M ammonium acetate buffer, pH 6"S, at a rate of 8 ml per hr.
contained the y-like antigenic activity (Figs. 6 and 7). The same technique was applied on the subfractions aI-a b obtained by chromatography of fraction V on Biorea 70 with ammonium acetate but%r; only a6 reacted with a antigen of Ethiopian N. haje venom. By immunoelectrophoresis, c3 (2 " 5 fig, y-like) and a6 (10 kg, a-like) subfractions obtained after Biorea appeared to be pure. TOXICON 197 Vol. l1
_ ~--~ .
Ouchferluny techn ique : FI-FY after Sepadex F
~-~
~`- ~ F ~ ^~ F ~~
F
"w~rr -, . .c~-.sti . . , _ ,~+. .. .. . . . .a,~a.._ ._ . . .+_si74: .,r3.: .
S .Onti N .nig .
,S .ant i .N. ..nig .
Fractions=5N.g in 5Y.1
aEth .nig=1~25Kg in 5~.1 r Eth .haje=2~5 Ng ir 5N .1
FIG . 2 . REACTION PATTERNS BETWEEN FRACTIONS I-V OF EGYPTIAN N. l1CJjE VENOM AND ETHIOPIAN x ANTIGEN OF N. I1pjC AND y ANTIGEN OF N. J7%gfiCOIIiS VENOOS .
Immune serum anti N .
Jrigricollis in trenches. Note identity reaction pattern and a antigen ; fraction IV and y antigen .
Ouehterlony technique : S .onti .N . niq .
o,b
in 9= 5/J.g 1~25~.g
=
between fraction V
o, b Fractions of F~ after Biorex
5~.1 in 5~1
Ouchterlony technique : C~-Cy. Fractions of FIQ after Biorex S .a nt i . N . nig r
_
CI
_r ~,+,r-~--~.
r
..~ ,:" _--.,.
.rEth .nig =1~25f1.q = I ~ 25N.g C i -C y
Ca
.1~".
Ca
..~ c 4
~+-
~ r .~,n . c
~ .. ~~h" ,
in 5fcl in 5p. I
7
FIG. 6. REACTION PATTERNS BETWEEN SUHFRACTIONS a AND b OF FRACTION IV (OBTAINED HY CHROMATOGRAPHY OF FRACTJON IV ON BIOREX iO WITH PHOSPHATE HUFFER~ AND y ANTIGEN FROM ETHIOPIAN N . 71181'iCOIIlS VENOM.
Note identity reaction between b and y antigen .
FIG. î. REACTION PATTERNS BETWEEN SUBFRACTIONS ~i -~ e OF FRACI'lON IV (OBTAINED BY CHROMATOGRAPHY OF FRACTION IV ON BIOREX iO WITH AMMONIUM ACETATE BUFFER AND y ANTIGEN FROM ETHIOPIAN N. 1llgriCOIhS VENOM.
Note identity reaction between c, and y antigen .
TOX/CON J97A l'ol. 11
f.~ .49R
Egyptian Cobra Venom
499 a
Amm. acetate Amen . acetate OßM 0~1 M
Aenm . 0" 35 M
acetate
7
E c
m 31 N T t 0 C
~° O
v
4 O
2 02
r
4
' 11 i~~3~4 ~6 , I 'I I I 11 I 1 I ~; 1 0 lo so so 40 0o eo ro eo eo loo I lo Iso Iso No leo leo 170 roo No . of tubes
FIO .
S.
CHROMAlbORAPHY ON
TIIB
BIORFJC 7O COLUMN os N. hgJe verloM.
OF
2OO mg LYOPHIIIZBD FItACrFON V
Elution was with 0"l-0" 5 M ammonium acetate buffer, pH 6" 5, at a rate of 8 ml per hr . AcknowledpenrentThe author expresses his gratitude to Professor Dr . PAUL BoQUe'r, for his wise counsel, supply of essential chemicals, apparatus and techniques while this work was performed in his section at Larches, France . Thanks are also extended to Y. IzARn and C. DuMAReY for th~r assistance with the biochemical and immunological techniques. REFERENCES BoQver, P., IZARD, Y., JOUANNEI', M. and MEAUME, J. (19C~ Essais de séparation des antigènes du venin de NaJa nigrtonllirper filtration sur $ephadex. Annls Inrt. Pastas, Porta 111, 719. BoQue~r, P., IzARn, Y., MSAVMS, J. and JOUANNEr, M. (1967) Etude despropriétés enzymatiques et toxiques des fractions obtenues par filtration du venin de Naja nigrtcollts sur Sephadex. Arptls Inst. Parlera, Paris 112, 213. HoQvEr, P., PotLLetnt, G., DAUMARSY, C., IZARD, Y. and RoNSSeRARY, A. M. (1973) An attempt to classify the toxic proteins of Elapidae and Hydrophidae venoms . Toxicon 11, 333. GRASAR, P. and Wn = *"~~, C. A., J. R. (1955) Méthode immunoekctrophorEtique d'analyse de IIIElanges de substances antigéniques . Btochim. blophys. Acts 17, 67. JovANNer, M. (1968) L'analyse immune-electrophorétique appliquée aux venins de serpents. Toxtcon 5,191. KARL.S70N, E., ARNBERO, H. and EAa>~t, D. (1971) Isolation of the principal neurotoxins of two Ngja ngja subspecies. F .ra. l. Biochem. 21, 1 . UIJCH'IERLONY, O. (1948) In vitro method for testing the toxin producing capacity of diphtheria bacteria. Acts. path . microbtol. scmld. 25, 186. P'ORATH, J. (1959) Fractionation of polypeptides and proteins on dextlsn gds. Clintca chien. Acts 4, 776. PORATH, J. (1960) Gel filtration of proteins, peptides and amino acids. Btochinr. btophya. Acts 39, 193.
TOXICON797~ Yor.IZ
FRACTIONATION OF EGYPTIAN COBRA VENOM A. KAMEL* Physiology Department, Faculty of Medicine, Ain-Shams University, Cairo, Egypt (.lccepted for publirnlion
17 Febru~y 1974)
Abstract-Five toxic fractions were isolated from Egyptian Ngja hgje venom by filtration using Sephadex G-75. Applying immunotüffusion techniques, fractions four and five were found to contain y(cardiotoxic} and a(neurotoxic}like antigenic activity, respectively. The maximum phospholipase A activity was recorded in fraction three. Quomatographic puriScation of fractions four and five, using Biorex 70 and various buffers, showed that the best resolution was obtained with ammonium atxtate, at pH 63 . The Snal concentrated fractions were submitted to immtmoekctrophorais as a test of their purity . INTRODUCTION
isolated only 2 major fractions from crude and dialysed Egyptian cobra venom. One had neurotoxic properties while the other was hemolytic. The present study was carried out to further fractionate this venom using various techniques. Lethality, and some immunological and enrymatic properties of the different fractions were also studied. KtiALIFAt
MATERIALS AND METHODS
A pool of freeze-dried Naja haje venom was used. Filtration on Sephadex G-75
Two glass columns measuring 2~5 x 100 cm were prepared for gel filtration (PORATH, 1959, 1960) using Sephadex G-75 (40-120 nm, Pharmacia Uppsala-Sweden) prepared with 0~2 M ammonium acetate buffer, pH 6~5. One g of venom was dissolved immediately before use in 6 ml of the buffer and then filtered through MilliporeO filters (04700, SS 3 ftm) . The rate of the effluent fluid was adjusted to 20 ml per hr (4°C). Chromatography on Biorex 70
Fractions IV and V obtained from venom filtration on Sephadex G-75 were fractionated on analytic grade cation exchange resin Biorex 70 (200-4U0 mesh, sodium type, Uppsala Sweden). The Biorex was prepared for chromatography in a glass column of 1 ~2 x 50 cm (KAttissoN et al., 1971) using in one run phosphate buffer and in the other acetate buffer, both at pH 6~5. The chromatography began with a low concentration (0~1 M) of buffer and increased gradually. All experiments were performed at 4°C and the rate of efliuent fluid was adjusted to 8 ml per hr. " This work was performed at the Pasteur Institute (Larches-France) in the Service des Recherches sur les Venins . t M.D. Thesis, Ain~hams University, Faculty of Medicine, Cairo, Egypt. 495 TOXICON r97~ Yo1. r7
496
A. KAMEL
The protein concentration in the eluate was determined spectrophotometrically at 280 nm . The fractions were lyophilized, weighed and stored at 4°G. Lethality, immunochemical and phospholipase studies ~so of the Sephadex fractions was assessed by injecting different concentrations i.v . to 20 g white mice (20 animals per dose). The mortality rate was recorded during 24 hr following the injection, and the i,nso calculated according to BoQusr et al. (1967) . The phospholipase A activity of these fractions was also tested (BoQusr et al., 1967). The method which is specific for phospholipase A activity depends upon hydrolysis of ovolecithin by the venom phospholipase A enzyme into lysolecithin, which is responsible for hemolysis of a suspension of horse red cells. Double diffusion in gel was carried out by the method of OUCHTERLONY (1948) as previously described (BoQusr et al., 1966, 1973 ; JouAnxgr, 1968). In the first series the crude venom was allowed to react with different types of antisera in order to choose the one most suitable for precipitation with the different fractions. The fractions were compared with proteins of different types. (a) Gamma and alpha proteins of Ethiopian N. nigricollis and N. haje venoms, respectively, extracted according to the method of IZARD et al. (unpublished observations) by ion exchange chromatography of the venoms (one run on Biorex 70, followed by filtration on Sephadex G-75). The y is a cardiotoxin, while a is a neurotoxin . (b) T3 of N. naja (siamenesis) extracted according to the method of KAxLt ssoN et al . (1971) by ion exchange of the venom (one run on Biorex 70 followed by filtration on Sephadex G-50), it is a minor neurotoxin with a curariform-like activity. (c) Lot X of Ethiopian N. nigricollis extracted according to the method of Dumarey et al . (unpublished observations) by ion exchange chromatography of the venom (one run on Biorex 70 followed by filtration on Sephadex G-50). It is a toxic basic phospholipase. Immuncelectrophoresis was performed on the final concentrated fractions according to the method Of GRABAR and WILLIAMS (1955), as modified by BoQusr et al. (1966) . RESULTS AND DISCUSSION Filtration on Sephadex G-75
The venom was separated into 5 fractions (Fig . 1) . The weights (mg) obtained after gel filtration of 1 g of venom were 225, 133, 155, 110 and 335 for fractions one through five, respectively. The corresponding intravenous Lnso values in mice (mg/kg) were 035, 0~4, 0~5, 015 and 0~2. All the fractions were soluble in distilled water and physiological saline solution, while fraction 1V was the most potent and fraction III the least potent. Fraction III was the most powerful hemolytic (phospholipase A) producing 100 per cent hemolysis at a dose of 1 hg/~ . Complete hemolysis was produced by fractions 1V and II at doses of 30 and 100 Elg/ml, respectively. Only 20 per cent hemolysis was caused by fraction V even at a dose of 100 fig/ml. Fraction I was devoid of hemolytic activity up to a dose of 100 pg/ml. By double diffusion according to Ouchterlony the total venom reacted maximally with Ethiopian anti Naja nigricollis serum, so this antiserum was used in the subsequent studies. There was an identity reaction pattern between fraction IV and y antigen of Ethiopian N. nigricollis venom and between fraction V and a antigen of Ethiopian N. haje venom, indicating the presence of y-like antigen in fraction IV and a-like antigen in fraction V (Fig. 2). The immunological relationship between fraction lv and y antigen of N. nigricollis T10XICON J97~ Yob !1
Egyptian Cobra Venom
497
8'0 TS
E c 0 m N
"N
T0 6~ 6 6'0 5'S 5' 0 4~ 5
m
O U t
a O
3'~ 2tf 2'0 l' S l' 0 0"5 0
luiL_ L
fla . 1 . Get Fn.rw~rlox ox
S
t
10 I S 20 2S 30 3S 40 40 SO SS 60 6S 70 7S 80 No. of tubes
~8
serzunex G-75
COLUMN OF veNOM.
1 g tYOrztn.>rz® eaYrnwN N.
hgje
Elution was with 0~2 M ammonium acetate buffer pH 6"S at a rate 20 ml per hr.
venom indicated that this fraction belongs to the group of cardiotoxic substances of 60 amino acids, containing 8 half cystines (BoQusr et al., 1973), while the same relation between fraction V and a antigen of N. haje venom showed that it belongs to a curarelike proteins of 61-62 amino acids carrying 4 S.S. bridges (HoQvEr et al., 1973) . There was no reaction between the five fractioas and T3 or Lot X antigens of N. naja and N. nigricollis venoms . Chromatography on Biorex 70
One hundred mg of fraction IV was separated into two sûbfractions a and b. Subfraction a (65 mg) was obtained at 0"2 M and b (29 mg) at 0"5 M phosphate buffer pH 6"5 (Fig. 3). Using ammonium acetate buffer, 100 mg of fraction IV was separated into five subfractions; cl (43 mg) and c2 (10 mg) were obtained at a buffer concentration 0"2 M, while c3 (15 mg), c4 (8 mg) and cs (22 mg) were obtained at a concentration of 0~5 M (Fig. 4). Better separations could thus be achieved with ammonium acetate buffer at pH 6"5. By chromatography of fraction V (200 mg) on Biorex 70 using ammonium acetate buffer at pH 6"5, six subfractions were obtained ; al (55 mg) and aZ (22 mg) were extracted at 0"1 M, a3 (14 mg), and a4 (12 mg) at 0~2 M, as (8 mg) and a6 (85 mg) at 035 M concentrations of buffer (Fig. ~. By double diffusion according to Ouchterlony, the subfractions (a,b) and (ct -cs) obtained by chromatography of fraction IV on Hiorex 70 with phosphate and ammonium acetate buffers, respectively, were reacted with y antigen of Ethiopian N. nigricollis venom. Only b and c s gave an identity reaction pattern with the y antigen denoting that they alone TOXICON 197~ Yol. 11
49B
A. KAMEL
l'S
E c O m N c m v ô u
o
s
~o u zo zs so 3s ~o so eo ~o so ao ioo ia izo po i~o iso No . of tubea
iO COLUMN OF I00 mg LYOPHiI1Z® FRACTION of N. lrgje vENOM. Elution was with 02-0"S M phosphate buffer at pH 6"S at a rate 8 ml per hr .
FIG. 3 . CHROMATOGRAPHY ON THE BIOREX
IV
No . of tubas
FIG. 4. CHROMATnGRAPHY ON THE BIOREX 7O OOLUMN OF of N. llgje vENOM.
100 mg
LYOPHIIJZHD FRACTION IV
Elution was with 02-03 M ammonium acetate buffer, pH 6"S, at a rate of 8 ml per hr.
contained the y-like antigenic activity (Figs. 6 and 7). The same technique was applied on the subfractions aI-a b obtained by chromatography of fraction V on Biorea 70 with ammonium acetate but%r; only a6 reacted with a antigen of Ethiopian N. haje venom. By immunoelectrophoresis, c3 (2 " 5 fig, y-like) and a6 (10 kg, a-like) subfractions obtained after Biorea appeared to be pure. TOXICON 197 Vol. l1
_ ~--~ .
Ouchferluny techn ique : FI-FY after Sepadex F
~-~
~`- ~ F ~ ^~ F ~~
F
"w~rr -, . .c~-.sti . . , _ ,~+. .. .. . . . .a,~a.._ ._ . . .+_si74: .,r3.: .
S .Onti N .nig .
,S .ant i .N. ..nig .
Fractions=5N.g in 5Y.1
aEth .nig=1~25Kg in 5~.1 r Eth .haje=2~5 Ng ir 5N .1
FIG . 2 . REACTION PATTERNS BETWEEN FRACTIONS I-V OF EGYPTIAN N. l1CJjE VENOM AND ETHIOPIAN x ANTIGEN OF N. I1pjC AND y ANTIGEN OF N. J7%gfiCOIIiS VENOOS .
Immune serum anti N .
Jrigricollis in trenches. Note identity reaction pattern and a antigen ; fraction IV and y antigen .
Ouehterlony technique : S .onti .N . niq .
o,b
in 9= 5/J.g 1~25~.g
=
between fraction V
o, b Fractions of F~ after Biorex
5~.1 in 5~1
Ouchterlony technique : C~-Cy. Fractions of FIQ after Biorex S .a nt i . N . nig r
_
CI
_r ~,+,r-~--~.
r
..~ ,:" _--.,.
.rEth .nig =1~25f1.q = I ~ 25N.g C i -C y
Ca
.1~".
Ca
..~ c 4
~+-
~ r .~,n . c
~ .. ~~h" ,
in 5fcl in 5p. I
7
FIG. 6. REACTION PATTERNS BETWEEN SUHFRACTIONS a AND b OF FRACTION IV (OBTAINED HY CHROMATOGRAPHY OF FRACTJON IV ON BIOREX iO WITH PHOSPHATE HUFFER~ AND y ANTIGEN FROM ETHIOPIAN N . 71181'iCOIIlS VENOM.
Note identity reaction between b and y antigen .
FIG. î. REACTION PATTERNS BETWEEN SUBFRACTIONS ~i -~ e OF FRACI'lON IV (OBTAINED BY CHROMATOGRAPHY OF FRACTION IV ON BIOREX iO WITH AMMONIUM ACETATE BUFFER AND y ANTIGEN FROM ETHIOPIAN N. 1llgriCOIhS VENOM.
Note identity reaction between c, and y antigen .
TOX/CON J97A l'ol. 11
f.~ .49R
Egyptian Cobra Venom
499 a
Amm. acetate Amen . acetate OßM 0~1 M
Aenm . 0" 35 M
acetate
7
E c
m 31 N T t 0 C
~° O
v
4 O
2 02
r
4
' 11 i~~3~4 ~6 , I 'I I I 11 I 1 I ~; 1 0 lo so so 40 0o eo ro eo eo loo I lo Iso Iso No leo leo 170 roo No . of tubes
FIO .
S.
CHROMAlbORAPHY ON
TIIB
BIORFJC 7O COLUMN os N. hgJe verloM.
OF
2OO mg LYOPHIIIZBD FItACrFON V
Elution was with 0"l-0" 5 M ammonium acetate buffer, pH 6" 5, at a rate of 8 ml per hr . AcknowledpenrentThe author expresses his gratitude to Professor Dr . PAUL BoQUe'r, for his wise counsel, supply of essential chemicals, apparatus and techniques while this work was performed in his section at Larches, France . Thanks are also extended to Y. IzARn and C. DuMAReY for th~r assistance with the biochemical and immunological techniques. REFERENCES BoQver, P., IZARD, Y., JOUANNEI', M. and MEAUME, J. (19C~ Essais de séparation des antigènes du venin de NaJa nigrtonllirper filtration sur $ephadex. Annls Inrt. Pastas, Porta 111, 719. BoQue~r, P., IzARn, Y., MSAVMS, J. and JOUANNEr, M. (1967) Etude despropriétés enzymatiques et toxiques des fractions obtenues par filtration du venin de Naja nigrtcollts sur Sephadex. Arptls Inst. Parlera, Paris 112, 213. HoQvEr, P., PotLLetnt, G., DAUMARSY, C., IZARD, Y. and RoNSSeRARY, A. M. (1973) An attempt to classify the toxic proteins of Elapidae and Hydrophidae venoms . Toxicon 11, 333. GRASAR, P. and Wn = *"~~, C. A., J. R. (1955) Méthode immunoekctrophorEtique d'analyse de IIIElanges de substances antigéniques . Btochim. blophys. Acts 17, 67. JovANNer, M. (1968) L'analyse immune-electrophorétique appliquée aux venins de serpents. Toxtcon 5,191. KARL.S70N, E., ARNBERO, H. and EAa>~t, D. (1971) Isolation of the principal neurotoxins of two Ngja ngja subspecies. F .ra. l. Biochem. 21, 1 . UIJCH'IERLONY, O. (1948) In vitro method for testing the toxin producing capacity of diphtheria bacteria. Acts. path . microbtol. scmld. 25, 186. P'ORATH, J. (1959) Fractionation of polypeptides and proteins on dextlsn gds. Clintca chien. Acts 4, 776. PORATH, J. (1960) Gel filtration of proteins, peptides and amino acids. Btochinr. btophya. Acts 39, 193.
TOXICON797~ Yor.IZ