331

Biochimica et Biophysica Acta, 534 (1978) 331--340 © Elsevier/North-Holland Biomedical Press

BBA 37921

NAJA HAJE HA]E (EGYPTIAN COBRA} VENOM

PURIFICATION, SOME PROPERTIES AND THE AMINO ACID SEQUENCES OF FOUR TOXINS (CM-7, CM-8, CM-9, AND CM-10b} *

FRANCOIS JOUBERT and NICO TALJAARD

National Chemical Research Laboratory, Council for Scientific and Industrial Research, P.O. Box 395, Pretoria 0001 (Republic of South Africa)

(Received Octobert 31st, 1977)

Summa~

Four toxins (CM-7, CM-8, CM-9 and CM-10b) were purified from Nala hale hale (Egyptian cobra) venom by gel filtration on Sephadex G-50 followed by ion~xchange chromatography on CM~ellulose. They each contain 60 amino acid residues and are cross-linked by four intrachain disulphide bridges. The complete primary structure of the four toxins have been elucidated. The toxici- ties, the immunochemical properties, the sequences and the invariant amino acid residues of toxins CM-7, CM-8, CM-9 and CM-10b resemble the corre- sponding properties of the cytotoxin group.

Introduction

The Egyptian cobra (Naia hale), consisting of three sub-species, is distributed throughout Africa and also occurs in Arabia [1]. The sub-species Naia hale hale, is very common to North-Eastern Africa; Nala hale annulifera is distrib- uted throughout Rhodesia, Eastern Transvaal, Zululand and Naia hale anchietae occurs chiefly in Southern Angola, Northern South West Africa, Botswana and North-Western Rhodesia, while Naia hale arabica inhabits Western and Southern Arabia [1]. To date, the complete primary structure of two short neurotoxins [2,3] and 14 cytotoxins [4--8] from the venom of Naja hale annulifera have been elucidated. Besides the toxins, this venom contains also a

* S u p p l e m e n t a r y d a t a t o t h i s a r t i c l e a~e d e p o s i t e d w i t h , a n d c a n b e o b t a i n e d f r o m : Elsevier Scient i f ic P u b l i s h i n g C o m p a n y , B B A D a t a D e p o s i t i o n , P .O . B o x 1 5 2 7 , 1 0 0 0 BM A m s t e r d a m , T h e N e t h e r l a n d s . Reference s h o u l d b e m a d e t o N o . B B A / D D / 0 7 3 / 3 7 9 2 1 / 5 3 4 ( 1 9 7 8 ) 3 3 1 . T h e s u p p l e m e n t a r y i n f o r m a - t i o n i n c l u d e s : c h r o m a t o g ~ a m s o f t h e t r y p t i c a n d c h y m o t r y p t i c d iges t s , a m i n o ac id c o m p o s i t i o n s a n d s e q u e n c e s o f the trypt ic p e p t i d e s , a m i n o ac id c o m p o s i t i o n s o f the c h y m o t r y p t i c p e p t i d e , a n d N - t e r m i n a l s e q u e n c e s , o f t h e r e d u c e d a n d S - c a r b o x y m e t h y l a t e d t o x i n s CM-7, CM-8, CM-9 a n d C M - 1 0 b .

332

number of low-molecular-weight basic polypeptides, usually of low toxicity and of completely distinct immunochemical properties. Thus, amino acid sequences of 5 polypeptides [3,6,9,10] which were reminiscent of either a short neurotoxin or/and cytotoxin, have been reported. From the venom of Naja hale, obtained from Miami Serpentarium (Miami, Fla., U.S.A.) or Ethiopia, a group of workers [11,13] purified several neurotoxins, established the partial amino acid sequence of some of them and elucidated the complete sequence of one long neurotoxino

The present study is concerned with the purification, some of the properties and primary structure of four cytotoxins from the venoms of Naja haje haje, a sub-species of the Naja haje complex, obtained from North-Eastern Africa.

Experimental procedure

Desiccated Naja haje haje venom from North-Eastern Africa was supplied by D. Muller, Professional Snake Catcher (Pry) Ltd., 215 Barkston Drive, Blairgowrie, Johannesburg, 2001. The source of the trypsin, ~-chymotrypsin and chemical reagents have been described previously [3,6]. The physico- chemical methods, the toxicity determinations by intravenous injection; the immunochemical examination by the Ouchterlony technique; the.reduction and S-carboxymethylation; the digestion with trypsin and ~-chymotrypsin; the fractionation of enzyme digests by chromatography on DEAE-cellulose and paper chromatography and/or high-voltage paper electrophoresis, the amino acid analyses of the toxins and of the peptides; the sequence determination of reduced and S-carboxymethylated toxins and peptides by Edman degradation with a Beckman sequencer or manually and the nomenclature of the peptides have all been detailed in previous communications [3,6].

Results

Purification and some properties of toxins CM-7, CM.8, CM-9 and CM-10b. The elution profile obtained for the separation of Naja haje haje venom on

Sephadex G-50 in 0.2 M ammonium acetate (pH 6.5) is shown in Fig. 1. One major and two minor peaks were evident of which the S¢ peak exhibited toxi- city. Peak Se was further fractionated on CM-cellulose using a linear ammonium acetate (pH 6.5) gradient. The elution pattern, depicted in Fig. 2, affords 12 fractions and indicates that toxicity is widely spread. Final purification of toxic fractions CM-7, CM~ and CM-9 was achieved by rechromatography on CM- cellulose columns at pH 5.0 as above. When fraction CM-10 was rechromatog- raphed on a phosphate-cellulose column at pH 6.5, two major toxic fractions (CM-10a .and CM-10b) were separated. In the present communication toxins CM-7, CM-8, CM-9 and CM-10b were selected for further study. The toxins appeared to be homogeneous by chromatography, amino acid analysis, N-ter- minal end group determination and immunodiffusion.

Some of the properties of the four toxins are summarised in Table I and the amino acid composition of the toxins are given in Table II. No free sulphydryl groups could be demonstrated in the intact toxins.

333

1,5

IC

o.~_ ~ , , ,~ ~_

0 ~ I I I I I I 300 600 900 Elu(~te vol. (ml)

Fig. 1 Gel f i l t r a t ion of NaSa hale hale v e n o m . C ~ d e v e n o m (5 g) was l o a d e d on the S e p h a d e x G-50 c o l u m n (3.8 X 150 cm) ~ d e l u t l o n e f f ec t ed w i ~ 0.2 M a m m o n i u m ace t a t e (pH 6.3) a t a f low ra te of 60 ~ / h . The c o l u m n t e m D e r a t u ~ w ~ 20~C and ~ e e lua te w ~ m o ~ t o r e d a t 280 nm. The ~ t e ~ k ~ d i c a t a s t o x i c i t y of t he f ~ c t i o n s .

Amino acid sequence of reduced and S-carboxymethylated toxins CM-7, CM-8, CM-9 and CM-10b

The tryptic and chymotrypt ic digests of the reduced and S-carboxy- methylated toxins were fractionated on columns of DEAE-cellulose as illu-

E

1.5

1.0 CM-3

CM~2 I C M - 4 t5 l

O ~ ! ~ ~ ~ 0

I I I

CM-7~ ~CM-8

c I Y I c~-~ ~

il/ / 2.0 4.0 6D

Eluete vol (liters) •

Fig. 2. C h r o m a t o g r a p h y of f r ac t ions S c on CM-cel lulose. F r a c t i o n S c (4 .0 g) was l oaded on the c o l u m n (3.8 X 50 cm) and e lu t i on e f f ec t ed b y a l inea~ g rad i en t of 0 . 0 5 ~ ) . 8 M a m m o n i u m ace t a t e s o l u t i o n s (pH 6.3) over 12 1 a t a f low ra t e of 200 m l / h . The c o l u m n t e m p e r a t u r e was 5°C and the e lua te was m o n i t o z e d at 280 nm. The as te r i sks i n d i c a t e t o x i c i t y of t he f rac t ions .

334

T A B L E I

SOME OF T H E P R O P E R T I E S O F T O X I N S CM-7, CM-8, CM-9 A N D CM-10b

Molecu la r weights were calculated from the amino acid sequences .

T o x i n Molecu la r Tox ic i t y LD50 Serological weight (#g /g m o u s e ) group

CM-7 6 8 3 1 2.8 -+ 0 .5 I I CM-8 6 7 4 7 2.6 + 0.5 II CM-9 6773 2.2 + 0 .4 II CM-10b 6 8 3 0 2.3 -+ 0.3 II

strated in the supplementary data. Peptides were further purified, where neces- sary, by methods indicated in tables of the supplementary data, which also list the amino acid composition of the peptides. The amino-terminal sequences of the reduced and S-carboxymethylated toxin, determined using the Beckman sequencer and additional sequence studies on the tryptic peptides are summa- rised in the tables of supplementary data.

The complete amino acid sequence of reduced and S-carboxymethylated toxin CM-7 is shown in Fig. 3. Sequencing the amino-terminal segment of the toxin by means of the sequencer, provided directly the position of peptides T-1 to T-4 and C-1 to C-5 or C-5b, and established overlaps for T-5 and T-6. Since

T A B L E II

A M I N O A C I D C O M P O S I T I O N O F T O X I N CM-7, CMoS, CM-9 A N D CM-10b

Samples were hydro ly sed for 24 h. Values are given as m o l o f residue per tool of tox in .

A m i n o acid CM-7 CM-8 CM-9 CM-10b

Analys i s Sequence Analys i s Sequence Analys i s Sequence Analys is Sequence

Asp attic acid 5.9 6 Threon ine 3.0 3 Serine 0.9 1 Glutamic acid 2.1 2 Proline 5.6 6 Glycine 2.1 2 Alanine 2.0 2 Hal f -cys t ine * 7.7 8 Val ine 5.0 5 Methionine 3.5 4 Isoleucine 1.9 2 Leucine 3.9 4 Tyros ine 2.9 3 Phenylalanine 0.9 1 Lysine 7.7 8 Histidine 1.0 1 Arginine 1.0 1 T r y p t o p h a n 0.8 1 Free su lPhydry ! 0 0

Tota l 60

5.8 6 5.9 6 5.8 6 3.0 3 3.0 3 3.0 3 3.8 4 1.0 1 1.0 1 1.9 2 1.2 1 1.3 1 4.5 5 5.7 6 5.6 6 2.0 2 2.0 2 2.1 2 1.0 1 2.9 3 1.7 2 7.8 8 7.9 8 7.6 8 6.7 7 4.9 5 5.5 6 2.0 2 3.5 4 2.7 3 1.0 1 1.9 2 1.9 2 4.0 4 3.8 4 3.5 4 2.8 3 2.9 3 2.6 3 1.0 1 1.0 1 0 .8 1 8.0 8 7.8 8 8.5 9 1.0 I 1.0 1 1.0 1 0.9 1 1.0 1 1.2 1 0.8 1 0 .8 1 0.7 1 0 0 0 0 0 0

60 60 60

* Determined as S - c a r b o x y m e t h y l c y s t e i n e .

335

I0 20 H2N-Leu-L~s-~ys-~s-G~n-Leu-~a~-Pr~-Pr~-Phe-Trp-Lys-Thr-C~s-Pr~-G~u-G~y~Lys-Asn-Leu-

r

• - - T - l a ~

4 c-~

¢ C-la

~----C-I~

T-I ~ T-2

T-~b )

~- 4 " C - 3

C - 2 " ~ C-3a

~ T-3

Sequencer

30 40 Cys-Tyr-Lys-Met-Tyr-Me~-Va~-A~a-Thr-Pr~-Mec-I~e-P~-Va~-LYs-Arg-G~y-Cys-I~e-Asp-

--~-3 ~ T-t~

g T-4a

~-3--~X---C-4 "~ C-5 ,

~C-3b--> ¢ c-5a )<

• C-5c ~

-Sequencer

~ T - 5 ~

~ T-Sa

C-Sb

50 60 va1-Cys-Pr~-Lys-As~-Ser-A~a-Leu-~a1-Lys-Tyr-Het-Cys-Cys-As~-Thr-Asp-Lys-~ys-Asn-~H

T-6 ~ T-7 ~

T-Sa ~

C-5 ~ C - 6 ' ~ C - 7 ~ ~,~ ~

~-5~ ~

Fig. 3. The comple te amino acid sequence of reduced and S-ca~boxymethylated tox in CM-7 from NaSa ha.ie hoje v e n o m .

peptide T-7 was the only peptide not terminating in either lysine or arginine, this peptide was assigned as the carboxy-terminal peptide of toxin CM-7.

Fig. 4 reveals the complete amino acid sequence of redured and 8-carboxy- methylated toxin CM-8. The known amino-terminal seqhence of the toxin established directly the alignment of peptides T-1 to T-4 and C-1 to C-5 or C-5a and furnished overlaps for T-5 and T-6. Peptide T-7 contained no C-terminal arginine or lysine and must be derived from the carboxy-terminal of toxin CM-8.

The complete amino acid sequence of reduced and S~arboxymethylated toxin CM-9 is illustrated in Fig. 5. Sequencing the amino-terminal segment of the toxin established the position of T-1 to T-4 and C-1 to C ~ and provided overlaps for T-5 and T-6. Furthermore, alignment of T-7 as the carboxytermi- nal peptide was confirmed by the fact that this was the only tryptic peptide not terminating in either lysine or arginine.

Fig. 6 depicts the complete amino sequence of reduced and S~arboxy- methylated toxin CM-10b. The known amino-terminal sequence of the toxin allowed peptides T-1 to T-5 and C-1 to C-6 to be placed in that order from the

336

I0 20 H2N-Leu-Lys-Cys-Hi s-Gln-Leu-Val-Pro-Pro-Phe-Trp-Lys-Thr-Cys-Pro-Glu-Gly-Lys-Asn-Leu-

{ T-1 ~ T-2 )4 T- 3-----

~ T-la~ T-Ib ~

~ C-I ~" C-2 ~ C-3 ~-~

~ C-Ia ~ ~ ' C-3a •

~ S e q u e n c e r

30 40 Cys-Tyr-Lys-Met-Tyr-Met -Val-Ser-Ser -Set -Thr-Val-Pro-Val-Ly s-Arg-Gly-Cys-I le-Asp-

T-3 ~ T-4 ~ T-5

~. T-4a ~ T-Sa ~

--C-3 -~-~C -4 ~ C-5

(-c-3b-~ ~ C-~a

S e q u e n c e r > 50 60

Ya l-Cys-Pro-Ly s-Asn-Ser-Ala-Leu-Va l-Lys-Tyr-Va l-Cys-Cys-Asn-Thr -Asp-Lys-Cys-Asn-OH

T-5 ~ T-6 ~ T-7 >

T-5a ~

~-5 ~ c-6 ~ c-7 ~

C-Sa )

Fig. 4. The complete amino acid sequence of reduced and S-caxboxymethylated tox in CM-8 from Naja haje haje venom.

NH~-terminus. The position of C-6 established overlaps for T-6 and T-7. Peptide T-8 was assigned COOH-terminal since it was the only tryptic peptide not terminating in lysine or arginine.

Discussion

The properties of four major toxins, viz., CM-7, CM-8, CM-9 and CM-10b, from Naja haje haje venom have been studied. The four toxins contain 60 amino acid residues and each comprise 8 half~ystine residues (Table II). Since no free sulphydryl groups could be detected, they are cross-linked by four intra- molecular disulphide bridges. The amino acid composition of the toxins are characterised by a high lyaine (8--9 residues) and a low arginine {1 residue) con- tent, a high content of hydrophobic amino acids, such as valine, leucine and methionine, and a low content of glycine (2 residues). A high proportion of the amino acids for the four toxins differ by not more than one or two residues. Furthermore, the number of a~partic acid, threonine, glycine, leucine, tyrosine, phenylalanine, histidine, arginine and tryptophan residues for the four toxins

3 3 7

i0 20 H2N-Leu-Ly s-Cys-tlis-Gln-Leu-Val-Pro-Pro-Phe-Trp-Lys-Thr-Cys-Pro-Ala-Gly-Lys-Asn-Leu-

< T-I'

(---T-laX T-ib

< c-i >( c-2 ~

4---C - i a------~ <

~ Sequencer

30

>

T-2 ~ ~ T-3

C-3

C-3a >

40 Cys-Tyr-Lys-Met-Tyr-•et-Va•-A•a-Thr-Pr•-•et-••e-Pr•-Va•-Lys-Arg-G•y-Cys-••e-Asp-

~ T - 3 ~J,~ T-4 ~ . T-5

~ T-4a ~ T-Sa

---c-~ .-.~ C-4 X c-5 X c-6

~ - 3 ~ ( C-5a ~

~equencer ~

50 6O Val-Cys-Pro-Lys-Asn-Ser-A1 a-Leu-Va1-Lys-Tyr-Met -Cys-Cys-Asn-Thr-Asp-Lys-Cys-Asn-OH

T-~ ~ T-6 ~ ( T-7 •

T-Sa ~ • T-7a ~ T-7~ ~ f ~ ~

C-6 ~-~ C-7 ~ C-8 )

F~. 5. The complete ~ o ~cid sequence o~ Ceduced ~ d S ~ b o x y m e t h ~ a t e d t o x ~ CM-9 ~om N~a h~e h~e venom.

are invariant. The toxins appear to resemble the cytotoxin group, if criteria of amino acid composition are used [14] .

The complete primary structure of toxin, CM-7, CM-8, CM-9 and CM-10b were established (Figs. 3--6) by using trypsin and chymotrypsin digestions. The proteolytic enzymes in general exhibited their anticipated specificities. Apart from the cleavage of Ash 55--Thr 56 bond of toxin CM-9, trypsin exhibited normal specificity only lysyl and arginyl bonds being hydrolysed.

Concerning the biological properties of the toxins, their toxicities, viz., LDs0 values (Table I), are of the same order as values which were established for the cytotoxin group [4,15--18]. The immunochemical properties of the toxins were investigated by the Ouchterlony technique. A single distinct precipitin band was found for each toxin. Further, the toxins were compared with a long neurotoxin (~), a short neurotoxin (~) and a cytotoxin (VII1) fromNaja nivea venom [18,19]. Complete crossing of the precipitin bands was observed for the long and short neurotoxins but the cytotoxin revealed complete coalescence with each of the four toxins. Consequently, the four toxins are immunochemi- cally related to the cytotoxin (cardiotoxin) group II, as classified by Boquet et al. [201.

To date, the complete primary structure of 26 cytotoxins and of two cyto-

3 3 8

iO 20 H2N-Leu-Lys-~vs-His-Lys-Leu-Va~-Pr~-Pr~-Phe-Trp-Lys-Thr-Cys-Pr~-G~u-G~y-Lys-~sn-Leu-

• T-~ ~

< T-la~T-Ib ~

~ C-I ~ ~

T-2 ~ T-3 ~ T-4-- ~ ~

C-2 ~ C-3 ~ f ~

4 c-2~ ~

~ S e q u e n c e r

30 40 Cys-Tyr-Lys-Met-Tyr-Met-Val-Ala-Thr-Pro-Met-lle-Pro-Val-Lys-Arg-Gly-Cys-lle-Asp

~T-4 ~ T-5

( T-Sa ~

~ c-~ ~ ~-~ ;~ c-6

~ C-Sa .~.~ C-5~ ~

Sequencer ~

50

~ T-6

T-6s

6O Va•-Cys-Pr• -Lys-Asn-•er -A•a-Leu-Va•-Lys-Tyr-Va•-Cys-Cys-Asn-Thr-Asp-Lys-Cys-Asn-•H

T-6 ~-~ T-7 ~l~ T-8 ~

T-6a ~

~ C-7 ~ C-8 ~ C-6 ?.~ ~

Fig. 6. The comple te a m i n o acid sequence o f reduced and S - c ~ r b o x y m e t h y l a t e d t o x i n CM-1Ob from Naja haje hoje v e n o m .

toxin homologues have been reported from several species of the genus Naja (see Joubert [6,8]). In Fig. 7 the amino acid sequence of toxins CM-7. CM-8, CM-9 and CM-10b from Na]a haje haje venom are compared among themselves and also to some of known cytotoxin sequences. What is immediately apparent is the high degree of homology within the group, consisting of CM-7, CM-8, CM-9 and CM-10b from Naja haje hale venom. The sequence of toxin CM-7 differs from that of CM-9 in only a single position; glumatic acid (position 17) replaces the alanine in toxin CM-9. Further, comparing the sequences of toxin CM-7 and CM-10b reveal an exchange of glutamine (position 6) and methionine (position 53) for a lysine and valine, respectively. The sequences of the four toxins, from Naja haje haje venom are also very homologous to those of the cytotoxin from Naja haje annulifera and Na.ia nivea venom (Fig. 7). In addition, the invariant amino acid residues of the cytotoxin group was found to be also invariant in the four sequences of Naja hale haje venom.

Regarding the structure vs. function relationship, Ryden et al. [21] observed for the primary structures of neurotoxins and cytotoxins from venoms of elapid or hydrophid snakes, that eleven residue positions are invariant. In the sequences listed in Fig. 7 this is found to be so and these residue position corre- spond to cysteine at positions 3, 15, 22, 39, 43, 54, 55 and 60, tyrosine 23,

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glycine 38 and proline 44. These invariant residues are probably necessary for determining the general folding of the peptide chain and are called the "struc- turally invariant" residues.

In conclusion, the toxicities, the immunochemical properties, the primary structures and the invariant amino acid residues of toxins CM-7, CM-8, CM-9 and CM-10b from Naja hale haje venom, indicate that they can be regarded as belonging to the cytotoxin group.

Acknowledgements

The authors are indebted to Mrs. A. Ruelle for her assistance with the Beckman sequencer and to Mrs. L. Copperthwaite for her skilful assistance with experimental work.

References

1 Broadley, D.G. (1968) Amodia 3, 1--3 2 Botes, D.P. and Strydom, D.J. (1969) J. Biol. Chem. 244, 4147--4157 3 Joubert , F.J. (1975) HoppeoSeyler's Z. Physiol. Chem. 356, 53--72 4 Wiese, H.K., Carlsson, F.H.H., Joubert , F.J. and Str~dom, D.J. (1973) Hoppe-Seyler's Physiol. Chem.

354, 1317--1326 5 Joubert , F.J. (1975) Hoppe-Seyler's Z. Physiol. Chem. 356, 1893--1900 6 Joubert , F.J. (1976) Eur. J. Biochem. 64, 219--232 7 Joubert , F.J. (1976) Hoppe-Se~,ler's Z. Physiol. Chem. 357, 1735--1750 8 Joubert , F.J. (1977) Hoppe-Seyler's Z. Physiol. Chem. 358, 79--96 9 Joubert , F.J. (1975) HoppeoSeyier's Z. Physiol. Chem. 356, 1901--1908

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