Snake Venom Toxins

THE AP\IIKO ACID SKQUESCES OF TWO TOXIXS FROM DEXDROASPIS POLI’LEPIX POLI’LEPIS (BLACK MAMRA) VENOM

(Received for publication, November 12, l!)il)

SUMMARY

The venom of the black mamba (Dendroaspis poZyZepis) was fractionated by chromatography on Amberlite CG-50. Two of the toxins were purified, and their amino acid se- quences were determined. Toxin QI is a 60-residue protein cross-linked by four disulfide bridges; it is novel among snake venom toxins in containing 4 tyrosyl residues as op-

posed to the usual single tyrosyl residue. Toxin y is a ‘72- residue protein with five disulfide bridges and is the first snake venom toxin isolated to contain 2 tryptophanyl resi- dues. Toxins LY and y have subcutaneous LD50 values of 0.09 and 0.12 pg per g of mouse, respectively.

The black I~~al~~biL (Dev~dronspis polylepis) is popularly the most dreaded snake in .Ifrica, l)resumably because of the reportedly short death time of \-ictims, the size of the snake, and the fierce- ness of its attacks. Very little ia knows ahout its venom and even less about the toSill ill the venom. Zaki et al. (I) used gel filtration on Sepliades C-100 and electrfq~horesia on cellulose ace- ta.te strips to indicate the lwesence of a toxic cwnl~or~ent of t,he venom. Strvdorn :uvl Hates (2) used ion eschange chromatog- raphy 011 Xmberlite (‘G-50 :1.11(1 gel filtratiotl tlirou~lll Sq)hadrs G-50 to obtain sevewl flwtioii~.

Recently a number of :rmi~) twid .sequences of snake venom tosins were coull)leted. Tl~rsr were all isolatetl from venoms of the genera iVcr;ia (3-y),’ Ilen~cha~~rs (8), :mtl Lalicaudn (9) alld appear to form an homologous series. 1%~ illcbluding toxins from other Keller:,, sucll as Dmdronspis, it should be possible to estab- lish meanii~gful l~liplo~enetic relationships between the various toxins, as well as thr rrlationship of strucature and function. Therefore the studies lxeaeiited here concern the elucidatioln of the amino acid sequences of two of the ~nincipnl toxins of Dendro- nspis polylepis veiwm.

MSPERIRlI~ST.~L PROCEDURE

Desiccated Dendroaspis polylepis polylepis rellom wns ob- tained from D. Muller, Professional Snake Catcher, (By) Ltd.,

1 D. P. B&es, mpnblished results.

Ion f?xchange Chrol~lafogmphU-~\lnbel,litr (‘G-50 rrsill (l)ritisll Drug Houses) was regenerated and equilibmted wit11 0.1 11 an- monium bicarbonate solution before each run by the nwthod of Hagihara ct al. (10) as described (2, 7). The resin was ~xi~krtl ill columns (130 x 3.8 cm) a.nd equilibrat,ed \vith 2 eolunm \-olunrr~ of 0.1 31 ammoniun~ bicarbonate solution. D. polylepis n-l~)lr venom (10 g) was dissolved iu 100 ml of 0.05 11 ammoninm l)icxl~-

bonate solution and was applied to the col~unn. A lilwar gradi- ent, of 18 1iterS from 0.1 &I to 0.475 M mllllloniilm bic:~ihon;ltr

solution was used to elute the column at a flow rate of 250 ml I’el hour. The column temperature was kept at so, and the &late was monitored at 280 urn.

Carbo~~?-Inetli~lcellulose (Whatman, C.“W32) was lwel~ai~~l :LS recommended by the manufacturers and equilibrated wit11 0.1 11

ammonium bicxrbonate solution. Tqophilized tosilr y l)rqx~r:~~ tion from Sepliades (Fin. 10) ws applied to :I cwlumt~ of c~:~rboq- met~hylcellulose (20 X 1.9 cm) and &ted with :I lilrwr gutlirnt

of 6 liters froni 0.1 a1 to 0.25 11 ;~ninlo~~i~iin I)ic*:irbolclte solutioll. The pumping rnt,e WIZ; 100 ml 1~ lwur, :~nd thr cBluel,t was monit~ored at 280 nm.

Gel E’iZtration-The Sepliadrs (I’harnxlci:~) c~olu~iins ww ~xts- pared as recoinmeided by t’he rn:bllut;i~tlu,e~. ~01~ I)twtrill

separations three cwlumns (3.X X 150 cm) of ;iepli:~drs (i-50 ww connected in series as I)reviou+- described ii, 11). 1‘1~ &ant. was 0.2 M ammonium bicarl~onate solut,ioii at pH 8, and the rlw tion rate was 50 ml per how. For peptide sepa1’ations three columns (0.9 x 170 cm) of Sephndes G-25 iu 0.05 nl anlnlolliunl acetate solution at l)H 7 were utilized. The sample \olrimr clicl not exceed 1 ml, and the columns were eluted at a rate of 15 ml per hour; 3-ml fractions were collected after mollitorina at 240, 260, and 280 nm with a Beckman Spectrochrom model 130.

Ultracentrifugation-~~ Spinco model E: analytiral ultraww trifuge was used for determination of the molecular neigllt of toxin Q by the rapid sedimentation equilibrium method of Yphantis (12). *An eight-channrl cell was fmplo~-ed wit11 yw tein concentrat~ions of 1.2, 0.9, 0.6, and 0.3yc (K/V) iit 0.2 JI

4029

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O 0 it

a

Rlaclc Mamba Toxins Vol. “47, x-0. 12

(bl

3 000 3 500 4000 4500

ELUATE VOLUME (ml)

FIG. 1. Gel filtration through Sephadex G-50 (450 X 3.8 cm) in 0.2 M ammonium bicarbonate solution. a, Fraction E from Am- berlite; b, Fraction F. a, 0, y, and B indicate the toxins in those fractions.

6

4

START OF GRADIENT

g

: 2

0 1 7 13

1

\ L I6

ELUATE VOLUME (LITRES 1

FIG. 2. Chromatography of 10 g of D. polylepis venom on Am- berlite CG-50. ,4 linear gradient of 18 liters from 0.1 to 0.475 M ammonium bicarbonate was applied as described under “Experi- mental Procedure.” The asterisks indieat,e toxicity of a fraction. A t.o K refer to fractions which were pooled as indicated by the bars on the abscissa.

sodium acetate buffer (pII4.5) at a rotor speed of 24,000 rpm and a temperature of 20”. Partial specific volumes were calculated from the amino a.cid compositions (13), Prior to ultracentrifuga- tion the protein solutions were equilibrated by dialysis overnight

f 000 2 000 3000

ELUATE VOLUME (ml)

FIG. 3. Final purification of toxin y by chromatography on carboxymethylcellulose as described under “Experimental Pro- cedure.”

TAHLE I

Amino acid compositions of toxins 01 and y yiwn as residues per molecule

Lysine 6.13 Histidine. 2.83 Arginine. 5.01 Aspartic acid. 4.87 Threonineb. 4.87 Serinec.......... 3.79 Glutamic acid. . 5.35

Proline. 1.76 Glycine 4.96 Alanine. 0.99 Half-cystined 7.95 Valine. 1.88 Methionine...... 0.00 Isoleucine 3.09 Leucine 0.00 Tyrosine 3.61

Phenylalanine. 0.00 Tryptophan. le

Total

Toxin a

.-

6

3 5 5

5 4 5 2

5 1

8 2

I -

From analysis’

FEWtl 3equence:

9.11 0.00 3.91

6.88 5.78 3.74

6.29

3.86 5.06 4.04

10.04 2.58 0.00

2.45 1.05 0.96

2.75 2e

9

4 7

6 4

6 4 5 4

10 3

60 72

Toxin y

(D Hydrolysis with 6 N HCl at 110” for 18 hours. b Corrected for destruction by 4%. c Corrected for destruction by 8%. d Determined as S-carboxymethyl cysteine. e Determined by difference spectroscopy as described under

“Experimental Procedure.”

against 2 liters of the sodium acetate buffer, with 23/32 Visking

tubing previously boiled for 15 min in distilled water (4). Reduction and X-Carboxymethylation of Toxins-Tosins o( and

y were reduced with P-mercaptoethanol and S-carboxymethyl- ated with iodoacetate as described (4). When dithiothreitol (14) was used as reducing agent, the same degree of reduction was obtained.

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Issue of June 25, 1972 D. J. Xtrydom

TABLE II Tryptic peptides of toxin 01

4031

/1 aj B iin,

T-i

--

0.94(1

1.73(2

0.65(1

0.99(1

1.99(2

1.05(x

1.28(1

C.80(1

0.79(1

3.5

ix

2.03(2

~1 1) -1 .) 1) -1 -1

) )

3’

2:

3.37(1

1 .O 9

j.2 '_j 1 2. 18.2

j

WC

11

Blue

-

i.OO(l X87(2 1.02(1

i.68(2

@.99(1

0.12

1.88(2

2.15(2

1.63(:

1.04(1

1.88(;

1.95(;

0.24

0.83(1 0.83(1

8

3.5

iue

13

5.5

3lue

T-3

Tozicity Test-Column eluates were checked for toxicity, and toxicity of the pure fractions was determined, by subcutaneous injections into white mice weighing 20 to 25 g. Death within 24 hours was taken as indicating toxicity.

.lmino Acid Analyses--Proteins and peptides were hydrolyzed with constant boiling HCl at 110” for 18 hours as described (4). Values for the serine and threonine contents of the peptides were corrected by 8% and 4%, respectively, to compensate for destruc- tion during hydrolysis. Half-cystine was determined as S-car- bosymethylcyst~eine after reduction and S-carboxymethylation.

E&nation of Trypfophan Content-The tryptophan content of t.osins o( and y were determined by using ultraviolet difference spectra between these toxins and toxins 01 of Nuja huje and Q( of ll--crja nivea, respect’ively. The latter two toxins have been char- acterized completely with respect to amino acid composition and sequence (4, 5). Their similarity in size to toxins rr and y of D. polylepis venom enabled the contributions to the ultraviolet spectra by the invariant tyrosyl and tryptophanyl residues to be subtracted from the ultraviolet spectra of toxins LY and y. With the tyrosine content of both toxins (Y and y known from amino acid analyses, the t.ryptophan contents were easily obtained.

T-3: I

1)

: !)

-li

1) !I

)

1 -

T-4a

0.93(11

1.07(l)

O.@(l)

3

28.3

18.5

7

5

17.5

54.5

3lue

T-5

c.95(1

1.04(1

i.Ol(l

3

i9.4

18.5 i

5.5

'he

Ii

1

2

Y

-

T-G

0.97(1

0.98(1

1.06(1

O.Y7(1

1.50(2

1.86(2 2.88(3)

2.05(2)

l.OY(l

?.O;i(Y

1.04(l)

1.09(l)

0.92(l)

2.10(;,)

1.70(r)

0.74(l)

0.99(l)

6 6 7

i0.4 13.8 6.2

-8.5

5

ioutral

16.5

4cidic

-4

'6

cllow

6

:reen ( L

6.5

eutra.1

5

lue

T-7 T-8

l.OO(l 1.94(2)

0.94(l)

In some cases the tryptophan content of peptides was deter- mined by a combination of pronase and aminopeptidase M diges- tion, followed by analysis on the amino acid analyzer.

Digestion with Proteolytic Enzymes-Digestion with trypsiu and chymotrypsin was carried out at 37” in 2y0 ammonium bicarbon- ate solution at pH 8. Tryptic digests were carried out with 1 y0 (w/w) trypsin for 2 hours. Toxin 01 was digested with 2% (w/w) chymotrypsin for 3 hours, and toxin y digested exhaustively with 3% (w/w) chymotrypsin for a total of 7 hours. Thermolysin (lo/ w/v) digestion of peptides was carried out at 50” in 2% am- monium bicarbonate solution at pH 8. Papain was used for digestion of peptides at pH 6.3 as described (4).

Asparagine and Glutamine-Asparagine and glutamine con- tents of peptides were determined by examining the electropho- retie mobilities of appropriate peptides at pH 6.5. Alternatively, it was determined by digestion with aminopeptidase M or a com- bination of pronase and aminopeptidase M, and analysis of the enzymatic hydrolysates on the automatic amino acid analyzer, Blank runs were included as a check on autodigestion.

Puriification of Pepticks-Peptides were purifred and their homogeneity tested by descending paper chromatography and

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4032

TAr3LE III Chymotryptic peptides of toxin a

Vol. 247, No. 12

C-i c-i‘ C-j I I 1 I j 0.92(l) j

0.96(l) / ; I T.Oi(i)

7-

l.OY(l)

1.:13( ?) :.7..:;-:

0.11 i ;. ,= .: ,

0.::) .

- I I-

!

C-j% / c-4

I LOO(l) i Lrn(1)

O.&l) j 0.14

1.:1(Z) : 1.84(Z)

i.O5(1) j 1.05(l)

1.78(2) ! 0.29

i.73(2) j 1.70(z)

T-07(2) 1 i.96(2)

C-5

E.OO(2)

c.77

0.31

G.iS

.--u(2)

O.-E(?)

:.sc(:) 1.:4(z)

2.:

C.%(i)

o.:.u l.->(i)

.‘.lO(?)

: -ii . .

O.J.7

1.35( )

1.7ti(.')

4.03(4)

1 I

1 It?

i .c);(: )

'.. ( )

C.j8(1) :

I

I

I

1.11(Lj

:.55(r) : 1.52(2)

1.~0(1)

:.22(i)

2.98(l)

c.;‘!

5.76(l)

.L ,

10.0

Tj--

0.96(l) i.OS(l) 1.50(l) : :.>i(..)

+"( -1)

o.w(lj

9

‘,tj.!,

T Y ~

16

i i

‘2.; : Il.:,

::.r : Thr

4 '1 .

i.:

!c..

IL::

2.5

12.'~

/ I

I

Tr~~~tophan-ContaillilIg peptides were revealed by the 1’:lwlich reagent (16) and were observed under ultraviolet light brl’o~~ ninhydriu coloring.

COOI/-imrnird A wirm =I&! il?Lnlll~~-COOII-terl~~ill:ll :nnino acids of peptides and reduced ;n1(i S-canbos~tllet,l~~-l:~t~~~~ toGIl< were tleternliued by the trit,ium Meling tt~chuiclue of AI:r tsuo el al. (I 9) as modified by Haylett et tel. (20).

i~omenclature-l’el~tides derived from tryl)t,ic :IIK~ ohymotr~~~- tic digests of t.osins ar and y are prefised T :wd (y, resljectiwly, and are numbered consequently accordiug t.o their posit ion:: in the polypept.ide chain. Pept.ides derived from the original lqtidc:: by further degradatiou are similarly distinguislletl by appendiug TL (thermolysin) or P (papaiu) to the symbol for the parent prp- tide. In all tables the numbers given in pnrent,heses after the analytical values signify the assumed int.egral \-alues for thr residues per molecule of pure peptide. Analyt.iicnl \-aluen Iowr than 0.1 residue are not listed.

The two groups of highly lethal t,orilla iu snake venon~~ arc

2 The abbreviations used arc: DNS- or dawyl-, 5-dimethyl- aminonaphthalene-1-sulfonyl-; Cys(Cm), S-c:lrbos~lncth!l~~s- teine; PTH-, phenylthiohyd:tnt.oin-.

150 250 350 450

ELUATE W (ml)

FIG. 4. Pructionation of t.he chymot.ryptic digest of reduced and S-carboxymethvlated tosiu 01 (i.5 pmoles) by gel filtration through Sephades G-25 (500 X 0.0 cm). The different fractions cont.nined the peptides as indicated. Vt indica.tes the total vol- ume of the column.

high volta.ge paper elect.rophoresis as described (7). The solvent systems for paper chromatography were Solvent I, butan-l-ol- acetic acid-water (40:6: 15, v/v) and Solvent II, butan-l-ol- pyridine-acetic acid-water (15: 10: 3 : 12, v/v). Paper electro- phoresis was performed at pEI 1.9, 4.5, and 6.5, Peptides were located on paper with the collidiile-ninllydrin rea.gent (15).

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hue of ,June 25, 1972 D. J. Strydom

TABLE IV Amino acid sequence of tryptic and chymotryptic peptides of reduced and S-carboxllmethylated toxin a

Peptide Residues

T-l l-11

T-2 12-15

T-3a 16-24

T-3 16-25

T-4 26-28

T-5 29-31

T-6 32-37

T-7 38-43

T-8 44-60

C-l

C-;’

C-3

C-3a

C-4

C-5

C-5a

C-6

C-7

l- 4

5-1c

11-14

u-23

15-113

24-26

24-27

27-30

31-51

C-7a 34-51

C-8 52-60

Sequence*

(Arg, Ile, Cys(Cm) , Tyr, Asx, His, Glx, Ser, Thr, Thr, Rrg)

Ala-Thr-Thr-Lys -7 Se:-Cys(Cm/)-Glu-Glu-Asn-Ser-Cgs(Cz$Q-Lys -7-7 Se:-Cys(C!m/)-Glu-Glu-Asn-Ser-Cys(Cm)-Tyr-Lys-Lys

q-Trp-Arg

Asp-His-Arg

q-Thr-Ile-Ile-Glu-Arg r-17 a,-Cgs(Cm/)-Gig-Cys(CIn)-Fro+Lys

Val,-Ly?-Pro;Gly-Va;-Gly-Il?-(His, Cys(Cn), Cys(Cm),

Glx, Ser, Asx, Lys, Cys(Cm), Asx, Tgr)

Arg-Ile-Cys(Cm)-w

Asn-His-Gin-Ser-Thr-Thr -7/7/\ Arg-Ala-Thr-Thr

A+-Ala_;Thr;Thr_-Ly$- r Ser-Cys(Cm)-Glu-Glu-Asn-Ser-Cys(Cm)-?Cyy

Lq-Ser;Cgs(Cm/)-Glu-Glu-Asn-Ser-Cys(Cm)-Tyr

Lys-Lys-T&r

Lys-Lys-Tyr-Trp

Trp-Arg-Asp-His

Arg-Gly-Thr-Ile-Ile-Glu-Arg-Gly-Cys(Cm)-Gly-Cys(Cm)-

Pro-Lys-Val-Lys-Pro-Gly-Val-Gly-Ile-His

Ile-Ile-Glu-Arg-Gly-Cys(Cm)-Gly-Cys~Cm)-Pro-Lys-Val-Lys-

Pro-Gly-Val-Gly-Ile-p

Cys(Cm_)-Cys(Cm/)-Gln;Se~-As~-Ly~-Cys(Cr$-As+-'l&

4033

0 The half-arrows indicate the extent’ of the Edman degradations and the results of the COOH-terminal amino acid determinations.

called ‘%ort toxins” (with 1 c’ lair1 lengths of 60, 61, and 62 reai- dues) and “long toxins” (with chain lengths of 71 and ‘i2 resi- dues). The 1). polylepis toxins are arbitrarily designated (Y, @, y, etc. acrording to their abundance ilt the vettorn, toxin cr being the ntwt ahut~dattt.

Fraction&ion of TI-hole Benom-Cllt,omatograi~~t~- of D. polyle- pis venom on Atnberlite CG-50 gave the chrotnatogram of Fig. 2. Toxicity was widely spread among the fractions as is indicated on t.he clworttu.togram. The same effecta noticed with fractions of D. crngusfieeps venom (a), were found for Fractions B, C, I, and Ii, namely Ion: toxicity, long death times, a “fluffed” appearance, and festering of the eyes. This places these fractions in a differ- ent rategory from the other toxic. fractions. Fraction E caused dent,h within 4 t,o 4) min, by subcutaneous injection of a large overdose. Such short death times have not been found for any of the otlter toxins from snake \-enoms so far examined, the short- est death times being in the range of 7 to 8 min. Fractions E attd F (containing toxins (Y and y, respectively) were lgophilized.

FwQ?cntion of Toxin a--The lyophilized fraction E was dis-

TABLE V

Amino acid sequence o.f peptide a-C-8 (Residrrc~s 6.8 to 60)

Sequence, Cys(Cm)-C?s(Cm)-C:llt-Ser-Asp-I,~s-C~s(Cnl)- Asn-Tyr

COOH-terminal Edman degradation,

Steps 1 to 9

Papain pept,ides

P-l

P-2

Tyr DNS-Cys (Cm), DNS-Cys(Cm), PTH-C:In,

DNS-Ser; DNS-Asp; DNS-Lys; DNS- Cys(Cm)-DNS-Asp; DNS-Tyr

Purified by paper electrophoresis at pH 1.9, 50 Volts per cm, 60 min.

Ser 1.02 (l), Asp 0.98 (l), Lys 1.00 (1) (mobility at pH 1.9, 21.5 cm). Amino- peptidase M digest: Ser 0.89, Asp 0.31, Lys 0.36.

Lys 0.80 (I), Cys(Cm) 0.43 (I), Asp 1.00 (l), Tyr 0.78 (I), Ser 0.14 (mobility at pH 1.9, 16J cm). Amimopeptidase WI digest,: Lys 1.18, Cys(Cm) 0.92, Ser + Asn 1.04, Asp 0.33, Tyr 0.90

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4034 Black Mamba Toxins Vol. 247, No. 12

10 20

y: ~,N-Arg~Thr-CYs-Asn-Lys-Thr-Phe-Ser-Asp-Gln-~er-LyS-Ile-Cys-Pro-Pro-Gly-Glu-Asn-~~e-Cys-Tyr-Th~-L~~-Thr-Trp-

T( T2 T3 T4

T!o Ttb -p

Cl c2 c3 c4 ) ..- 30 40 50

CYS -ASP-A10 -Trp-Cys-Ser -Gin -Arg -Gly -Lys-Arg-Vol -Glu-Leu-Gly-Cys-Ala-Alo-Thr -Cys-pro -Lys-Vol -Lys -Alo -Gly _

T4 T5 T6 T7 T8

Pa T60

c5 C6 c7 C8 C60 C6c C70

C6b C6d

60 70

VOl -Glu - Ile -LYs -Cys-Cys -Ser -Thr-Asp-Asp-Cys-Asp-Lys-Phe-Gin -Phe-Gly-Lys- Pro-Arg-OH

T8 T9 T10 -

C8 c9 Cl0 c70

10 20

a: H,N- Arg-Ile -CyS -Tyr -Asn -His -Gin -Ser -Thr-Thr-Arg-Alo-Thr-Thr-Lys -Ser-Cys-Glu-Glu -Asn-Ser -Cys-Tyr -Lys -Lys -Tyr -

TI T2 T3 T4

T3a - c2 c3 c4 I c5

c30 c50

30 40 50

Trp -Arg -Asp -HIS -Arg-Gly -Thr -Ile -1le -Glu-Arg-Gly-Cys-Gly-Cys-Pro-Lys-Vol-Lys -Pro-Gly -Val-Gly --Ile -HIS - Cys-

T4 T5 T6 T7 T8

C6

c50

60

Cys -Gin -Ser -Asp-Lys-Cys -Asn-Tyr - OH

TA

c7

C7a

C8

C8

FIG. 5. The amino acid sequences of toxins 01 and 7. The tryptic and chymotryptic peptides which were found are indicated

solved in 50 ml of 0.2 M ammonium bicarbonate solution and eluted through Sephadex G-50 (three columns, 3.8 x 150 cm). Three peaks were obtained, as is shown in Fig. la. All three of these fractions were toxic, the most retarded fraction being pure toxin CX, at a yield of 6.8 ye by weight of venom.

Putification of Z’ozin y-The lyophilized Fraction F was eluted through Sephadex G-50 in a similar manner as Fraction E above. Both of the fractions obtained from G-50 (Fig. lb) were toxic. The least retarded peak contained toxin y. After rechromatog- raphy of this fraction through carboxymethylcellulose, pure toxin y was obtained (Fig. 3) in 1.6% yield.

Toxicity of Toxins a and y-Subcutaneous LDsO values of 0.09 and 0.12 fig per g of white mouse were found for toxins (*: and y, respectively.

Molecular Weight and Bmino Acid Composition of Toxins a! and 7-A molecular weight of 7050 f 20 (extrapolated to zero pro- tein concentration) was found for toxin (Y by the rapid sedimenta- tion equilibrium method of Yphantis (12). The amino acid analyses of toxins cy and y are presented in Table I. The differ- ence spectrum of toxin a and toxin o( of Naja haje venom showed

that mamba toxin a! contained 3 more tyrosyl residues than Seja haje toxin (I! and also only 1 tryptophanyl residue. The spertrum of 1 tryptophanyl residue remained when the spectrmll of t)orin (Y of Naja nivea venom was subtracted from the spectrum of toxin y. Toxin y therefore contains 2 tryptophanyl residues. No S-carboxymethylcysteine could be demonstrated in hydrolysates of unreduced S-carboxymethylated toxins (Y and y.

The Tryptic and Chymotryptic Peptides of Reduced and S-Car- boxymethylated Toxin a-Dansyl-Edman degradat’ion of toxin o( yielded Arg-Ile-Cys(Cm)-Tyr-Asx as NHz-terminal sequence. Tritium labeling of the reduced and S-carboxymethylated toxin gave tyrosine as COOH-terminal amino acid.

The tryptic digest of toxin OL was fractionated by paper chroma,- tography with Solvent II. Peptides T-3, T-4, and T-6 required no further purification. Peptides T-l and T-2 mere separated from each other by electrophoresis at pH 1.9, while the fraction containing Peptides T-3a, T-5, T-7, and T-S was fractionated by electrophoresis at the same pH, to yield pure Peptides T-3a and T-5. Peptides T-7 and T-8 had the same mobility at pH 1.9 and were separated by electrophoresis at pH 4.5. The amino acid

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TAIIL

I; VI

Tryp

tic

pept

ides

of

re

duce

d an

d S-

carb

oxym

ethy

loted

to

xin

2

T-4

- I - ! *I -1

I! i 1 I 0

-

T-l

T-la

T-

lb

T-2

T-5

T-5a

T-

6 T-

6a

T-7

T-8

Amin

o ac

id

Lyr

i.ne

Argi

nino

C:irb

oxg~

neth

glcy

::i.c

jnc;

A::p

ar!:f

c

ncj

d

Thre

onin

c

Scrin

e

Glu

tam

ic ac

id

Prol

ine

Glyc

inc

Alan

ine

Valin

e

Isol

eucin

e

Leuc

inc

Tyro

sine

Phen

ylala

ninc

Tryp

toph

nn

TOta

l

Yiel

d ($

)

Mob

ility

(cm

)

PRpe

relcc

tr[)p

hore

nis

pI1

l.gb

Pape

rchr

olnat

oera

phy

301v

ent

IC

Solvf

nt

IIC

Color

wi

th

Ninh

ydrin

-Col

lidin

e

T-7

0.95(

l)

1.43(

2)

1.04(

l)

0.87(

l)

0.16

0.99

(l)

1.67

(Z)

1.01

(l)

0.14

1.30(

2)

0.78(

l)

~2

-5.0

;7-5

.l .5

.7.8

BlUC

T-9

T-10

1*03

(l) 1.

08(l)

0.92

(l)

).61(

3)

?.98(

3)

1.13(

l)

).88(

l) 1.

11(l)

1.02

(l)

1.16(

l)

3 5.8

7.5

0.99

(l)

0.88

(l)

0.85

(l)

0.99

(l)

1.0?

(l)

5 ‘9.9

I7 1.

8

5.6

Blur

:

1+10

(l)

0.87(

l)

i.O8(

1)

0.82

(l)

1.91

(l)

O.%

‘(l)

1.61

1(2)

1.00

(l)

0.27

0.19

0.86(

l)

4 7

6.8

!3.9

53

j/l.

2.5

6.5

Blue

4.5

BlW

0.22

0.99

(1

1.2G

(i

0.98

(1

0.90

(1

0.90(

1

1.04(

1

0.17

0.96

(1

+"(2

.O 7.2

5.5

Gray

1.05

(l)

0.95

(l)

2 0.1

8 2.8

8 Yello

w-

Gre

ell

1.11

(l)

0.88

(l)

1.03

(l)

7 7.2

2 1.2

1.2

Ycll3

x-

Gree

n

0.95(

l)

0.79(

l)

1.95(

.7)

0.20

0.97(

l)

0.23

1.09

(l)

0.86

(l)

1.11

(l)

1.95(

2)

0.98(

l)

0.90(

l)

7 Blue

1.10

(l)

1.88

(2)

0.47

1.05

(l)

0.23

1.07

(l)

1.15

(l)

1.05

(l)

l-69(

2)

0.78

(l)

0.13

0.88

(l)

1.5

3.8

Blue

0.96

(l)

1.07(

l)

1.08

(l)

0.94

(l)

1.00

(l)

0.95

(l)

2.5

0.5

Blue

1.05(

l)

0.27

0.14

0.18

0.33

0.11

0.27

0.22

0.96(

l)

0.10

0.20

8 .4.5

Blue

.) !

) -1

‘1 3

~

9

i a

Ehrli

ch

posit

ive.

See

Tabl

e VI

II an

d te

xt.

b 40

v/

cm.

3 ho

urs.

' 18

ho

urs.

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4036

2

4

is N

a

C

c4 \

C7a

i50 300

ELUATE VOLUME

FIG. 6. Fractionation of the chymotryptic digest of reduced and S-carboxymethylated toxin y (7.4 rmoles) by gel filtration through Sephadex G-25 as described in Fig. 4.

compositions and some properties of t,hese peptides are sum- marized in Table IT. The chymotryptic digest of toxin o( was fractionated by gel filtration through Sephades G-25. The elu- tion diagram is presented in Fig. 4. Fractions S2, 53, S5, and S8 were further fract’ionnted by paper chromatography in Solvent II, Fraction S4 by electrophoresis at pH 6.5, and Fraction S6 by paper chromatography in Solvent I. This resulted in the purif% cation of all of the peptides except Pept,ide C-3a of Fraction S2 \Thich had to be further purified by electrophoresis at pH 1.9. The amino acid composition and some properties of the chymo- tryptic peptides are summarized in Table III. The complete amino acid composition of toxin cy could be accounted for with both the tryptic or the chymotryptic peptides.

Sequence studies on the tryptic and chymotryptic peptides of toxin (Y are summarized in Table IV.

The sequence of Peptide C-l is obvious from the NHz-terminal sequence of tosin (Y. The tryptophan residue of Peptide C-5a was destroyed upon acid hydrolysis of t’he tritium-labeled peptide so that the radioact.ivity of t,his residue could not be determined. Neither lysine nor tyrosine was labeled and, together with the unique composition of Peptide C-5, when compared to Peptide C-5, the sequence must be as stated. The sequence of Peptide C-6 follows from Pept)ides T-4 and T-5. The amides of the 2 aspartyl residues in Peptide C-8 were determined on peptides isolated from a papain digest of Peptide C-8. The sequence studies on this peptide are summarized in Table V.

Complete Sequence of Toxin a-The unique alanyl residue pres- ent in peptide C-3a, together with its high glutamic acid content, showed this peptide to be an overlap for Peptides T-2 and T-3. Peptide C-5a with it’s unique lysyl-lysyl sequence presents an overlap for peptides T-3 and T-4. The occurrence of the unique tryptophanyl residue in Pept,ide C-6 furnishes the overlap be- tween Peptides T-4 and T-5. If Peptides T-6, T-7, and the sequenced 7 residues of Peptide T-8 are coupled, we find that the resultant peptide has the amino acid composition of Peptide C-7 minus arginine and the COOH-terminal histidine of Peptide C-7. When Peptide C-7a is esamined in conjunction with these, we

find that only one unique sequence is possible for Peptides ‘M, T-7, and T-8. The COOH-terminal histidine of l’eptides C-7 and C-7a must follow the last isoleucyl residue of the sequenced half of Peptide T-8 and must be followed by the known sequence of Peptide C-8 which accounts for the rest of the amino acid von1- position of Peptide T-8. The arginyl, glycyl, and threon$ residues by which Peptide C-7a is shorter than Peptide (r-7 pro- vide the evidence for placing Peptide T-6 to the NI-IQ-ternlinnl end of C-7. Peptide T-8 must provide the COO%ternlinal part of Peptides C-7 or C-7+ leaving Peptide T-i to be between l’elj- tides T-6 and T-8.

The result is that we have three regions of toxin LY that overlap to the extent of only 1 residue (arginine) with each other. Pep tide T-l must be NHz-terminal from its resemblance t,o the S& terminal of the tosin. The region T-6-T-i-T-X ends in tyrosine and therefore has to be COOH-terminal. De&r two single amino acid overlaps, therefore, the sequence of tosin o( :W sl~om-~l

in Fig. 5 is unambiguous. Tryptic and Chgmotryptic I’cplides 01 Retlw~d nerd S-Cwl~o.r,t-

methyylated Toxin y-Edmnn degradation on reduced :md S-cnr- boxymethylated toxin y yielded t,lle NH?-terminnl sqwnw of Arg-‘l‘hr-Cgs-(C:m)-has-1,~s.Tllr-l’lle. ?\‘o C’OOII-terniin:il amino acid could be demonstrated by the tritium ltlbeling proce- dure. The tryptic digest of reduced and S-~arl)o?n-nletlU-I:lted toxin y was fractionated by paper electrophoresis at, pl1 1.9. Peptides T-l, T-la, T-5, ‘I-5a, T-6a, T-7, :md T-10 were pure. Peptides T-2 and T-6, and T-lb and T-S were sepnlatrtl, and Peptide T-4 was purified by paper chromatography in Sol\-ent I. Paper chromatography in Solvent II was used to separate l’rl~- tides T-3 and T-9. The amino acid compo&ion and Some prop erties of these peptides are summarized in Table VI. The thy- motryptic digest of toxin y was fractionat.ed by gel filtrat.ion through Sephadex G-25 as is shown in Fig. 6. Two of the sis fractions were pure peptides, namely Fraction Sl (Peptide CTa) and Fraction S5 (Peptide C-4). Peptides C-3 and C-8 in Frac- tion S2 were separated by chromatography in Solvent I. Frac- tion S3 yielded four subfractions by electrophoresis at pH 1.9. The fastest migrating fraction was pure Peptide C-10. The other three fractions were fractionated by chronlnt~ogral~ll~ ill Solvent 11 to yield pure peptides C-6, C-6b, C-tic, :md C-7. Fraction S4 was fractionated into five major fractions upon chromatography in Solvent 1. Three peptides were pure, i.c~. Peptides C-l, C-2, and C-(id. Peptides C-61) :~nd (‘-10, \vhich were also found in Fraction S3, were separated, and I’el)t,ide C-6a was purified by chromatography in Solvent, II. Fractioll S6 contained peptides C-4, C-5, and C-9, which were aepar:ltetl 1)~ electrophoresis at $1 I .9. The amino acid composition and some properties of the chymotryptic peptides are presented in Tal:le VII. The full amino acid composition of toxin y could 1~ :IC- counted for by both the tryptic and chymotryptic peptides.

Sequence studies for most of the tryptic peptides are ~11- marized in Table VIII. Peptides T-6a and T-6 (which is Peptide T-6a + Arg) were pooled and digested with papain. One of the pure peptides obtained from this digest had the composition Thr 0.87 (l), Cys(Cm), 0.91 (l), Pro 1.11 (l), and Lys 1.10 (1). Two steps of dansy-Edman degradation ga.ve Thr-Cys(C’m). Lysine must be the COOH-terminal of Peptide T-63 (tryptic specificity) and therefore also of this papain peptide. Together I\-it11 the Edman degradation studies on Peptide T-Fa (Table VII), the complete sequence of Pept,ide T-6:1 is as above. The amidr in- tent of Peptide T-9 was ascertained by nluillol’rl’ticl:lse 11 digt+

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4038 Black Mamba Toxins

TABLE VIII

Amino acid sequence of tryptic peptides of toxin y

Vol. 247, No. 12

Peptide

T-l l-5

T-la 1

T-lb 2-5

T-2 6-12

T-3 13-24

T-4

T-5

T-5a

T-6

25-34 Thr-Tr?-Cgs(Cm/)-As?-Ala;(Trp, Cys(Cm), Ser, Glx, Arg)

35-36 s--Lys

35-37 &Lq(Lys, Ax)

37-48 (Arg, Val, Glx, Leu, Gly, Cys(Cm), Ala, Ala, Thr, Cys(Cm), Pro, Lys)

T-6a 38-48

T-7 49-50

T-8 51-56

T-9 57-65

T-10 66-72

Residues Sequence*

A+%-Q&r&A&+Lys

Arg

(Thr-Cys(Cm)-Asx-Lys)

Thr-Phe-Ser-Asx-Glx-Ser-Lys ---I--

Ile;Cys(Cm/)-Pro;Prt-Gl?-Glx;Asx;(Ile, Cys(Cm), Tyr, Thr, Lys)

Val-Glx-Leu-Gly-Cys(Cm/)-Al%-Al?-Thr;(Cys(Cm), ---

Pro, Lys)

*-Lys

Al?-Gl?-Val;Gl$+-Lys

Cys(C?)-Cys(Cm/)-Se?-Thr;As?-Asp-Cgs(Cm,)-A+-Lys

We-Glx-Phe-Q-&Pro-F -I- 7,

&The sequence of residues in brackets were not determined. The half arrows

indicate the extent of Edman-Dansyl degradation.

4 Peptide T-8 is neutral at pH 6.5, thereby proving the existence of glutamyl

instead glutaminyl in this peptide.

G NO DNS-amino acid could be demonstrated at position X. Either arginine or

lysine can therefore occupy positions X or Y.

tion of a papain peptide derived from Peptide T-9. This indi- Complete Sequence of Toxin y-The sequence work on the whole

cated the absence of asparagine and the presence of 3 aspartyl toxin joins Peptides T-l and T-2 at the NHn-terminal portion of residues. toxin y. Peptide C-3 overlaps Peptides T-2 and T-3, whereas

The sequences and sequence studies of the chymotryptic pep- Peptide C-4 forges the link between Peptides T-3 and T-4. The tides of reduced and S-carbo.xymethylated toxin y are sum- overlap for Peptides T-4, T-5a, and T-6 (in this order) is provided marked in Table IX. The sequences of Peptides C-l and C-2 by Peptide C-6. Together with Peptide C-6d, Peptide C-Ta

were obvious from the sequence studies on reduced and X-car- equals Peptides T-6 + T-7 + T-8 + T-9 + Phe. This phenyl- boxymethylated toxin y. Peptide C-3 was digested with papain, alanine can only come from Peptide T-10. Peptides C-7a TL1 and from studies on the papain peptides, which are summarized and -TLz provide the overlap for Peptides T-7 and T-8. Peptide in Table X, the complete sequence followed. Two peptides were T-6 has to provide the NHz-terminal part of Peptide C-7a. The isolated from a thermolysin digest of Peptide C-7a. Peptide amino acid composition of Peptide C-8 shows that T-8 and T-9 C-7a TL1 (Val 1.02 (l), Lys 1.06 (l), Ala 0.92 (1)) had COOH- are joined, so that the sequence of Peptide C-ia must be con- terminal alanine, and Peptide C-7a TL2 (Val 0.99 (l), Lys 0.98 tained in the sequence T-6-T-7-T-8-T-g-T-10. Thus a unique (l), Ala 1.10 (l), Gly 0.93 (1)) had COOH-terminal glycine. amino acid sequence can be written for toxin y as is presented in These two peptides gave an overlap for Peptide T-7 and T-8. Fig. 5.

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Issue of June 26, 1972 D. J. Strydwn

TABLE IX

Amino acid sequence of chymotryptic peptides of reduced and S-carboxymethylated toxin y

Peptide Residues

C-l

C-2

c-3

1-4 Arg-Thr-Cys(Cm)-$&

5-7 Lys-Thr-g

8-22 Ser-A~Gln-Ser-Lys-Ile-Cys(Cm)-Pro-Pro-Gly-Glu-Asn- -

Ile-Cys(Cm)-T+

23-26 Thr;Ly+Thy-Tr.p

27-30 Cys(Cm,)-As?-Ala;Trp

31-40 (Cys(Cm), Ser, Glx, Arg, Gly, Lys, Arg, Val, Glx)-Leu -

31-33 cy3(cT:)-ser-G$~

31-36 Cys(Cm)-SW-Gln-Arg-Gly-@

35-40 Gly-Lys-Arg-Val-Glx-@

37-w Arg-Val-Glu&-Lou

41-50 Gly-Cys(Cm)-Ala-Ala-Thr-Cys(Cm)-Pro-Lys-Val-@

41-66 Gly-Cys(Cm)-Ala-Ala-Thr-Cys(Cm)-Pro-Lys-Val-Lys-Ala-

Gly-Val-Glu-Ile-Lys-Cys(Cm)-Cys(Cm)-Ser-Thr-Asp-

Asp-Cys(Cm)-Asp-Lys-z

51-66 Ala-Gly-Val-Glu-Ile-Lys-Cys(Cm)-Cys(Cm)-Ser-T~r-Asp-

Asp-Cys(Cm)-Asp-Lys-Phe

67-68 GlrkPhe

69-72 Gly-Ly&$-Pro,-&-$~

*The half arrows indicate the extent of Edman-degradation or the results of

c-4

c-5

C-6

C-6a

C-6b

C-6C

C-6d

C-7

C-7a

C-8

c-9

c-10

Sequence+

tritium labelling of the carboxyl terminal amino acids.

&These amides were assigned according to electrophoretic mobilities.

ego DNS-amino acid could be demonstrated here, but the rest of the data

allows Lysine to be unequivocally assigned to this position.

s!+ These amides were assigned by aminopeptidase M digestion.

@Determined as free amino acid after three Edman-degradation steps.

DIsCUssION mum death time of 7 to 8 min is customary with all of the toxins

Considering the data presented here, it is obvious that toxin a! so far examined in this laboratory, but toxin cy of black marnba

of D. polylepis venom is a 60-amino acid residue polypeptide chain venom killed mice in 4 to 43 min. This had tended t,o confirm

with a molecular weight of 6907. This differs from an estimate the Sephadex estimates for the molecular weight of this toxin. of 3500 to 4000 based on the elution from Sephadex G-50 (2). We cannot account for the physiological action of this tosin, but The death time of mice after subcutaneous injections of toxins is the retardation on Sephadex is explained by adsorption of the probably dependent on the diffusion rate of the tosins. A mini- toxin because of the exceptionally high content of aromatic amino

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4040

TABLE X Amino acid sequence of Peptide y C-3

Sequence: Ser-Asp-Gln-Ser-Lys-Ile-Cys(Cm)-Pro-Pro-Gly-Glu-Asn-Ile-Cys(Cm)-Tyr

F-l P-2 P-3 P-4 c \/ >--

T-2 T-3

Vol. 247, No. 12

Edman degradation

COOH-terminal

Papain peptides

P-l

COOH-terminal of P-l

P-2

P-3

COOH-terminal of P-3

P-4

Ser-Asp

TY~

Purified by paperchromatography in Solvent II

Ser 1.89 (2), Asp 0.97 (l), Glu 1.03 (l), LYS 1.00 (1)

[mobility 6 cm] Aminopeptidase iY digest: User + Gln

2.52, Asp 0.91, Glu 0.0, Lys 1.09

Lye.

Ile 1.03 (l), Cys(Cm) 0.84 (l), Pro 2.00 (2), Gly 0.97

(l), Asp 0.35, Glu 0.33, Tyr 0.23 [Mobility 23 cm]

Glu 1.09 (l), Asp 1.05 (l), Ile 0.86 (l), Cys(Cm) 0.67

(l), Ser 0.13, Gly 0.22 [Mobility 15 cm]

Cys(Cm)

Tyr [Mobility 28 cm 3 authentic Tyr, color with

Ninhydrin-Collidine, brown]

acid residues (as compared \vith other Elupidae toxins). That even :I medium of high ionic strength (0.3 RI potassium phosphate, I = 0.6) camlot prel-ent this adsorbance is surprising, although the adsorbance of aromatic compounds to destran gels at low ionic strength is known (21, 22). A similar effect has recently been noticed with a protease from rat mast cells (23).

TIP use of Sephatles G-25 columns for t,he initial separat’ion of hnme of the enzyme digests proved to be more advantageous than l~:~~~er electropliore&- :md paper chromatography. Ammonium avetatr IV:W used :ls ~1 relatively volatile buffer rather than the illore volatile acetic avid. Neutral so1Utions were preferred be- cause ol the smaller risk of deanlidation of peptides during chro- mntogra1)hy. The Ion- ionic strength of the 0.05 ti1 ammonium acetate eluent facilitated the separation of tryptophanyl peptides from the reht, by adhorption to the destran gel. Since the spec- trol’llotolnetl,ic system used at, 210, 260, and 280 nm for monitor- ing the eluent flow \vas not compatible with pyridine solutions, this solvent wns not, considered.

On both of the reduced and S-carboq-methylated toxins, tryl’sin exhibited norn~al specificity. Only lysyl and arginyl bouds were hydrolyzed, whereas the COOH-terminal bonds of Lyn-45, Lys-57, and Arg-1 of toxin 01 and Lys-70 of toxin y were not hydrolyzed. *Irg-1 of toxin a is KHz-terminal and should therefore not be suscel)t,ible to attack by the endo- I)eptidasr, trypsin, alt~hough Arg-1 of tosin y was partially

hydrolyzed. Lys-45 of tosin 01 and Lys-70 of tosin y are fol- lowed by prolyl residues in the chain, and t,hus are not sus- ceptible to trypsin; the inertness of the lysyl bolld in l)osition 57 in toxin o( can be ascribed t,o the nspa’tyl and S-c:lrbox- ymethylcysteinyl residues 011 eit,her side of it,, as found for most of the Elapidnr toxins (3-8, 11). Chymotr\-pain h\-tlrolyzed peptide bonds to the (‘OOII-terminals of asparaginr, pIlen\-l- alanine, tyrosille, tryptophan, leuc%~e, lysine, and :lrgiuine in toxin y and those of tyrosine, threolline, tryptophau, and his- tidine in t,osin o(. Of these sitrs, those iilvolving lysine aiid arginine are iiiiu~ii:i1, :~lthougli thymot,ryptic hydrolysis of the peptide bond in :a lys1\-1-1y~;7-1 sequence has been found (4). The hydrolysis of l\rg(34)-Gly(35) aud Lys(50)-Ala(51) iI1 toxin y can only be ascaribed to residual trgptic activity in the chymotrypxin. The hydrolysis of the Y&(30)-Arg(31) bond of toxin OL by chymotrypsin is quite suq)rising since thi:: bond, which is present in all of the Elapidar short tosins, has not been found to be susceptible in any of the earlier cases.

The COOH-terminal trit’ium labeling method of RIatsuo et al. (19) ~~-as useful in the sequence studies. All of the COOH- terminals of the chymotrptic peptides were readily identified, except for the COOH-terminal arginine of t,osin y and its cor- responding COOH-terminal peptide. This might be att,ributed to steric factors introduced by the ~)enultimnte prolyl residue,

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al

= Na

ja

Haje

2.

m

niv

ea

5 8

= L.

se

mifa

scia

ta

"b"

2 =

a niv

ea

3 9

= b

nivea

9

3 =

E.

haem

acha

tus

IV

10

= pI

. m

elan

oleu

ca

b 4

= 3.

ha

emac

hatu

s II

11

= N.

m

elan

oleu

ca

d 5

= N.

ni

gric

ollis

0

6 =

i?.

E.

atra

co

brot

oxir~

12

=

i. po

lylep

is ^I

13

= pa

po

lylfp

is :

7 =

x. s&

iZcia

ta

"a"

A Al

a E

Glu

H Hi

s L

Leu

P Pr

o s

Ser

w TR

P c

cys

F Ph

e I

Ile

M Ne

t Q

Gln

T Th

r Y

TYK

D As

p G

Gly

K Ly

s K

Asn

R Ar

g V

Val

Thes

e le

tters

co

rresp

ond

to

stan

dard

ab

brev

iatio

ns

as

indi

cate

d in

J.

Biol

. ch

cm.

243

(196

8)

3557

-355

9 -

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Black MawLba Toxins Vol. 247, No. 12

which could prevent the oxazolone formation during the la- beling procedure.

The sequences of these two toxins have a few interesting features. The occurrence of homodipeptides (i.e. places in the sequence where a residue is immediateIy repeated) is quite high in other toxins; toxins a! of N. huje and N. nigricollis have

nine such pairs, and N. melanoleuca toxin dl has 10 homodi- peptide sequences. These have been suggested to have some structural significance (4). Although toxin c11 of D. polylepis has sis pairs of homodipeptides, this is appreciably lower than other toxins and it does seem as if the relatively high level of occurrence of this type of oddity in snake venom toxins is of little critical significance in structure-function relationships since these homodipeptides are found at different positions in the chains of toxins from different species. The part of the chain between the second and third half-cystinyl residues needs an extra deletion when compared with the known short toxins of 61- and 62amino acid residues. Together with the sequence of the erabutoxins in this region, this part of the sequence of toxin cr differs so much from the corresponding region of the other Elapidae toxins (which are mutually re- markably similar in this respect) that we can conclude that its structure has little or no influence on the toxicity of the pro- tein. This does not necessarily mean that this region is without influence on the structure of the active site of the toxins, since it is conceivable that these different toxins can have different physiological activities at the molecular level and still retain comparable lethalities. The “extra” 3 tyrosyl residues (as compared with the average known toxins containing one tyro- sine), are accommodated in three novel positions in the chain, replacing His-4 or Phe-4, appearing next to the invariant tryp- t,ophanyl residue in a position which seems to be extremely variable, and appearing as the COOH-terminal residue, re- placing a basic amino acid or asparagine. Tyrosyl residues have therefore now been found in six different positions in the short tosin chains.

Tosin y differs considerably from the other two long toxins of known amino acid sequence; the main differing portions are the 20 NHp-terminal residues and the 7 residues at the COOH- terminus. The most prominent features of this toxin are the occurrence of a second tryptophanyl residue and an extra amino a.cid between half-cystines two and three as compared with e.g. Naja nivea toxin LY.

The occurrence of a second tryptophanyl residue in toxin y yields the first positive evidence that the rrlong’r line of toxins was genetically derived from a “short” line as was postulated by Strydom (24). When the alignment chart of the protero- gl-yphae toxins (Table XI) is inspected, a gap of four positions (alignment positions 33 to 36) is seen in the short toxins. Cor- responding t,o this gap is the sequence Trp-Cys-Ser-Gln in toxin y. The sequence of the preceding 4 residues of toxin y is Trp- Q-s-Asp-Ala. Such a situation could easily have come about as follows. Bearing in mind the existence of seryl residues in position 30 (alignment chart) of the erabutoxins (9), H. haemachatus toxin -I (8) and N. wzelanoleuca toxin d,i this posi- tion could conceivably contain a cysteinyl residue (single-base

change) in a short toxin. If the gene coding for this tosin underwent unequal crossing over to the extent of 12 bases, we

29 33 would find the sequence TrpCys-Asp-His-TrpCys-Asp-His

38 Arg-Gly in the resultant protein. The 2 cysteinyl residues could then form a disulfide bridge. In actual fact, in toxin a of Naja r;ivea venom, they do form a disulfide bridge (5), the rest of the disulfide bridges being identical with those of the

erabutoxins (25) and cobrotoxin (26). Normal mutations would then lead to the sequences we find today. In the long toxins of N. nivea and N. melanoleuca the second tryptophanyl residue has been replaced by a phenylalanyl residue.

AeknewZedgmentsI wish to thank Drs. F. J. Joubert and T. Haylett for stimulating discussions during these studies and for their criticism of this paper and Dr. F. J. Joubert for a sample of Nuju niuea toxin o(. Messrs. R. Erasmus and J. N. Taljaard, Miss P. G. Das, and Mrs. M. Morgan are thanked for technical assistance.

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Daniel J. StrydomFROM DENDROASPIS POLYLEPIS POLYLEPIS (BLACK MAMBA) VENOM

Snake Venom Toxins: THE AMINO ACID SEQUENCES OF TWO TOXINS

1972, 247:4029-4042.J. Biol. Chem. 

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