THE Jourtsa~ OF BIOLOGICAL CHEMISTRY Vol. 244, Ko. 15, 1r;sue of August 10, pp. 4147-4157, 19F9

Printed in U.S.A.

A Neurotoxin, Toxin a, from Egyptian Cobra

(hjcz haje haje) Venom

I. PURIFICATION, PROPERTIES, AND COMPLETE AMIn’ ACID SEQUEKCE

(Received for pltblication, November 19, 1968)

D. P. BOTES AND D. J. STRYDOM

Fv.nn the National Chemical Research Laboratory, Council for Scientijc and Industrial Research, P. 0. Box 395, Pretoria, Republic of South Africa

SUMMARY

A neurotoxin, designated toxin LY, has been isolated from the venom of the Egyptian cobra (Naja haje haje) by gradient chromatography on Amberlite CG-50, and has been further purified by gel filtration on Sephadex G-50. Homogeneity was verified by free boundary electrophoresis, acrylamide gel electrophoresis, sedimentation velocity, amino acid analysis, and end group analysis. Toxin LY has a sedimenta- tion constant, s& ,,,, of 1.18 S, a diffusion constant, D20,zu of 14.07 x 1OF cm2 set-I, electrophoretic mobilities of 6.02 X 10-5, 2.94 x 10-5, and 2.84 X lop5 cm2 set-1 voltV1 at pH 5.0, 6.8, and 8.3, respectively, and a formula weight of 6834.6. Ultracentrifugation studies indicate that the reduced, S-carboxymethylated toxin is capable of dimer formation. Chemical studies show that toxin (Y is a small, basic protein consisting of a single polypeptide chain of 61 amino acid residues, cross-linked by four disulfide bridges. Alanine, methionine, and phenylalanine are totally absent. The complete amino acid sequence of the neurotoxin was determined by analyzing tryptic and chymotryptic peptides of the S-carboxymethyl derivative of the neurotoxin. Align- ment with the partially determined structure of the probably homologous neurotoxin o( from Naja nigricollis indicates eight amino acid differences between the two neurotoxins.

Studies of the amino acid sequences of a number of proteins, in particular the homologous cytochromes c and hemoglobins, not only opened a remarkable phylogenetic field of study at the molecular level, but also provided a valuable tool in the study of structure-function relationships. Data which recently became available on the properties of a number of snake venom neuro- toxins (l-4) led us to expect an analogous degree of similarity among these substances. They are all small basic proteins, similar or identical in function, exceptionally rich in cystine, and void of two or more of the more hydrophobic amino acids.

The only snake venom neurotoxin of partially determined pri- mary structure is that from the black neck spitting cobra, Nuja

nigricollkl This paper describes the isolation and purification of a neurotoxin from Naja haje haje venom and some of its prop- erties. In addition, the complete amino acid sequence of the neurotoxin is described.

EXPERIMENTAL PROCEDURE

N. huje huje venom (dried in a desiccator over calcium chloride) was obtained from Transvaal Snake Park (Pty) Ltd., I’. 0. Box 97, Halfway House, Transvaal, and D. Muller, Professional Snake Catcher (Pty) Ltd., 215 Barkston Drive, Blairgowrie, Johannesburg, South -4frica. Trypsin was obtained from Sera- vat Laboratories, Cape Town, as a twice crystallized, diphenyl- carbamyl chloride-treated, salt-free preparation (Batch 336h). It had an activity of 3200 Sational Formulary trypsin units and 5 National Formulary chymotrypsin units per mg (5). Lu-Chy- motrypsin (three times crystallized, Batch CD1 6100-l) and papain (twice crystallized, Batch PAP 5582) were obtained from Worthington. Thermolysin (crystalline thermophilic bacterial proteinase, No. T7JC51) was obtained from Daiwa Kasei Com- pany Ltd., Osaka, Japan. DEAE-cellulose was a microgranular preparation, column Chromedia DE 32, from Whatman. Pyri- dine, N-ethylmorpholine, and oc-picoline were redistilled before use. All other reagents were of analytical grade.

Chromutogrnphic Methods-The Sephadex G-50 gels (Phar- macia, Uppsala) were prepared and packed in columns (66 x 2.5 cm) as recommended by the manufacturer. The ascending filtration method was used, at a flow rate of 30 ml per hour at 5”.

Amberlite CG-50 resin (British Drug Houses, 200 mesh, Type II) was regenerated before each run according to the method for Amberlite XE-64 of Hagihara et al. (6). The resin was finally converted to the sodium form and equilibrated with starting buffer, i.e. 0.1 M sodium phosphate buffer2 at pH 7.3. Packing into columns was carried out under a pressure of 15 to 20 p.s.i., and the columns were equilibrated with 3 volumes of starting buffer. A Beckman gradient pump provided a linear gradient, and all the chromatographic runs were performed at 5”. The eluate was continuously monitored at 254 rnb with a LKB Uvicord or at 260 and 280 1nl.L with the Spectrochrom model 130,

1 EAKER, D., AND POR.~TH, J., Abstracts Seventh International Congress of Biochemistry, Tokyo, 1967, Supplement I.

2 The molarities of all the sodium phosphate buffers mentioned in this paper are based on the sodium ion concentration.

4147

by guest on May 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

the absorbance \~-a:: recorded, and fractions wcrc collected in a rcfrigtratcd fraction collector.

Desaliing and Equilibration of I’rofein Solutions-The protein n-as no~m~all\- desalted by gel filtration through Sephadcs G-50 in 0.2 11 pyridine-acetic acid at pI 1 5.8. Prior lo clcctrophoresis or ultl,acentrifug:ltion, the l)rotein solution wa> cyuilibrated hi dialysis overnight against 2 liters of the alyropriate buffw, with 23,/32 Visking tubing previously boilctl for 15 min in dihtillctl \T-atcr. I~et\wrll 10 :111tl El c; of the tosin was lost I,- this pro- cedure.

Prepnration of IZeduced, S-Carbox!J,riethylatrd Toxin-The pro- cedurr of (‘rcstficld, Moore, ant1 Stein (‘7) was :~do~~tc~l, with the we of 6 hI guanidinc Ii\-tlroc~hloi,itle iiiatcad of 8 al urea as tlena- turing agent. The rcwtion nlisturc wvi~ dialyzed in 23132 V&king tubing ilgaillst distilled \v:lter in a wmtinuous flow ai)- paratus for 42 hours I)rior to frrczr-drying of the reduced :NJ~

S-cnrbox)-lrieth?-l~~tetl tlrrivatiw. A yield of 5 mg of deli\-atire \vas obtainetl from 5.5 rlig of toxin.

htinzation o,f Free LbUlfhJdr~Jl Groups-The procedure of Crest- field, Aloor~~, and Stein (7), omitting p-lllerc:ii)toetli:lnol, was used.

Tests SOT Biological ~lctinities-Tosicit?- tests were performed on white mice, weighing 16 to 20 g, by subcutaneous injections of 0.1 nil or 0.2 ml of test solutions. I’hosphodiesterase and I>Nase activities were determined by the method of Bomnn and Kaletta (8), RXase according to the Ilrocedure of Iiapla~~ and Hcppel (9), protease b)- the Kunitz test (lo), and acctyl cholines- tcrase by the method of BjGrk and I~OIIGIII (11). l’hosl)holipnse A was assayed b\- a modification of the method of Boman and Kaletta (8), except that a 0.27; solution of ovolecithin in isotonic T\‘:tCl solution was substituted for the egg yolk in the substrate suspension. I’hosl)hollionoestcrnse and AY!JI’ase activities were determined as follows: 1.0 nd of a solution of adcnosine 5’. monophosphate or ATI’ (1 mg per ml) in 0.1 JI Tris buffer-O.01 M magnesiunl chloride, pH 8.7, was incubated with 0.1 ml of test solution at room temperature, and aliquots \\-cre taken at suitable illtcrvals and analyzed b\- thin layer chromatography according to the method of Randerath (la), with DEhE-cellulose with 0.02 N hydrochloric wid as dewloper.

UltracentriSugalion Spiuco model E ultracentrifuge was used for determination of sedimentation and diffusion constants and molecular weight. The sedimentation coefficients w-cre dr- ternlined at ljrotein concentrations of 2, 1.5, 1, and 0.5’2 in a 0.05 M sodium phosphate buffer at pI1 7.6 containing 0.1 RI sodium chloride. A rotor speed of 64,000 rpm, schliercn optics, and a synthetic boundary cell were used. The diffusion constant was determined with the use of the Rayleigh optical system and a synthetic boundary (bell. The low speed erjuilibrium method (13) was used to determine the molecular weight of the reduced and S-carbos~lneth?-lated derivative in 0.3 JI sodium phosphate buffer at pII 7.3. Equilibrium was achieved aftrr 24 hours at a rotor speed of 29,933 rpm. A partial specific: volume of 0.70, computed from the amino acid composition (14), was used for the calculatiow

Blecfrophoresis of Toxin-Fret boundary c+ctrophore& ex- periments were performed in a Beckman model 1-I electrophore- sis-diff usion instlumeut h protein concentration of 0.155; was used in the following buffers (I’,‘2 = 0.1): pH 5.0, 0.05 XI sodium a&ate-acetic acid + 0.05 M SaU; pH 6.8, 0.05 JI sodium phos- phate + 0.05 JI NAY; pH 8.3, 0.05 AI sodium phosphate-disodium tetraborate + 0.05 JI SaCl.

I’olyacryl:m~ide gel elect,rophoresis was performed at 1)II 4.3 by the mcthotl of Reisfcld, Len-is, and Williams (15)) with the use or :I. 15:;, gel, :~lrd ktailliug n-ith A1nlido schwarz 13 in 7.5y0 aque- ous acetic acid.

Digestion with Proteolytic Bnsynres-Digestion with trypsiu or c.hytnotr\-pain was carried out at room te:nl)erature (except whcrc otherwise stated) in 2 ‘;t SH,IICO, solution at pH 8.0 for the specifittl tinw. The substrntc WM dissoli-cd to a concentra- tion of I (; (\\-/v), L md enzyme \V:IS added in an rnz~-mc to sub- htrate ratio of 1 :I00 (\v/w). The reaction n-as stol)ped by the addition of acetic acid to :L 1)H of 3.5, iilld the digest was lyol)h- ilizetl. I )igcstion with thcrrnolysin proccedcd as abore, ht at a

tcnrpeixtulc of 40”. Papain digestions of peptidw w.w iler- formed as follows. 5 1 L. 0 utions of I)eptidc (3 pinoles/3 nil), in 0.2 M I)yridine-wctic acid buffer at I)H 6.0, were incubated ulder nitrogen with 100 pg of papain autl 2 ~1 of I1iercal)tocth:lllol. A1fter 16 hours at 40”, the rwction nlisturc n-:IS lyophilized.

Column Chromotogmphy of Digests-DI~:.1E-cellulo~e cohunns (150 x 1.9 cm) were usrd for chromatogml)hic fractionation of the tryptic :IIJ~ chymotryptic digests of the rrdwed nnd S-car- bosynethylatcd toxin. The DIME-cellulose \V:IS prepared as recommended by the manufacturers, and the columns were finally equilibrated with 3 colunl~l volumes of st,arting buffer. The digest was dissolved in a small volume of starting buffer, the 1111 was adjusted to 9.3 with ammonia solution, and the digest was applied to the column. A I~eckman modrl 131 gratlieut 1 jump provided a B-liter linear gradient for elution of the pep- t,ides. The starting buffer was ‘2i’-rthT-llnorl)holitle-cu-l)icoliiie- pyridine-water (15 :20: 10: 955, r/v) at pH 9.3 (IS), and the limit- ing buffer was 0.055 hl Iyridinium acetate at pII 4.0. For the fractionation of the tryptic digest, the limiting buffer n-:ls 0.045 III l)yridinium acetate, pII 4.0. The column effluent was moni- tored at 285 nip with :I Beckman 1)B sl)ectrophotometrr to identify tyrosinr- and tr!-l,tophan-coiit~liiiilig peptides, and frac- tions of 15 ml were collerted at a flow rate of 200 ml per hour. At the base of the column 1 .5qC of the effluent ww diverted to be monitored by a Technicon peptide Auto-Analyzer (16). The chart speeds of both the ;\uto-Analyzer and the spertrophotom- eter recordrrs \verc synchronized. Correlation of peaks and fractions \vas whieved by injecting a small volume of an amino acid solution into the sample line of the Auto-Analyzer while re- cording the ercnt on the spectrophotomcter recorder, before the front peptides had reached the base of the column. The pep tide frnctiolls lverc concentrntcd by rotary evaporation and stored in 5% acetic acid-57; propan-l-01 solutions at 4”.

Paper Chroma.tography md Hectrophoresis of l’epfidesPIIo- mogcneity of prptides was examined by descending paper chroma- tograljhy and high voltage paper electrophorcsis. The solvent systems for ,,alwr ~hrorn~~togr:~l)h~ were Solvellt I, butan-l-ol- acetic acid-n-atc,r (40:6: IS? v v) ; and Sol\cllt I I, hutnll-l-ol- pgridine-acetic acaid-water (15: 10:3: 12, v,‘v).

Electrophoresis was I)erformed on a Gilson high voltage elec- trophorator, model I), with Varsol as cooling medium. The buffer systems were (a) lyridine-acetic acid-water, pH 6.5 (100: 4:896, v/v); (b) pyridine-acetic acid-water, $1 4.5 (10: l-1:976, v/v); and (c) acetic acid-formic acid-water, $1 1.9 (100:29.5: 870, v/v). Voltages of 50 to 100 volts per cm were used for suitable periods. Whatman So. 3M;\I paper n-as used in all cases, and preparative separations IT-ere carried out Cth the same systems. Peptides were located on paper by the chlorine- tolidine method (Ii) and with the collidine-ninhydrin reagent

by guest on May 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Issue of August 10, 1969 D. P. Botes and D. J. Strydom 4149

(18). Tryptophan-containing peptides were revealed by the Ehrlich reagent (19).

Amino Acid Analyses-Samples of the pure protein prepara- tions (1 to 2 mg) were hydrolyzed with redistilled, constant boil- ing HCl in evacuated,‘sealed tubes at 110” for 24, 48, and 72 hours. Samples of peptides (0.02 to 0.1 pmole) were hydrolyzed with 0.25 ml of constant boiling hydrochloric acid in the presence of 25 mg of phenol (20) at 110” for 20 hours. The hydrolysis tubes were sealed under vacuum at 0.01 mm Hg after deaeration of the samples. The hydrolysates were analyzed on Beckman model 120B and 120C automatic amino acid analyzers, coupled to Beckman 125 integrators. Values for the serine and threonine contents of the peptides were corrected by 7y0 and 3%, respec- tively, to compensate for destruction during hydrolysis.

Tryptophan analysis on the protein was also carried out on the Beckman 120B analyzer, after hydrolysis with barium hydroxide by the method of Robe1 (21). The extraction step with IRC- 120 ion exchange resin was omitted.

Determination of NHn-terminal Residue of Neurotoxin-Amino end group analysis was performed by manual manipulation of the phenylthiohydantoin method of Edman and Begg (22) and by the l-dimethylaminonaphthalene-5-sulfonyl chloride method of Gray and Hartley (23) and Gray (24). The phenylthio- hydantoinamino acid was identified by thin layer chromatog- raphy with Solvent D of Edtian and Begg (22). The DNS-3 amino acid was identified by high voltage paper electrophoresis at pH 4.38 by the method of Gray (24). The results were con- firmed by the subtractive method after one Edman degradation step.

Structure Determination of PeptidesMost of the sequence de- terminations were performed by a combination of the Edman and dimethylaminonaphthalenesulfonyl methods. Before com- mencement of a new Edman degradation cycle, an aliquot of the residual peptide was removed for amino-terminal analysis by the dimethylaminonaphthalenesulfonyl procedure as described by Gray (24), except that the hydrolysis period of the DNS- peptide was limited to 4 to 5 hours under vacuum (0.01 mm Hg). The DNS-amino acids were identified by high voltage paper electrophoresis at pH 4.38 as described by Gray, with a Shandon flat plate apparatus. The identities of DNS-glycine, DNS- proline, and DNS-serine were confirmed by thin layer chroma- tography on coated silica gel plates (Merck) with the solvent system benzene-pyridine-acetic acid (40 : 10 : 1, v/v) (25). The phenylthiohydantoinamino acids were used only to identify asparagine and glutamine. Thin layer chromatography on silica gel plates (Merck, type FZ54) was used with the solvent sys- tem chloroform-methanol (9: 1, v/v) as developer (26).

Nomenclature-Peptides derived from tryptic and chymotryp- tic digests of the reduced and S-carboxymethylated neurotoxin are prefixed T- and C-, respectively, and are numbered consecu- tively according to their positions in the polypeptide chain. Peptides derived from the original peptides by further degrada- tion are similarly distinguished by appending -T (trypsin), -TL (thermolysin), or -P (papain) to the symbol for the parent pep- tide. In all tables the numbers given in parentheses after the analytical values signify the assumed integral values for the resi- dues per molecule of pure peptide. Analytical values lower than 0.1 residue are not listed. In Tables IV to IX, the chromato- graphic and electrophoretic mobilities and the color with ninhy-

3 The abbreviations used are: DNS-, l-dimethylaminonaph- thalened-sulfonyl-; Cys(Cm), S-carboxymethylcysteine.

I ’ /I

‘5 I - 280my , 1 , I

v 2 m

m a

---- 260mp

-0 0

.5 0

0 .I ” I

0 560 1000 1500

ELUATE VOLUME (ML.) -

FIG. 1. Chromatography of N. haje haje venom on Amberlite CG-50. Venom (0.5 g) was applied to a column (45 X 1.9 cm) of CG-50 and eluted with a l-liter linear gradient of sodium phos- phate buffer, pH 7.3, from0.1 to 0.5 bf. The effluent was monitored at 280 and 260 rnp.

drin-collidine are listed in brackets after the analytical values for each peptide. The chromatographic and electrophoretic mobili- ties, in centimeters under the specified conditions, are designated by ch and el, respectively, followed by parenthetical numbers in- dicating the solvent system (I or II) or buffer system (pH 1.9, 4.5, or 6.5) used. -, denotes a negatively charged peptide; +, positively charged; and 0, no electrophoretic mobility, apart from endosmotic flow. No color is mentioned if the ninhydrin-colli- dine color was the usual blue.

RESULTS

Isolation of Neurotoxin

It was found from preliminary experiments that the procedure of stepwise elution on Amberlite CG-50 of Karlsson, Eaker, and Porath (1) could not be applied to Egyptian cobra venom.4 A gradient elution technique was therefore adopted. In a typical large scale experiment, 20 g of venom were dissolved in 200 ml of the starting buffer, the solution was centrifuged, and the clear supernatant was applied to an Amberlite CG-50 column (130 x

3.8 cm). After initial elution with 500 ml of starting buffer, a 3-liter gradient of 0.1 to 0.5 M sodium phosphate buffer at pH 7.3 was applied. The flow rate was 300 ml per hour, and the effluent was collected in 20-ml fractions. The elution pattern obtained in a small scale experiment is shown in Fig. 1. The most toxic fraction was diluted with 1.6 volumes of distilled water and re- chromatographed on a fresh Amberlite CG-50 column under identical conditions. The toxic fraction was again diluted with 1.6 volumes of water and adsorbed on a small column of Amber- lite CG-50 (40 x 0.9 cm). Desorption with 0.5 M buffer concen- trated the toxin into a small volume of eluate. The concentrated solution was desalted on a Sephadex G-50 column (66 x 2.5 cm) in 0.2 M pyridine-acetate buffer at pH 5.8. At the same time phosphodiesterase activity, which was still associated with the toxin, was separated, the phosphodiesterase being eluted before the toxin (see Fig. 2). The resultant pure toxin a! was then freeze-dried to yield 1.7% by weight of the whole venom.

This lyophilized preparation of toxin cr is readily soluble in

4 To be published elsewhere.

by guest on May 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Neurotoxin a! from Egyptian Cobra Venom. I Vol. 244, No. 15

r

2 1.0 - Phosphadiesterose-

IY 0 u-l

s O-6 L

(Azm mox= 3.34)

0 200 400

ELUATE VOLUME (ML.)

FIG. 2. Separation of toxin 01 and phosphodiesterase (derived from 20 g of venom) on a Sephadex G-50 column (70 X 2.5 cm) in 0.2 M pyridinium acetate buffer at pH 5.8.

I.201

I I I I I

0 0.25 0.50 0.75 IO0

CONCENTRATION to/o) - FIG. 3. Concentration dependence of ~~0,~. The values of

SW,, on the ordinate are given in Svedberg units. Standard devia- tions are shown. The extrapolation to zero concentration was done by least squares analysis, with the use of a weight factor, Wi, Of I./S<‘.

water, in contrast to the N. nigricollis toxin a! isolated by Karls- son, Eaker, and Porath (1). It was found, however, that if the preparation of toxin cy was dialyzed salt-free before the Sephadex

step by the same method as for equilibration with buffer solu- tions, it became partly insoluble upon freeze-drying. When the phosphodiesterase fraction from the Sephadex filtration (Fig. 2) was lyophilized, it was found that the phosphodiesterase was the component that becomes partly insoluble. Concomitant with this was the loss in nearly all of the phosphodiesterase activity. It was therefore imperative to test for phosphodiesterase con- tamination of the toxin cr preparation before lyophiliiation.

The phosphodiesterase could be separated from the neurotoxin during the re-chromatography step by stepwise elution with 0.25 M phosphate buffer at pH 6.8. This procedure, however, resulted in a poorer resolution between the toxin and Fraction H (Fig. 1). The resultant contamination by Fraction H could not be removed during the desalting on Sephadex G-50. This Fraction H also

appeared to be the source of traces of methionine, alanine, and phenylalanine found in some preparations of toxin a.

Ultracentrijugation

In the sedimentation velocity experiments only one, sym- metrical peak was observed. The results are summarized in Fig. 3. Extrapolation of .QO,~ to zero concentration yielded the sedimentation constant &o,~ of 1.182 f 0.009 S. Combining this value and the determined diffusion constant (Dzo.~ = [14.07 i 0.011 x lo+ cm2 see-‘) in the Svedberg equation gave a molecular weight of 6800. This is in excellent agreement with the molecular weight of 6834.6 calculated from the amino acid composition.

The molecular weight of the reduced and S-carboxymethylated derivative was determined by low speed equilibrium ultracen- trifugation. Extrapolation to zero concentration by a rigorous least squares analysis yielded a molecular weight of 12,500 as- suming the partial specific volume to be the same as that of the neurotoxin. This indicates that the derivative associates at neutral pH to form dimers.

Ebctrophoresis

Free boundary electrophoresis at pH 5.0,6.8, and 8.3 produced a single, sharp boundary in each case. The mobilities of toxin (Y at these pH values were 6.02 x 10p5, 2.94 X 10B5, and 2.84 X

TABLE I

Amino acid composition of toxin a

Amino acid From analysiP

-- g/100 g protein

Lysine ................ Histidine ............. Arginine ............. Aspartic acid. ........ Threonine ............. Serine ................ Glutamic acid. ........ Proline. .............. Glycine. .............. Alanine ............... S-Carboxymethyl-

cysteine ............. Valine ................ Methionine ........... Isoleucine. ............ Leucine, .............. Tyrosine .............. Phenylalanine ......... Tryptophan. .......... Amide NH3. ......... Water .................

10.64

3.52 8.27

11.02 9.22” 4.806

11.85 4.96 3.89 0.0

16.47 7.5 1.52 1.1 0.0 0.0 4.50 2.9 1.64 1.1 1.74 0.8 0.0 0.0 2.55c 1.2n

0.25c

Total. ............... 96.83 61 -

residues/ residzres/ molecrrle ?nolecule

6.1 6 1.9 2 3.9 4 7.0 7 6.7 7 4.1 4 6.7 7 3.7 4 5.0 5 0.0 0

- I

a Average or extrapolated values from duplicate analyses of 24-, 4%, and 72-hour hydrolysates. Values as residues per mole- cule are based on a molecular weight of 7307 for the reduced and S-carboxymethylated toxin.

* Obtained by extrapolation to zero hours of hydrolysis. e Theoretical value. d Determined on an alkaline hydrolysate.

by guest on May 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Issue of August 10, 1969 D. P. Bates and D. J. Strydom 4151

low5 cm2 set+ volt?, respectively. A single zone was also found on polyacrylamide gel electrophoresis at pH 4.3.

Biological Activity

Toxin (Y has a subcutaneous LD60 of 0.105 pg per g of body weight (white mouse).

No phosphodiesterase, RNase, DNase, phosphomonoesterase, ATPase, protease, acetylcholinesterase, or phospholipase A ac- tivity could be shown in preparations of the pure toxin.

Amino Acid Composition

The results of the amino acid analyses are given in Table I. The values for serine and threonine were obtained by extrapola- tion to zero hours of hydrolysis. Half-cystine was determined as S-carboxymethylcysteine. Tryptophan was estimated on al- kaline hydrolysates. No S-carboxymethylcysteine could be de- tected in hydrolysates of the unreduced, carboxymethylated neu- rotoxin.

End Group Analysis

The application of the phenylthiohydantoin method in con- junction with the dimethylaminonaphthalenesulfonyl method on the toxin gave leucine as the amino-terminal amino acid. Analysis of the residual peptide confirmed that leucine is the amino-terminal amino acid, as only 0.03 residue of leucine re- mained.

Fractionation of Chymotryptic and Tryptic Digests

The chromatograms obtained for the fractionation of the chymotryptic and tryptic digests are shown in Figs. 4 and 5, re- spectively. In both cases the composition of the peptides ac- counted for the entire composition of the reduced and S-carboxy- methylated toxin. The amino acid compositions of the reduced and S-carboxymethylated neurotoxin and the chymotryptic pep- tides are given in Table II, and that of the tryptic peptides in Table III. All the chymotryptic peptides could be used for se- quence studies without further purification, as were all the tryp- tic peptides except T-4, which contained impurities, and T-5 and

PERCENTAGE OF GRADIENT - FIG. 4. Chromatography of an 8-hour chymotryptic digest of

the reduced and S-carboxymethylated toxin on DEAE-cellulose (DE-32). The digest (10 pmoles) was applied to a column (150 X 1.9 cm) of DEAE-cellulose and eluted with a linear gradient of 6 liters at a flow rate of 200 ml per hour. The starting and limiting buffers were at pH 9.3 and pH 4.0, respectively. Their composi- tion is described under “Experimental Procedure.” The effluent was monitored by a Technicon Auto-Analyzer, as described under “Experimental Procedure.”

PERCENTAGE OF GRADIENT - FIG. 5. Chromatography of a a-hour tryptic digest of the re-

duced and S-carboxymethylated toxin (20 pmoles) on DEAE- cellulose DE-32. Conditions were as in Fig. 1. The limiting buffer was 0.045 M pyridinium acetate at pH 4.0.

T-6, which were eluted together. Peptide T-4 was purified by paper chromatography in Solvent II, and Peptides T-5 and T-6 were separated by paper electrophoresis at pH 4.5.

Amino Acid Sequence of Chymotryptic Peptides

Peptide C-l (Residues 1 to 7): Leu-Glx-Cys(Cm)-(His,Asx, Gh, Gh)-The presence in this peptide of the unique leucyl resi- due of the toxin places Peptide C-l at the amino terminus. Ed- man degradation established the amino-terminal sequence Leu- Glx-Cys(Cm).

Peptide C-d (Residues 8 to 24): Ser-Ser-Gln-PrePro-Thr-Thr- Lys-Thr-(Cys(Cm),Pro,Gly,Glx,Thr,Asx,Cys(Cm))-Tyr (Ta- ble IV)-Five steps of Edman degradation established the se- quence at the amino terminus of the peptide. From chymotryp- tic specificity, tyrosine was placed at the carboxyl terminus. Digestion of C-2 with 1 y0 trypsin for 1 hour yielded two peptides. Peptide C&-T1 contains lysine which, from tryptic specificity, was placed at the carboxyl terminus, establishing the sequence of the two tryptic peptides of C-2 as Tl-Tz. The composition of &-TI, together with the amino-terminal sequence of Peptide C-2, es- tablished the full sequence of Peptide C&-T1 as Ser-Ser-Gln- Pro-Pro-Thr-Thr-Lys, the two threonyl residues being placed by difference. The amino terminus of Peptide C&T2 was threo- nine, giving the partial sequence of Peptide C-2 as shown above.

Peptide C-la (Residues 1 to 24): Leu-Glx-Cys(Cm)-(His, Asx, Glx, Glx, Ser, Ser, Glx, Pro, Pro, Thr, Thr, Lys, Thr, Cys(Cm), Pro, Gly, Glx, Thr, Asx, Cys(Cm))-Tyr-No sequence studies were performed on this 24-residue peptide, but its unique com- position provided an overlap between Peptides C-l and C-2, since it contains the only leucyl and tyrosyl residues in the molecule.

Peptide C-S (Residues 25 to B): Lys-LysArg-Trp-This pep- tide was Ehrlich-positive. Tryptophan was placed carboxyl- terminal from chymotryptic specificity. Lysine was found to be amino-terminal by the dimethylaminonaphthalenesulfonyl pro- cedure. An analysis of the residue after two degradative steps yielded only arginine. This established the sequence Lys-Lys- Arg-Trp.

Peptide C-4 (Residues 29 to 46): Arg-Asx-His-Arg-Gly-Ser- [Ile-Thr-Glu-Arg-Gly- Cys(Cm) - (GZy , Cys(Cm) , Pro, Ser) /Val- Lys] (Table V)-Digestion of this peptide with 1% thermolysin for 1 hour yielded three peptides. The specificity of thermolysin allowed CII-TL to be placed at the amino terminus, since it did not contain isoleucine or valine. The sequence of the three ther- molysin peptides of Peptide C-4 was therefore TLI-(TLt,TLs).

by guest on May 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

4152 Neurofoxin a from Egyptian Cobra T’erzom. I Vol. 244, So. 15

T.IIILE 11

Amino acid composition of reduced and S-carboxymethylaled neuroloxin and chymolr~plic peptitles

Amino acid Seurotoxin C-l 1

c-2 C-la /

c-3 I

C-4 I

C-5 -

Lysine. ........................ Histidine. .................. Arginine. .......................... S-Carbosymethylcysteine ......... Aspartic acid. ................... Threonine ......................... Serine .......................... Glutamic acid.. .................. Proline. ...................... Glycine, .......................... T-aline. ........................... Isolertcine ....................... Leltcine ........................... Tyrosine ........................... Tryptophan ........................

6.1 (6) 1.9 (2) 3.9 (4) 7.5 (8) 7.0 (7) 6.7 (7) 4.1 (4) G.7 (7) 3.7 (4) 5.0 (5) 1.1 (1) 2.9 (3) 1.1 (1) 0.8 (1) 1.2 (1)

1.05 (1)

0.76 (1) 0.96 (1)

1.08 (1) 1.00 (1)

1.95

1.06

(2)

(1)

(1)’

1.05 (1) 0.97 (1) 2.97 (3) 1.84 (2) 0.95 (1) 1.01 (1) 1.86 (2) 0.99 (1) 1.10 (1) 3.02 (3) 0.93 (1) 1.00 (1)

1.94 (2)

3.08 (3)

1.82 (2) 2.70 (3) 0.98 (1) 1.94 (2) 3.92 (4) 3.94 (4) 1.87 (2) 1.97 (2) 2.08 (2) 5.13 (5) 3.08 (3) 3.30 (3) 1.02 (1) 0.96 (1)

2.64 (3) 4.00 (4) 1.94 (2)

1.06 (1)

1.04 (1)

1.93 (2) 0.94 (1)

0.94 (1) 0.99 (1) 0.92 (1)

Total .............................. 61

Yield (7c). ........................

7 17 24 4 15 15

43 41 32 78 68 62

Mobility (cm) Electrophoresis at pH

1.9b ........................... 1.5b ........................... tj.5c ..........................

Chromatography in Solvent I ............................ II .........................

+11.1 +8.9 +9.2 +27.0 f13.9 +11.5 0 -Smear -Smear +11.0 +3.8 -Smear

-4.3 -3.8 -4.7 +14.9 +o.s -7.5

0.7 0 0 0.3 0 0 8.0 G.6 3.5 15.2 5.0 6.0

Color with ninhydrin-collidine .... Blue Gray Blue Blue Blue Blue

a l+Jhrlich-positive. b At 100 volts per cm for 20 min. c At 100 volts per cm for 30 min.

Digestion of Peptide C4-TL1 Jvith 1c;O trypsin for 2 hours yielded two peptides. The first step of the Edman degradation on Peptide Ch-TL1-TI yielded a basic DSS-amino acid which could not be identified unambiguously. The second step, how- ever, yielded DSS-aspartic acid. A1rginine was placed carboxyl- terminal from tryptic specificity. Dilute acid hydrolysis with 0.03 s I-IV1 for 18 hours at 110” (27) yielded free nspartic acid and free arginine, as shown by amino acid analysis. The seyuence Arg-Ass-His-kg was therefore established for Peptide Cd- TLI-Tl. Since glycine was the amino-terminal residue of Pep- tide CI-TL,-T,, and since T?, from tryptic specificity, had t,o be at the carboxyl terminus of C,-TL,, the complete sequence of Peptide C’?-TLI became apparent.

Three steps of the Edman degradation established the amino- terminal sequence of Peptide Cd-TL2 as Ile-Thr-Glx. Digestion of Peptide C4-TL2 with 1 yc trypsin for 2 hours yielded two pep- tides. Peptide Ch-TL2-T,, containing arginine, had to be the amino-terminal fragment of Peptide C,-TL2 from tryptic specific- ity. Since the amino-terminal sequence of Cd-TLn was already known, the complete sequence of the neutral peptide Cd-TL2-T1 is Ile-Thr-Glu-hrg. Edman degradation on Peptide Cd-TLz-Tz established the amino-terminal sequence Gly-Cys(Cm).

The sequence of Peptide Ck-TL3 must be Val-Lys, since valine

was found to be amino-terminal. The foregoing studies estab- lished the partial sequence of Peptide C-4 as given above.

Peptide C-5 (Residues 4’7 to 61): LysGly-Ile-Glu-Ile-A sn- Cys(C?~~)~Cys(C?~z)-Thr-Thr-~lsp-Lys-Cys(C1^IL)~~lS~--I1S1Z (Ta- ble VI)--Five steps of the Edman degradation established the amino-terminal sequence Lys-Gly-Ile-Glu-Ile. Tryptic diges- tion of this peptide (1 y0 trypsin for 2 hours) failed to effect a split at, any of the lysine residues. Digestion with 37, thermolysin for 22 hours produced three peptides, C5-TL1, TL2, and TLI. The sequence Lys-Gly was found for Peptide CS-TL1, which places it at the amino-terminal end of Peptide C-5. Since C&,- TL2 was foulid to be acidic at pII 6.5 and had isoleucine in the amino-terminal position, the sequence of this peptide was Ile- Glu. Although pure as judged by the chromatographic and clectrophoretic criteria, on amino acid analysis Peptide C5-TL3 was found to be a mixture of the two peptides, C5-TL3 and TLs,, as judged from the contents of glutamic acid and isoleucine. Two steps of t,he Edman degradation confirmed this when DNS- isoleucine was found before the first step and both DNS-glutamic acid and DSS-:lspnrtic acid were identified before the second step.

Digestion of Peptide C-5 with papain produced several pep- tides, of which four were purified. The unique composition of

by guest on May 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Issue of August 10, 1969 D. P. Botes and D. J. Xtrydom 4153

.knino acid T-l T-2 T-3 T-4 T-5 T-7 T-8

Lysine ..................... Histidine. ................... Arginiiie. ................... S-Carbosymethylcysteine. ... Aspartic arid ................ Threonine .................. berine .................... Glutamic acid. .............. Proline. .................... Glycine. ................... Valine ...................... Isoleucine .................. Letlcilie. .................... T?-rosine Tryptophan. ................

1.07 (1) 0.92 (1)

1.10 (1) 1.02 (1) 1.92 (1) 1.95 (2) 4.06 (4) 2.17 (2)

1.04 (1) 0.91 (1)

1.09 (1) 1.00 (1

_-

)

1

1.11 (1) 1.92 (2) 0.95 (1) 1.00 (1)

1.04 (1)

0.97 (1) 2.07 (2) 0.97 (1) 1.96 (2)

1.00 (1) 1.09 (1) 1.04 (1)

2.13 (2)

0.98 (1) 1.02 (1) 1.02 (1)

0.98 (1)

1.02 (1)

1.05 (1)

1.24 (1) 1.92 (2) 0.97 (1)

3.05 (31 3.88 (4) 2.05 (2)

1.09 (1)

1.04 (1)

1.98 (2) 1.01 (1)

0.89 (1)

Total ................... 15

84.9

10

75.0

2

61.6

0.38 (1

2

Yield is). .................. 28.9

3 6 8 15

55.6 37.7 74.8 27.1

hIobili ty (cm) Elertrophoresis at pH

1.9”. .................. 4.5” ................... 6.5~ .....................

Chroma: ography in Solvent I ........................ II .......................

+6.4 +5.0 +18.6 +11.9 +8.5 f8.0 +7.0 +7.0 0 -Smear +15.5 +7.0 +7.0 +1.5 -Smear -Smear

-0.5 -1.5 +18.5 +6.5 +4.0 0 -0.9 -1.7

0.3 4.0 0.9 13.2 1.1 7.6 4.5 0.7 2.5 6.4 7.1 21.6 5.2 13.5 6.5 2.9

Color \vit h ninhydrin-collidim Blue Blue Blue Blue Green-gray Yellow Green Blue

-

TABLE III

Amino acid composition of hyptic peptides

Edman degradation, Steps 1 to 5 DXS-Ser, DNS-Ser, phenylthiohydantoin-Gln, DNS-Pro, DNS-Pro Tryptic peptides Purified by paper chromatography with Solverrt I

T-l Ser 1.97(Z), Glu 1.09(l), Pro 2.20(2), Thr 2.04(2), Lys 0.93(l) [ch (I), 1.51 T-2 Thr 1.88(2), Cys(Cm) 1.58(2), Pro 1.12(l), Gly 1.01(l), Glu 0.99(l), Asp 1.02(l), Tyr 0.91(l)

Edman degradation on T-2, Step I [ch (I), 11.5; yellow-green]

DXS-Thr

-

- a ht 100 volts per cm for 20 min. b i2t 100 volts per cm for 30 min.

TABLE IV

Amino ncid sequence of Peptide C-2 (Residues 8 to 24)

Sequence: Ser-Ser-Gln-Pro-Pro-Thr-Thr-Lvs-Thr-(Cys(Cm),Pro,Gly,Glx,Thr,Asx,Cys(Cm))-Tyr

Peptide C’j-Pi placed it at the amino terminus of Peptide C-5. Edman degradation on C,-P2 established the sequence Ile-Asn- C~a(C’nl)~(lys(Cm)-Thr-Thr. Since this peptide contained iso- leucine, it had to be adjacent to Peptide C&Pi. Peptide C,-P,, ~-as identical with Pz escepb for an additional aspartyl residue, which could therefore be placed carboxyl-terminal to Peptide Cj-l’,. Seutrality of the peptide AspLys (C,-P,) showed this asparty- residue to be present as the free acid. The composition of Cj-Pq placed it, by difference, at the carboxyl-terminal end of Peptide C-5. Edman degradation gave Lys-Cys(Cm)-Asx as the amino-terminal sequence of C5-Ph. Neutrality of this pep- tide at p1-I 6.5 allowed t,he amides to be assigned, so that the com- plete sequence of Peptide Cs-Pd is Lys-Cys(Cm)-Asn-Asn. The

sequence of peptide (‘-5 was therefore established as that given above.

The presence of the sequence Cys(Cm)-Cys(Cm) in Peptide C-5 was confirmed in the native neurotoxin according to the method of Lindley and Haylett (28) with a slight modification.

Native neurot,oxin (0.2 pmole) was performic acid-oxidized and hydrolyzed with constant boiling HCI for 30 min at 110”. The

hydrolysate was subjected to paper electrophoresis at pH 1.9. The peptide which migrated almost twice as far as authentic cysteic acid t.oward the cathode was cut out and eluted. On hy-

drolysis, only cysteic acid was found. The identity of this pep-

tide, yellon--brown with ninhydrin, is therefore dicysteic acid,

by guest on May 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

4154 Neurotoxin (Y from Egyptian Cobra Venom. I Vol. 244, No. 15

TABLE V

Awlino acid sequence of Peptide C-4 (Residues 29 to 46)

Sequence: Arg-Asx-His-Arg-Gly-Ser[Ile-Thr-G1u-Arg-Gly-Cys(Cm)-(Gly,Cys(Cm),Pro,Ser)/Val-Lys]

+--TL1 I I TLz - +-TLa

-Tl- +T,--1 -T~F [c ‘I-,-----+ +-t[Acidl+----+

Thermolysin peptides

TL TLz

Edman degradation on TL2 Steps 1 to 3

TL Edman degradation on TLP, Step 1

Tryptic peptides of TL, T1

Edman degradation onTX, Steps 1 to 2

T2 Edman degradation on Tf, Step 1

Tryptic peptides of TLs

T1 T2

Edman degradation on Tz, Steps 1 to Z

Purified by paper chromatography with Solvent IT Arg 2.03(2), Asp 1.00(l), His 0.86(l), Gly 1.11(l), Ser 1.02(l) [ch (II), 6.51 Ile O.S9(1), Thr 0.96(l), Glu 0.99(l), Arg 1.00(l), Gly 2.05(2), Cys(Cm) 1.80(2), Pro 1.12(l),

Ser 1.13(l) [ch (II), 101 DNS-Ile, DNS-Thr, DNS-Gin

Val 0.97(l), Lys 1.03(l) [ch (II), 161 DNS-Val Purified by paper chromatography with Solvent II Arg 2.01(2), Asp 1.11(l), His O.%(l) [ch (II), 61 DNS- derivative of a basic amino acid, DNS-Asp Gly 1.02(l), Ser 0.98(l) [ch (II), 9.8; yellow] DNS-Gly Purified by paper chromatography with Solvent II Ile 1.07(l), Thr 1.04(l), Glu 1.00(l), .4rg 0.89(l), [ch (II), 16.Sl Gly 2.14(2), Cys(Cm) l.%(2), Pro 0.91(l), Ser 1.14(l) [ch (II), 7.8; yellow] DNS-Gly, DNS-Cys(Cm)

TABLE VI

Amino acid sequence of Peptide C-5 (Residues 47 to 61)

Sequence: Lys-GIy-Tle-G1u-Ile-Asn-Cys(Cm)-Cys(Cm)-Thr-Thr-Asp-Lys-Cys(Cm)-Asn-Asn

+TL+ +TL+j< TL, >

< TL ,

+---PI- c------P?-

I( PZ3 @------P4-

Edman degradation, Steps 1 to 5 Thermolysin peptides

TLI Edman degradation, Step 1

TL2 Edman degradation, Step 1

TLr + TLa

Papain peptides

P, P2

Edman degradation, Steps I to 5 P 23 P3 P4 Edman degradation, Steps 1 to 3

di-DYNS-Lys, DKS-Gly, DNS-Ile, DNS-Glu, DNS-Ile Purified by paper electrophoresis at pH 1.9, 50 volts per cm, 1 hour, and pH 6.5, 100 volts

per cm, 20 min Lys 1.03(l), Gly 0.97(l) [eZ (1.9), 1341 di-DNS-Lys Ile 1.04(l), Glu 0.95(l) [el (1.9), +16; el (6.5), -G] DNS-Ile Ile l.GG (>l), Glu0.68 (<I), Asp3.%(4), Cys(Cm) 3.17(3),Thr2.05(2),Lys 1.01(l) [el (1.9),

f12.51 Purified by paper electrophoresis at pH 1.9 (50 volts per cm, 1 hour) and pH 4.5 (100 volts

per cm, 30 min) and by chromatography in Solvents I and II Lys 1,05(l), Gly 1.01(l), Ile 0.97(l), GluO.97(1) [ch (I), 13.6; el (1.9) +31] Ile 1.00(l), Asp 1.11(l), Cys(Cm) l.%(2), Thr 1.83(2) [ch (I), 13.6; el (1.9) +12] DNS-Ile, phenylthiohydantoin-Asn, DNS-CysiCm), DNS-Cys(Cm), DNS-Thr Ile 0.81(l), Asp 1.99(2), Cys(Cm) 1.70(2), Thr 2.02(2) [el (1.9), +14.1] Asp 1.03(l), Lys 0.97(l) [ch (II), 5.0; eI (4.5), f1.01 Lys 1.00(l), Cys(Cm) 0.97(l), Asp 2.07(2) [ch (I), 1.6; el (6.5), 0; el (1.9), +12.5] di-DNS-Lys, DNS-CJ-s(Cm), DNS-Asp

which could only arise from a Cys-Cys seyuence in the native

neurotoxin.

Amino Acid Sequences of Tryptic Peptides

Peptide T-l (Residues 1 to 15): Leu-Gln-Cys(Cm)-His-dsn-

Gln-Gln-Ser-Ser-Gln-Pro-Pro-Thr-Thr-Lys (Table VII)-This

peptide, from its unique composition, provided further proof for

the overlap between chyrnotryptic peptides C-l and C-2. Digestion with papain yielded several peptides, a number of

which were purified. Peptides T1-P1 and PI, accounted for the first three residues, the sequence of which was established in pep- tide C-l. The neutrality of Peptide T1-P1 allowed the amide to

be assigned. Edman degradation established the sequence of Peptide T,-P2, as His-Ass-Glx-Glx-Ser. Peptide T1-P2h was found to be neutral at pH 6.5, showing all the acidic residues in it to be amidated as follows: Asn-Gln-Gln-Ser. Peptide T1-P2 was found to be identical in composition with P-2a but for an addition of the only carboxymethylcysteine residue in T-l, which therefore provided the overlap between T1-P1, and Pz.

The carboxyl-terminal part of Peptide T-l is obviously identi- cal {Tith Peptide C2-T1, the sequence of Jvhich has already been established. The sequence of Peptide T-l is therefore as given above.

Peptide T-6 (Residues 16 to 25): Thr-Cys(Cm)-Pro-Gly-Glu-

by guest on May 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Issue of August 10, 1969 D. P. Botes and D. J. Strydom 4155

T.WLE VII

Amino acid sequence of Peptide T-l (Residues 1 to 15)

Seqrxnce: Leu-Gl~~-Cys(Cm)-His-Asn-Gln-Gln-Ser-Ser-Glr~-Pro-Pro-Thr-Thr-Lys

-PI-+ P*’ I -I’,- 1 ltP,+

-p’a- ’ TP

zn----+ I --pa, I +---P*ll- 1

-

Papain peptides

PI P1, 1’2

P P:, l’:dman tlegradation, Steps 1 to 4

Prb P,

p,, pr

Plwified by paper chromatography with Solvent II and paper electrophoresis at pH 1.9 (100 volts per cm, 30 min)

Leu 0.91(l), Glu 1.09(l) [ch (II), 30; el (6.5), O] Leu 0.96(l), Glu 1.16(l), Cys(Cm) 0.89(l) [ch (II), 211 CJ-s(Cm) 0.50(l), His 0,92(l), Asp 1,08(l), (;lu 2.00(2), Ser 1.12(l) [ch (II), 3.2; el (1.9),

+16.i] His 0.91(l), Asp 1.01(l), Glu 2.11(2), Ser 0.97(l) [ch (II), 7.3; el (1.9), +18] I)XS-His, UNS-Asp, DNS-Glu, IINS-Glu .4sp 0.96(l), Glu 2.10(2), Ser 0.93(l) [ch (II), 7.3; el (6.5), 0; el (1.9), +11.5; yellow] Ser 0.97(l), G1r1 l.OG(l), Pro 1.81(2), Thr 0.97(l) [ch (II), 16; el (6.5), 0; el (1.9), $24; gray-

green] Ser 1,12(l), Gl\r 1.08(l), Pro 2.12(2), Thr 1.80(2) [ch (II), 18.8; el (1.9), +29; gray-green] Lys

T.\BLE VIII

flnlino acid sequence of peplide T-2 (Residue 16 to 25)

Sequence: Thr-Cys(Cm)-Pro-Gly-Glu-Thr-Asn-CysiCm)-Tyr-Lys

EdmaIl degradation, Steps 1 to 7

Papaill prptides PI

Ktlman degradation, Steps 1 to 3 I’?

Edman degradation, Steps 1 to 3

DNS-Thr, I>NS-Cys(Cm), DNS-Pro, DNS-Gly, phenylthiohydantoin-Glu, DNS-Thr, phenylthiohydantoin-Asn

Purified by paper electrophoresis (100 volts per cm) at pH 1.9 (30 min) and 4.5 (25 min) G1r1 0.86(l), Thr 0.86(l), Asp 1.10(l), Cys(Cm) 1.04(l), Tyr 1.05(l), Lys 1.10(l) [el (4.5),

+1.8; el (1.9), +12.5] I)NS-GIII, DNS-Thr, DNS-Asp Asp 0,95(l), Cys(Cm) 0.97(l), Tyr 0.83(l), Lys l.OSil) [el (l.5), +1.8; el (l.O), +14.5] l>NS-Asp, DNS-Cys(Cm), DNS-Tyr

Thr-. tsn-Cys(Cm-Tyr-Lys (Table VZIZ)-This peptide, cow taking the unique tyrosyl residue, is identical in composition with peptide (‘2-T2 but for an additional lysine residue, which had to be carboxyl-terminal from tryptic specificity. The carboxyl- terminal sequence is therefore Tyr-Lys. Edman degradation e>tnblished the amino-terminal sequence Thr-Cys(Cm)-Pro- GIy-Glu-Thl.-Asn. Digestion with papain yielded several pep- tides, two of which were purified. Three steps of Edman degra- dation completed the sequence, since the two pcptides were overlapping, both containing tyrosine and lysine.

Peptide T-3 (Residues 26 and 27): I;ys-.lrg-One step of Edman degradation established the sequence Lys-Arg.

Pepfide T-4 (Residues 28 altd $9) Trp-ilrg-This peptide was Ehrlich-positive. Arginine was placed at the carbosyl terminus from tryptic specificity, giving the sequence Trpkg. Peptide T-4 provided an overlap for Peptides C-3 and C-4.

Peptide T-i; (Residues SO to 32): Asp-His-drg-Step orle of the Edman degradation yielded phenylthiohydantoin aspartic acid. .kginine was placed carbosyl-terminal from tryptic specificity, which ekblished the sequence .\sp-His-Arg.

Peptide T-6 (Residues SS to 58): Gly-Ser-Ile-Thr-Glu--4rg- The unique composit,ion of this peptide provided the overlap for the thermolysin peptides CI-TL1 and TL2.

Peptide T-7 (Residues 39 fo 46): Gly-Cys(Cm-Gly-Cys(Cm)-

Pro-Ser-Val-Lys (Table IX)-Since valine is unique in the chain and is present in Peptide C-4, Peptide T-7 provides an overlap between thermolysin peptides CI-TL2 and TLa. The obvious relationship between Peptides T-7 and Ct-TLz-Tz (see Table V)

Amino acid sequence of Peptide T-7 (Residues 39 to 46)

Sequence: Gly-Cys(Cm)-Gly-Cys(Cm)-Pro-Ser-Val-Lys

Papain peptides Purified by paper electrophoresis at pII 4.5, 100 volts per cm, 25 min

P1 Gly 2.14(2), Cys(Cm) 0.89(l) [el (4.5), -13.51

P2 Cys(Cm) 1.00(l), Pro 1.01(l), Ser 1.03 (l), Val 0.97(l), Lys 0.99(l) [el (4.5),

+11 Edman degradation, DNS-Cys(Cm), DNS-Pro, DNS-Ser,

Steps 1 to 4 DNS-Val

gives Peptide T-7 the amino-terminal sequence Gly-Cys(Cm) and the carboxyl-terminal sequence Val-Lys because of tryptic specificity. Digestion with papain produced two peptides. The composition of Peptide TimI’ established the amino-terminal se- quence of T-7 as Gly+2ys(Cm-Gly. Four steps of the Edman degradation ascertained the sequence of T;-P2 as Cys(Cm-Pro- Ser-Val-Lys, thereby establishing the sequence of Peptide T-7 as given above.

Peptide T-8 (Residues 47 to 61): LysGly-Ile-Glu-Ile-Asn-

Cys(Cm)-Cys(Cm)-Thr-Thr-Asp-LysCys(Cm)-Asn-Asn-Five

steps of the Edman degradation established the amino-terminal sequence Lys-Gly-Ile-Glu-Ile. This peptide is therefore identi- cal with chymotryptic Peptide C-5, since their amino acid com- positions (Tables II and III) and amino-terminal segments cor-

by guest on May 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

I N.h: H2N-Leu-Gln-Cys-Hi~-Asn-Gln-GlnC’Ser- ~er-GPn-~ro-~ro-~hr-~hr- Nn: HZN-Leu-Glu-Cys-His-Asn, G~x,G/x,.%~, Ser, G/x, pm, pm, ~hr, ThI;

20 T C T

N.h Ly~~Thr-Cy~-Pro-Gly-G~u-Thr-A~n-Cy~-Tyr~Ly~~Ly~-Arg?Trp’Arg’

Nn. LYS- Thr-CyS, Pro,Gly, G/x,Thr, As& Cys, Tyr-LyS-Lys-Vol-Trp-Arg-

N.h: At:-His-ArgTLGly-Ser-Ile -Thr-Giu-ArgIGly-z:-Gly-Cys-Pro-Ser-

NIT Asp-His-Arg-Gly-Thr-Ile-Ile-Glu-Arg-Gly-Cys,G/y, Cys, Pro,Thr,

50 N.h Vol - LysC~Lys-Gly-Ile-Glu-I,e-Asn-Cys-Cys-Thr-Thr-Asp-Lys-Cys-

N.n. Val Lys, Pro, Gly,Tle,Lys-Leu-Am-Cys,Cys.Thr, Thr, Asx, Lys-Cys-

N.h: ,“p, -As”-OH

N.n: AW-ASP-OH

FIG. G. Amino acid sequence of A-. haje neurotoxin (N./L.) and partly inferred amino acid sequence of N. nigricollis (X.n). The parts of the N. nigricollis toxin sequence in italics were assigned by similarity to the N. haje toxin sequence.

responded. This tryptic peptide, which does not terminate in lysine or srginine, must, by its composition, be at the carboxyl terminus of the molecule.

Alignment of Peptides

From the sequences of the tryptic and chgmotryptic peptides, it was found possible to derive the complete sequence of the neu- rotoxin. Overlapping peptides were found for all the tryptic and chymotryptic peptides except for the carbosyl-terminal segment of the molecule, where trypsin and chymotrypsin produced the same peptide. However, since it was possible to align all the other peptides, this peptide, by virtue of the fact that it is the only remaining peptide to account for the complete composition of the neurotoxin, had to be carbosyl-terminal (see Fig. 6).

DISCUSSIOX

On the basis of its monodispersc behavior during sedimenta- tion velocity, free boundary electrophoresis, and polyacrylamide gel electrophoresis experiments, the neurotoxin was regarded as being homogeneous. Furthermore, only one amino-terminal amino acid, could be demonstrated (in 977, yield, based on a molecular weight of SSOO), while the complete absence of methio- nine, nlanine, and phenylalanine and the near integral values of all the other amino acids also indicate homogeneity.

The results support a structure for N. huje huje toxin cy of a single polypeptide chain containing 61 amino acid residues and having four intramolecular disulfide cross-links. Confirmation of this comes from the linear amino acid sequence.

Tosin (Y exhibits little tendency to undergo association at neu- tral pH, as is seen from the slight concentration dependence of the sedimentation constant at pH 7.6 (Fig. 2). Gel filtration in 57, acet.ic acid, however, gives rise to an asymmetrical peak having a diffuse front and sharp tail, which indicates a tendency toward concentration-dependent association. Samples of the leading and trailing portions of the peak showed no significant differences in their amino acid compositions.

The use of DEAE-cellulose rather than the conventional Dowex resins proved to be very reliable as an initial separation method for the chymotryptic and tryptic pept,ides. The volatile

buffer system developed by Schroeder and Robberson (16) loi chromatography on Dowes 1 could be adopted without modifi- cation for use on DEAE-cellulose. Since only a two-chamber linear gradient device was used, a rather abrupt change in 111-I around 7.5 n-as observed, ai; expected. This, however, ~-a:: with- out a deleterious effect on the separation of the peptides. L+u% from the fact that most of t,he peptides did not require any fur- ther purification, one of the most attractive features of the method is its rapidity. An elution rate of 200 ml per hour for a co1u11m of dimensions 1.9 x 150 cm could be n~nint:~inetl. This is an order of magnitude higher than the conrcntional flow rates for similar size Dews columns.

Chymotrypsin exhibited the usual sllecificity. Splits were ob- served predominantly at the carboxyl groups of tyrosine :uld tryptophan, the neurotoxin being devoid of phenylalanine. The carbox)- peptide bond of the unique amino-terminal lewyl resi- due WLS not hydrol\-zed. Although the protein contains two histidyl residues, no peptides containing carboxyl-terminal histi- dine were isolated. Cleavage occurred at the carboxyl group of one of the four glutaminyl residues, that at position 7. Scission at the carboxyl group of one of the six lysyl residues was in strik- ing contrast to the normal specificity of chymotrypsin. This split, which occurred between two lys)-1 residues in positions 46 and 47, is similar to the chymotryptic split observed in kangaroo cytochrome c between the lysyl residues in positions 86 and 87 (29), in bovine cytochrome c between the lysyl residues in l)osi- tions 86 and 87, and 87 a11t1 88 (30), and ill the estrncellular nu- clense from Staphylococcus aureus between the lysyl residues in in positions 5 and 6 (31).

Tryptic specificity was observed throughout, esccpt for the lysyl residue at position 58 at which no hydrolysis took place. Eaker and I’orathl reported a 20’;; split in this position for the N. nigricollis neurotosin. Thermolysin effected a split at the amino terminus of valine and isoleucine in I’eptides C-4 and C3. It proved to be very useful in the elucidation of the amino acid sequence of this part of the molecule.

A striking feature of the sequence is the frequency with which a residue is immediately repeated. Kc’0 less than nine such pairs occur, accounting for 18 of t,he 61 residues of the molecule. In fact, the homodipeptides of lysine and threonine occur twice.

The region of the molecule from Residues 23 to 39 i.s particu- larly interesting. Xot only are most of the basic, bulky side chain residues located in this region, but it is also completely free of prolyl and cystingl residues. This uncross-linked loop, pos- sibly projecting outward from the molecule because of its hy- drol)hilic character, is the only region in the molecule where, potentiall\-, a considerable degree of a-helical structure could be present. Also noteworthy is the extremely basic nature of this region of the molerule. All four arginyl residues, two of the six lysyl residues, and one of the two histidines are present in this region.

The only other snake venom neurotoxin which has as yet been studied in some detail is that. from the black neck cobra, J. nigricollis.' The amino acid sequences of N. haje and S. nigri- co&s neurotosins are compared ill Fig. 6. The sequence of the portions of the N. nigricollis neurotoxin in italics has not been determined but, assuming identity of structure and minimal variability between the two neurotosins, has been assigned on the basis of the sequence of the N. haje ncurotosin.

It is evident that on this basis a remarkable degree of simi- larity between the two ncurotosins exists. Disregarding the

by guest on May 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Issue of Auglst 10, 1969 D. I’. Botcs and D. J. Strydm 4157

glut;~tllinc-glutatllic :rcifl diffcre1lce in l)ositioi1 2, which could

have resulted fro111 seco11dary dcarnidation, the two neurotosins are itlrntical from the amino terminus to position 26 and also in thf% cahosyl terminal sequences from positions 52 to 61. In the rcgio11 f~o111 b)ositioas 27 t,o 51, seve1I suhstitutio11s ale ob-

served, four of which are radical. All substitutions fxel)t those at positio11s 2T (kg ----f Val) and 47 (LJ-s + Pro) can be csplainrd in tcrmh of :I single base replacement in the coding tril)let.

The possible participation of any as yet invariant. part of the molecule in the biological function or the significance of the wri- a1It l)ositio11s will hare to await the elucidat,ion of the sequences of other probably homologous neurotoxins, preferably of species in other genera where more substitutions nre likely to be found.

dc~nowledgl,~ents-~~~,‘e wish to thank Dr. F. ,J. Joubert for his constructive advice during the course of this work, ;\Ir. T. r\;.

van der \Valt for the execution of the ultracelltrifllgatioii esperi- merits, Mr. ,J. S. Taljaard for the quantitative amino acid xx& yses, and ?\Ir. AI. A. C. Uurns for pcrformiup the Tiselius elec- trophoresis experiments.

1.

2. 3. 4.

REFERENCES

KrlRLSSON, b:., EaI<ER, I>. L., .ZND POR.ITH, J., I<i0chi,,l. ni0- phys. Ada, 127, 505 (1966).

SaSAKI, T., Yakz~gak?~ Zasshi, ‘77, 845, 848 (1957). TAMIYA, N., ,\ND ARAI, H., Biochem. J., 99, 624 (196G). T~MIYA, N., ARAI, II., AND SATO, S., in F. 11:. RUSSEI, AND I’. 11.

S.~~XDERS (Editors), Animal torins, Pergamon Press, New York, 1967, ,. 249.

HCHXVEHT, G. W., AND T.~KEN~IU, Y., Biochim. Biophys. Acta, 16, 570 (1955).

I121~~~.\~.l, B., MoRIK~~~, I., T.\G.~~A, K., ANI) OKUNUKI, K., Biochem. Prep., 6, 1 (1958).

CRES’L’FIELD, A. M., MOORE, S., reed STEIS, W. II., J. BioZ. Chem., 238, G22 (1963).

BOMAN, H. CT., END K.~LETTA, U., Biochim. Biophys. Acta, 24, 619 (1957).

9.

10. 11.

12.

13.

14.

15.

16.

17. 18.

19. 20.

21. 22. 23. 24.

25.

26.

27.

28. 29.

30.

31.

KAPLAN, H. S., AND HEPPEL, I,. A., J. Bd. Chem., 222, 007 (1956).

KUNITZ, M., J. Gen. Physiol., 30, 291 (1947). BJ~RK, W., AND BOMAN, H. (i., Biochim. Biophys. Acta, 34,

503 (1959). ~‘LANDERATH, K., Thin-layer chromatography, Academic Press,

New York, 1964, p. 192. I:ICH~RDS, E. G., AND SCHACHRI.\N, II. K., J. Phys. Chews., 63.

1578 (1959). COHN, E. J., AND EDSALL, J. T., Proteins, umino acids and

peptides, Reinhold Publishing Corporation, New York, 1943, p. 370.

I~EISFELD, R. A., LEWIS, U. .J., .~ND WILLIA~MS, D. E., i\:uture, 195, 281 (1962).

SCHROEDER, W. A., AND I~OIIIOGRSON, B., Anal. Chen~., 37, 1583 (1965).

I~EINDEL, F., AND Hopes, W., Chsm. Be?., 87, 1103 (1954). ~IAILKLAND, F. S., KREIL, (;., I~II~AI~E~u-~)~~~.~s, B., .\KD

S~~ITH, 15. L., J. Biol. Chem., 241, 4642 (1966). ~<.\sLEY, C. W., Biochim. Biophys. Acla, 107, 386 (1965). S,.\XGER, F., AND THOMPSON, E. 0. I’., Biochim. Biophys. Ada,

71, 468 (1963). I~OIIEL, E. J., Anal. Biochewz., 18, 406 (1967). I<:DM~N, P., AND BEGG, G., EUT. .I. Biochewz., 1, 80 (1907). (:u.\Y, W. R., AND HARTLEY, B. S., Niocheaz. J., 89, 379 (1963). Gnau, W. It., in S. P. COLO~ICIC .~ND N. 0. K.\PL.~~ (Editors),

dfethods in enzymology, Vol. 11, Academic Press, New York, 1967, p. 139.

MORSE, L)., AND HORECKER, I<. I,., Anal. Biochcwb., 14, 429 (1966).

BREXNER, M., NIEDERWIESER, A., AND P.~T.~KI, G., Expcri- e&u, 17, 145 (1961).

LIGHT, A., in S. P. COLOWICK .ZND N. 0. K~PL.G (Editors), Methods in enzymology, Vol. 11, Academic Press, New York, 1967, p. 417.

LINDLEY, H., AND HAYLETT, T., J. Mol. Biol., 30, 63 (1967). NOLAN, C., AND MARGOLIASH, E., J. Biol. Chews., 241, 1049

(1966). NAKASHIMA, T., HIGA, H., MATSU~~R~, II., BENSON, A. II.,

.~ND YASUNORU, K. T., J. Biol. C&m., 241, 1166 (196(i). TANIUCHI, H., ANFINSEN, C. B., AND SODJA, A., J. Biol. Chem.,

242,4752 (1967).

by guest on May 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from

D. P. Botes and D. J. StrydomSEQUENCE

PURIFICATION, PROPERTIES, AND COMPLETE AMINO ACID ) Venom : I.Naja haje haje, from Egyptian Cobra (αA Neurotoxin, Toxin

1969, 244:4147-4157.J. Biol. Chem. 

  http://www.jbc.org/content/244/15/4147Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/244/15/4147.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on May 23, 2018

http://ww

w.jbc.org/

Dow

nloaded from