Isolation of the Principal Neurotoxins of Two Naja Naja Subspecies - Karlsson - 2005 - European Journal of Biochemistry - Wiley Online Library

7/28/2019 Isolation of the Principal Neurotoxins of Two Naja Naja Subspecies - Karlsson - 2005 - European Journal of Biochemistry - Wiley Online Library

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Eur. J. Biochem. 21 (1971) 1-16

Isolation of the Principal Neurotoxins of Two Naja nuja SubspeciesEvert KARLSSON,enrik ARNBERC,nd David EAKER

Biokemiska Institutionen, Uppsala Universitet(ReceivedJuly 25,1970/April26,1971)

The principal neurotoxins of two commercial (Miami Serpentarium) preparations of venomfrom Na ja naja siamensis, or Thailand cobra, and Naja naja naja, or spectacled Indian cobra,are isolated rapidly and simply by ion exchange chromatography on shor t (20-30 cm) columnsof Bio-Rex 70 (or IRC-50) in volatile ammonium acetate buffers, followed by gel filtration onSephadex G-50.The Na ja naja siamensis preparation contains a single principal neurotoxin that accountsfor about one-fourth of th e weight of the lyophilized crude venom, or one-third of the total venomprotein. The Na ja naja nuja venom preparation contains approximately equal amounts of twoprincipal neurotoxins which together account for more than one-fourth of the total venomprotein.The three principal neurotoxins contain 71 amino acids in a single peptide chain cross-linkedby five disulfide bridges. The two lNaja naja naja neurotoxins appear to differ only by a serinelisoleucine substitution, while both appear t o differ from the principal Na ja naja siamensis toxinonly by two additional replacements: arginine for lysine and glycine for alanine.Three minor neurotoxic components th at are present a t levels of 1O l i o or less in the Naja nujasiamensis venom are also described. One of these is a 61 amino acid toxin containing four disulfidebridges and two residues of tryptophan. The other two contain 62 amino acids and four disulfidebridges. One of the latt er toxins appears to be nearly identical to the cobrotoxin of Naja najaatra venom.

Within the last few years, small basic proteinsexhibiting peripheral, curare-like neurotoxic ac-tivity have been isolated in pure form from the ve-noms of several species of cobra [l-71 and seasnakes of the genus L a t i c a h [8,9]. These curari-form neurotoxins are characterized by minimumlethal doses of 75-150 pg/kg in albino mice, and onthis basis appear t o be roughly an order of magnitudemore toxic than any other substance yet discoveredin the venoms from which they have been isolated.The work described in the references cited aboveindicates that the principal neurotoxic components inall of the several African and single island (Na ja najaatra) cobra venom studied contain 61 or 62 aminoacids in a single peptide chain cross-linked by fourdisulfide bridges an d are structurally homologous tothe Laticauda toxins.On the other hand, more than 90Q/, f all fatal orotherwise serious cobra bites involving human vic-tims occur in the Indian subcontinent and the main-land of Southest Asia, and no neurotoxins from any ofthe several Naja naja subspecies tha t predominate in

Enzymes. Carbovypeptidase A (EC 3.4.2.1); a-chymo-trypsin (EC 3.4.4.5); phospholipase A (E C 3.1.1.4); ribo-nuclease A (E C 2.7.7.16).1 Eur. J. Bioehem., Vo1.21

the latter regions have yet been distinctly charac-terized.I n the course of screening more than two dozenpreparations of cobra venoms from different sourcesby gradient chromatographic procedures similar tothat first described for Hemachatus hemachatesvenom [2], we observed that N aja naja venomsoriginating from Thailand, Cambodia, and variousregions of the Indian subcontinent differed strikinglyfrom the African (Hemachatus hemachates, Na ja haje,and Na ja nigricollis) and Iranian (N aja naja oxiana)venoms studied with regard to the overall chroma-tographic profile and, more importan t, the structuraland immunological character of the principal curari-form toxic components. All of the neurotoxic com-ponents isolated in our early screening work were sub-jected to immunological testing by Professor PaulBoquet of the Pasteur Inst itut e at Garches, who alsokindly supplied the Cambodian N aja naja venomfrom which the distinctly new F; antigen [lo]was first isolated. The N aja naja neurotoxins ofthe Faantigenic type were found to contain 71 aminoacids and five disulfide bridges. The F, toxins showedno immunological cross-reaction with the smallertype of neurotoxin isolated earlier [l], but using


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2 Naja w j a Neurotoxins Eur. J. Biochem.

purified material supplied by us, Boquet [lo] wasable to demonstrate the presence of small amountsof F,-type antigens in a number of African cobravenoms. Toxins containing 71 amino acids and five&sulfide bridges have recently been isolated fromNaja nivea [4] and Naja haje [6] venoms, wherethey occur a t very low levels compared t o the prin-cipal toxins of the 61 amino acid type.Although in all of the N aja naja venoms initi-ally studied the principal neurotoxins (whichgenerally accounted for more than loo/,of the weightof the respective crude lyophilized venoms) invariab-ly were of the 71 amino acid type, we observed a fewdiscrete differences in amino acid composition whichindicated that we were dealing with at least twodistinct genetic lines. However, all of these variouspreparations were designated only as Na ja naja,and it was not possible, afterwards, t o obtain specificinformation regarding the particular snakes fromwhich the venoms were taken.During the autumn of 1968 we discussed our re-sults with Mr. William Haast, the director of theMiami Serpentarium in Florida, who has had longpractical experience with the difficult Naja najaclassification problem in connection with the com-mercial production of defined venoms. Mr. Haastkindly gave us six different preparations obtainedfrom Naja naja snakes that differed either with re-spect to physical appearance (e.g. , morphology, hoodmarkings, or coloration) or geographic origin. Twoof the samples corresponded to large commerciallots obtained from (a) monocellate Naja naja sia-mensis cobras originating from Thailand, and (b)spec-tacled Naja naja naja snakes from India. For threevery practical reasons, we chose to describe here theisolation and characterization of the principal curari-form toxins of these lat ter two venom preparations:(a) these particular venom preparations are availableto anyone. (b) The principal neurotoxins are presentin such large amounts that the term principalcan be used without ambiguity. The Naja naja sia-mensis preparation is the richest source of an indi-vidual neurotoxin that we have yet encounteredamong cobra venoms. The principal toxin accountsfor one-third of the total protein content of thevenom, and is, in fact, by far the principal proteinconstituent as well. The two Naja naja naja toxinsdescribed are also conspicuous, each representingabout one-sixth of the total venom protein. (c) Bothof the principal Naja naja naja toxins are differentfrom the single principal Naja naja siamensis toxin,indicating a sharp genetic separation that correlateswith the distinct physical differences between thesnakes. Furthermore, these three neurotoxins col-lectively display all of the differences in amino acidcomposition tha t we have observed among the prin-cipal neurotoxins of all the Naja naja venomsstudied.

MATERIALS AND METHODSVenoms

The venoms of the monocellate cobra Naja najasiamensis (regular type, lot no. NSlS) and the spec-tacled Indian cobra Naja naja naja (lot no. 1234F)were obtained in t he lyophilized form from the MiamiSerpentarium Laboratories (Miami, Florida 33156,U.S.A.).

Ion-Exchange ChromatographyPreparation and In itia l Equilibration of the Resin.For use in the chromatography, the polycarboxylicacrylic-type cation exchange resin Bio-Rex 70 ,minus 400 mesh (sodium form, Bio-Rad cat. no.45 180) was settled repeatedly from 10-volume por-tions of tap water (1 lb of resin gives about 400 mlof packed bed in the initial overnight settling) untildecanted free of particles sedimenting slower than10 cm/h. Following removal of the fines the resin

was converted to the acid form by treatment with a10-volumeportion of 1 M HC1, washed with 10-volumeportions of water, 1 M acetic acid, water again, andthen converted to the ammonium form by treatmentwith 10 volumes of 2 M ammonia. Following a washwith distilled water, the resin was suspended in 6volumes of 0.20M ammonium acetate and understirring tit rated down to the desired pH, in this casepH 6.50, by addition of 0.20 M acetic acid. After sett-ling, the resin was suspended in 6 volumes of 0.20Mammonium acetate buffer, pH 6.50, and the pH ad-justed as before. This process was repeated, usuallytwo or three times, with fresh portions of bufferuntil the resin no longer altered the pH of the addedbuffer as measured af ter a t least 30 min of contactwith the resin, indicating that the resin was in com-plete equilibrium with 0.20M ammonium ion a t p H6.50.

On the basis of the ammonia content as deter-mined by quanti tative ninhydrin analysis of materialfrom freshly opened containers, commercial reagentgrade ammonium acetate (Merck, Darmstadt) isonly 93-95O/, ammonium aceta te.pl l of the ammo-nium acetate solutions used in the chromatographywere therefore prepared by appropriate dilution of5 M stock solutions (e.g., 410 g 94O/, ammonium ace-tate per liter) which were first standardized by nin-hydrin analysis at 10000-fold dilution. According toour measurements, the conductivities at 20 C ofneutral ammonium acetate solutions having totalammonia concentrations of 0.10 and 0.20M are8.90 an d 16.7 kohm-lcm-l, respectively. At 24 C,the corresponding values are 9.80 and 18.2kohm-1.

Columns. The ion exchange chromatography andgel filtration runs were done in glass or Perspex chro-matographic tubes closed at both ends by adjustableplungers fitted with porous Vyon disks [ill . The ion


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Vo1.21, No. 1,1971 E. KARLSSON,. ARNBBRG,nd D. EARER 3

exchange columns we repacked under flow from afunnel or extension tube using a 1 3 slurry of resin inthe 0.20 M ammonium acetate equilibration bufferof pH 6.50. When th e packing was complete the upperplunger was inserted and pressed firmly into the resinsurface, compressing th e bed 3-5O/,. This was doneto ensure that no dead space was present within thecolumn initially or developed in the gradient runbefore the ammonium ion concentration reached

Gradient Chromatography. For application to theion exchange column, the venom was dissolved in0.09M ammonium acetate to a concentration ofabout 5O/, (wlv). The Miami Serpentarium venomsstudied contain very little insoluble material, but thesolutions were centrifuged for 30min a t about2OOOOxg to remove a fine turbidity. With regardto the gradient separations described herein, columnsof the very fine (d, less than 35 pm) Bio-Rex-70 resinused can be charged with as much as 200 mg of venomper square centimeter of bed cross-section. A columndiameter of 3.2 cm (8 cmz) s thus used for 0.5-1.5 gof venom, while a 2-cm column (3.14 cmz) is con-venient for smaller samples. The rapid equilibrationtime and even packing afforded by the use of theminus 400 mesh resin fraction permits operationof the columns a t flow rates up to 15mlxh-l xcm-lwithout excessive band broadening. The 3.2 cmcolumns were routinely operated a t 80 mlxh-l,and the 2 cm columns at 40mlxh-l. The venom samp-les in 0.09 M acetate were applied to t he closed-bed ionexchange columns by means of a peristaltic pump.Prior to applying the sample, I4o I2ed volume of0.09 M ammonium acetate was pumped into the GO -lumn to displace the 0.20M equilibration bufferfrom the upper pa rt of the resin bed. Following appli-cation of the sample, elution was continued with0.09M ammonium acetate until all the weakly ad-sorbed components had emerged. Elution was thenbegun with a concave gradient of 0.14 M us . 1.40 Mammonium acetate. In the case of the gradient runsdescribed herein, the gradients were formed by meansof a Beckman Model 131proportionating pump fittedwith a standard concave program cam (Beckman partno. 324812). When the 3.2 cm bore columns wereused, the gear ratio was chosen such that one cycleof the gradient cam corresponds to a total deliveredvolume of 2 liters. Equivalent results are obtainedwith 2 cm bore columns using a 1 iter gradient.Since the Beckman gradient pump described aboveis not a common piece of equipment we should mentionhere th at with regard to the toxin separations describ-ed herein, the gradient provided by the Beckman324812 cam can be satisfactorily approximated by asimple two-cylinder device [12] wherein the mixingor output cylinder has twice the diameter ( i . e . ,fourtimes the cross-sectional area) of the other containingthe terminal buffer.

0.3-0.4 M.

1

The ion-exchange runs were monitored with aBeckman Model 130 Spectrochrome Analyzer pro-grammed for continuous strip-char t registration ofthe pH, conductivity, and absorbance of the columneffluent a t 260, 280, and 290 nm.Re-Equilibration of the Resin. The 1.40M ammo-nium acetate used as the terminal buffer in the gra-

dient runs with whole venoms seems adequate to dis-place all reversibly adsorbed protein from the resin,since no further release of protein was observed uponelution with 2 M ammonium acetate adjusted topH 11 with ammonia. Following elution with the1.40 M acetate the resin can therefore be used againwithout further washing.However, the enormous buffer capacity of the resinin the region pH 5.5-7.5 renders it impractical to tryto re-equilibrate the packed column with the stand-ard 0.20 M equilibration buffer of pH 6.50. Therefore,the resin was extruded from the column in the 1.40 Macetate and stirred with 5 bed volumes of distilled

water t o reduce the ammonium ion concentration toabout 0.2 M and the pH adjusted ;Go6.50 with 0.20 Macetic acid. The resin was then brought to completeequilibrium by successive treatment with 6-volumeportions of buffer as described above. If required,upward adjustment of the p H was done with 0.2 Mammonia.After 5 g-scale runs with crude venom, the resinwas thoroughly cleaned by cycling through the acidand ammonium forms. !To: avoid all possibility ofbackground contamination, th e final rechromatog-raphy of individual fractions, particularly the smal-lest ones, was done on fully regenerated resin.

Recovery of the Xeprated ProteinsThe separated fractions were recovered from thevolatile ammonium acetate buffers by lyophilizationin flat polyethylene refrigerator boxes covered byporous filter paper. To minimize losses of materialand the risk of allergic sensitization the transferringof dry material was avoided as much as possible.Therefore, the quanti tative transfer of material fromthe bulky containers used in the initial lyophilizationto smaller, accurately tared vessels more suitable forweighing and storage was done with small portionsof 0.01 M ammonium acetate. All of the neurotoxicfractions and most of the others were readily solublein the latter medium, but a centrifugation step wassometimes introduced here to remove all traces oflint and other particulate contamination. The frac-tions were then lyophilized again over citric acid andKOH. Convenient vessels for the second lyophili-zation and storage were wide-mouthed brown glasstab let jars of 30, 50, 75, or 100 ml capacity. Duringthe lyophilization the jars were covered with lintlessnylon bolting cloth (Monyl,50 micron mesh, Ziiricher


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4 Naja naja Neurotoxins Eur. J. Uiochem.Beuteltuchfabrik A.G., Ziirich, Switzerland) to pre-vent losses by entrainment.

Am ino Acid AnalysisFor amino acid analysis, samples were hydrolyzeda t 110 "C with 6.0HC1 (prepared by dilution of Merck

Suprapur HC1 with glass-distilled water) in thoroughlyevacuated (less than 5 pm Hg a t - 6 "C) and sealedPyrex ignition tubes. Norleucine was included asinternal standard "131.The hydrolysates were ana-lyzed by ion exchange chromatography as describedelsewhere[14].Carboxypeptidase digests were ana-lyzed using the lithium cit rate system of Benson et al.1151, n which glutamine and asparagine are resolved.Ultraviolet Abso rptio n

Ultraviolet spectra were run in distilled watercontaining 0.5 pmole/ml of norleucine, and the pro-tein concentration was determined subsequently bytotal amino acid analysis. Corrections for scatteringwere made by linear extrapolation of the absorbancesa t 380, 370, nd 360 nm.

Estimations of Molecular WeightsAnalytical ultracentrifugations were done withthe Spinco Model E instrument in 0.20M K2HP04-KH,PO,, pH 7.3, ontaining 0.4M NaC1. The densityat 20 "C was 1.0335gxcm-3. Part ial specific volumeswere calculated from amino acid composition data .Molecular weights were also estimated from gelfiltration dat a obtained with a 1.93x73.2 cm columnof Sephadex G-50 ( V t=214ml, V o=69.5& 0.3ml,

V i =110 ml) using 0.2M ammonium acetate aseluant a t a flow rate of 17 5 1.0ml/h. The distribu-tion coefficient, K D=(V e - V, ) /V i , was determinedas follows: V , was obtained a s the elution volume V ,of human y-globulin. Vi was calculated as the productof the water regain and the dry weight of the gel inthe column. The elution volume V e was obtained asV:,-0.5 'V,-Vd, where V , is the total volume ofeffluent collected from the star t of sample applicationto the apex of the peak, V s is the sample volume,which was 5.0 ml in all cases, and Vd is the total deadspace in the lines outside the column.The calibration data for the column are given inTable 1. The molecular weight values used for oc-chy-

motrypsin, ribonuclease A, and the Naja nigricollistoxin ct [5] ere calculated from sequence dat a. A plotof -log Kd vs. Mila[I61 gave a good straight linedescribed by the equation -log Kd =-0.16, as obtained by the method of least squares.The column used for these measurements will bereferred to hereafter as the calibrated G-50 column.Estimation of Free Sulfhydryl Groups

To 2.0mg samples of protein dissolved in 1.0mlof 0.20M (Na+) sodium phosphate buffer of pH 7.3was added 0.20ml each of 10 mM Ellman's reagent(5,5'-dithiobis-(2-nitrobenzoiccid)) [17] nd 25 mMEDTA (disodium salt) in the same buffer. The ab-sorbance a t 412nm was measured after the solutionhad stood for 1h a t rom temperature. The principalneurotoxin of the Naja naja s iamensis venom wasalso tested for free sulfhydryl groups with iodoaceticacid as described elsewhere [l].Reduction and Alkylation

The reduced and S-carboxymethylated derivativesof the toxins were prepared as described earlier [l].Sequential Degradation

Serial degradation by the direct Edman methodand the subsequent spectrophotometric estimationand thin-layer chromatographic identification of thcphenylthiohydantoin derivatives of amino acids wasdone essentially as described by Iwanaga et al. [18].Carboxy l-Terminal Residues

The C-termini of the three principal neurotoxinswere determined by hydrazinolysis of the intactmolecules under nitrogen in stoppered test tubes for24 h a t 80 "C, essentially according to Fraenkel-Conrat and Tsung [191.The C-terminal sequence of the minor componentNaja naja s iamensis toxin 7C was determined withcarboxypeptidase A as follows. To 0.1 pmole of thereduced and S-carboxymethylated toxin in 1O mlof0.2M NH,HCO, containing 0.2 pmole of norleucinewas added 0.96 mg of DFP-treated carboxypeptidaseA (Sigma) in 0.1 n12M NH,HCO,. After incubationfor 24h a t 37 C the solution was acidified with glacial

Table 1. Calibration data for analytical G-50 columnEquationof data: - og K a =10-3 Mrsls- .1 6Substance V. Ku -log Ru JI, M2ia

mla-Chymotrypsin bovine) 91.7 0.202 0.695 25200 86 0Ribonuclease A (bovine pancreatic) 112.6 0.392 0.407 13683 572Neurotoxin3 from Na ja na ja siamensis 132.7 0.575 0.240 7 820 38 4Neurotoxin a rom Naj a nigr iwl l is 140.4 0.645 0.190 6 786 358


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Vol.21, No.l,1971 E. KARLSSON,. ARNBERU,nd D. EAKER 5

acetic acid, shaken gently for a few minutes to liberateth e CO,, and lyophilized. The dry residue was takenup in 0.2M lithium c itra te buffer of pH 2.2 [la], theinsoluble material removed by centrifugation, andaliquots were analyzed directly using automatic aminoacid analyzers equipped with 6.6 mm or 15mmcuvets.Immunoelectrophoresis

Immunoelectrophoretic analysis of the principalNaja naja siamensis toxin was done at t he PasteurInstitute in Garches as described by Jouannet [20].Toxicity Assays

Toxicity was assayed by intravenous injection(cau-dal vein) into female white mice weighing 20-25 g.The injections were done in 0.1 or 0.2 ml of 0.9O/,NaC1. I n the initial screening of the chromatographicfractions the assay solutions were prepared, by weight,from the lyophilized material without allowance formoisture content. I n the case of the three principalneurotoxins and the minor component Naja najasiamensis to 7C, the concentrations were estab-lished accurately by spectrophotometry, using thepre-determined molar absorptivity values. I n thedetermination of LD,,, doses a t least three mice wereused a t each dose level.Phospholipase A

Assays of phospholipase A activity were done bytitration of fatty acids liberated from egg yolk asdescribed by Dole and Meinertz [21].Hemolysis Assa y of Direct Lytic Activity

Samples of the lyophilized venom fractions weredissolved in 0.90/, NaCl a t a concentration of 1 mg/ml.250 pl of each solution was mixed with 1.0 ml of asuspension of thoroughly wased human erythrocytesin 0.90/, saline buffered a t p H 7.4 with sodium phos-phate. The mixtures were then shaken gently for 5 h

Table 2. Recovery a d ome properties of Naja naja siamensisf ructionsThe fractions correspond to those shown in Fig.1Fraction Lyophilized Percentage H ~ ~ ~ -hospho-

product of venom LD1oo lysis lipaseAactivit.v

ABC1 and 235678910

mg17761283250

(200)13121211266

~

1

19.96.83.20.328.1(22.5)1.51.31.30.10.129.9

pg/kg mouse>3000>3000>3000>1000100

200>3000200>1000>1000x 2000

~

155150000

...

......50

+++0000

...

......0Total 824 92.5Crude

(774) (86.9)venom 890 275 +

a t 37 C. Following dilution with 3.0 ml of bufferand centrifugation the absorbance a t 540 nm wasmeasured against the appropriate blank. Three orfour tests were done in parallel with each sample.The initial erythrocyte concentration was chosen togive an absorbance of about 0.8 a t tota l hemolysis, asobt,ained by dilution with 3.0mlof water rather thanbuffered saline. The degree of hemolysis observed withthe venom fractions is expressed on a percentagebasis. The values given in Table 2 have been roundedoff to the nearest 5O/,.RESULTS

Naja naja siamensis VENOMThe chromatographic profile showninFig. 1 s typi-cal of results obtained in seven gram-scale runsdone with venom from the NS1S lot over a period of

A B C

Effluent ( m l )Eig.1. Gradient chromatography of 890mg Naja naja sia-mensis venom on a 3 . 2 ~ 2 2 . 5m column of Bio-Rex 70 at80 ml x h-1 as described in Methods. The solid line is the ab-sorbance a t 280 nm recorded from a flow cuvet of I0 mm opti-cal path, and the dashed curve shown for peak 3 is the tracerecorded from the tandem flow cell of 2.5 mm path length.Downard deflections of the conductivity (-.---) and pH(----) traces correspond to increases in the conductivity andpH, which are therefore seen to increase and decrease, re-

spectively, as the gradient proceeds. Zero on the volume scalecorrespondsto the s tar t of sample application, and the arrowindicates the initia tion of th e gradient. Zero on the conduc-tivity trace indicates the breakthrough of the 0.14 M bufferin the effluent and 50 indicates the mid-point (1 liter) of thegradient. The sudden jump in the conductivity trace a t abou t1730 ml is due to switching the meter to another measuringrange. The numbers 6.7,6.3, and 5.9 on the pH trace indicatethe effluent pH


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6 Naja m ja Neurotoxins Eur. 3. Biochem.

two years. The material balance for the run and somerelevant activities of the various fractions are givenin Table 2. The mouse assay data are based on theuncorrected weights of the lyophilized fractions, sincethe purpose here is only to locate fractions havinglethal doses a t least as low as tha t of the crude venom.At the dose levels indicated, none of the fractions A,B, C, 1 , 2 , 6 , 8 ,or 9 caused any visible distress in mice.Fraction 4was not tested.

Fractions A, B, and C contain phospholipase Aactivity, as evidenced by the liberation of fat ty acidsfrom egg yolk emulsions, bu t only fraction C shows asignificant tendency to hemolyze washed human ery-throcytes. Fractions A-C contain, in addition to pro-tein, most of the low molecular weight non-proteinconstituents of the venom, and fractions B and Chave absorption maxima at 260 nm, rather than inthe 278-280nm region observed with the otherfractions. The substances responsible for the highabsorbance a t 260 nm and other small non-proteinconstituents can be removed in a prior gel filtrationstep, and we know from experiments done with gel-filtered venom that the proteins eluting in the A-Cregion are also non-lethal at the highest dose level(3000pg/kg) used in the mouse assays of the compositefractions.Fraction 6 was inert in the three assays used.When chromatographed on the calibrated (2-50 co-lumn, this material eluted in a single symmetricalpeak with a Ka of 0.480,which upon insertion into theequation of Table 1 indicates a molecular weight of17400.Fraction 10, which was deliberately displacedfrom the column by an abrupt stepwise change to the1.4 M terminal buffer, is resolved into a single peakfollowed by a double peak if the gradient is allowedto continue, but re-chromatography of the wholefraction in various gradients on resin equilibratedinitially with 0.2 M ammonium acetate at p H 7.3,rather than p H 6.5, indicates the presence of a t leastfour principal components and a few very minor ones.The whole complex fraction 10 is devoid of phospho-lipase A activity, but causes extensive lysis of washedhuman red cells under the conditions described inthe Methods section. The moderate lethality of themixture appears to be due to to cardiotoxic action.Tests done elsewhere [21a] indimte that the mosttoxic (intravenous mouse LD,,, about I000 pg/kg)of the several subfractions th at we have obtained byre-chromatography of no. 10 is closely related to t hecardiotoxin y isolated from Naja nigricollis venomby Izard et al. [22] with regard to both activity andimmunological properties. Since neither fraction 10nor any of the several subfractions that we have ob-tained therefrom exhibit the type of neurotoxic activ-ity with which we are concerned herein, the additionalfractionation obtained upon continuation of thegradient is of no interest in the present context. The

sharp displacement step is therefore used here simplyto shorten the chromatogram, which would otherwiseextend to 2600 ml.The curariform neurotoxicity of the Naja naja sia-mensis venom thus appears to be confined to the peaks3, 5, and 7, which elute in the gradient run a t ammo-nium ion concentrations between 0.14and 0.3M. These

fractions account for 67O/, of the LD,,, units appliedto the column.Protein Recovery

As determined by total amino acid analysis ofmaterial from freshly opened bottles, the NSlS lotof Naja naja siamensis venom supplied by the MiamiSerpentarium Laboratories is 78 f O/, protein. Toobtain an accurate estimate of the total recovery ofprotein from the gradient column, 10Opl aliquotswere taken from each of the 220 fractions (fractionvolume 10.0 ml) collected in a run done with 0.5 gof venom. To the pooled aliquots thus representing1.Oo/ , of the total column effluent was added5.0 pmoles of norleucine and the solution was lyo-philized and subjected to total amino acid analysis.The amino acids thus determined corresponded to3.50 mg of protein, compared to a value of 3.98 mgobtained in similar fashion with a 1.00/, aliquot ofthe solution applied to the column. The latter valueindicates a protein content of 79.6O/, for the crudevenom, and 88 of the applied protein is recoveredin the column effluent. A similar value (92.50/,) forthe total material balance is obtained by summationof the weights oft he lyophilized fractions, as indicatedin Table 2. The good agreement is somewhat fortui-tous, however, since for the reason given below theweights of many of the lyophilized fractions are notstrictly comparable to that of the crude venom.

The values given in parenthesis for fraction 3 inTable 2 indicate the true protein content of t>he rac-tion as determined by amino acid analysis, which ac-counts for only 80/, of the weight (measured afterat least 12 h of equilibration with the laboratory air)of the material obtained after lyophilization of thefraction as described in the Methods section. Valuesof 80-8850/, protein are typical for preparations ofthis and related basic venom proteins, such as, forexample, the Naja nigricollis toxin a [I] and theHemachatus hemachatus toxins 3 and 5, and 12 [2]described earlier, upon lyophilization from ammoniumacetate, and apply also for the several other toxinsdescribed in this paper. Although these very basicproteins have isoelectric points i n the range of pH 9 to11 , such lyophilized preparations invariably give al-most perfect1y neutral solutions when dissolved inde-ionized water, indicating that the proteins arepresent in the form of acetate salts. Since all of theproteins eluting a t ammonium ion concentrations of0.14 M or greater in the gradient runs contain about


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Vo1.21, No.1, 1971 E.KARLSSON,. ARNBERG,nd D. EAKER 7

1.5 mequiv. of cationic groups per gram, completelyequivalent salts would contain about 7/, acetate.We know from the results obtained upon direct ana-lysis of l mg samples on the short column of the aminoacid analyzer, without prior acid hydrolysis, that thelyophilized preparations typically contain only,0.1-0.5/0 of free ammonia. If the acetate prep-arations are subjected to high vacuum (less than5 pm Hg) for two days over P,O, or are dialyzedagainst distilled water prior to the lyophilization step,the material obtained after equilibration with theaverage ambient humidity conditions is 88-90/,protein, most of the remaining 10-120/, apparentlybeing moisture.

The principal NeurotoxinFraction 3, which extends from 600 to 760 ml andhas a maximum a t 680 ml in the chromatogram shownin Fig. 1, accounts for 94O/, of the neurotoxic activitydetected in the column effluent and 29O/, of the total

protein content of the crude venom. The principalneurotoxin is obtained in pure form from fraction 3by gel filtration, as illustrated by the chromatogramshown in Fig.2 for an analytical sca.lerun done on thecalibrated G;--50column. I n round figures, the fourfractions TII I, TI I, T, and C account for 1,3,94,and20/,, respectively, of the protein applied to the column.The main component T eluting with a Kd of 0.575 isthe monomeric form of the principal neurotoxin.The retarded component C is an entirely differentneurotoxin, and is described below under MinorNeurotoxic Components. The components TI11 andTII, but not C, reappear if the principal componentT is lyophilized and run again. On the other hand,only peaks T and C are observed when a n aliquot ofthe ion-exchange fraction 3 is gel filtered directlywithout an intermediate lyophilization step. TI1 hasthe same amino acid compositions as T, and peakscorresponding to TI1 and the fully active monomer T

415 1

I !

Fraction no.Fig.2. Gel filtration on the calibrated G-50 column (Table 1in Methods) of 14 mg lyophilized material from fraction 3 ofthe gradient run shown in lGg.1. Elution was with 0.20Mammonium acetate at 16.8 mlxh-l

are obtained in a ratio of about 5 : when TI 1 is gelfiltered again after standing for three days a t roomtemperature in 0.2 M ammonium acetate a t p H 7.3.TI11 has no t been analyzed. According to the equationof Table 1 the Kd-ValUeS observed for the componentsTI1 (Kd=0.347)and TI11 (Kd=0.215) correspondto molecular weights of 15400 and 23800, respective-ly. On the basis of this and the other evidence givenabove we conclude that fraction TI1 consists entirelyof dimers of the principal toxin; TI11 is apparentlytrimers. The dimers and higher aggregates are un-doubtedly generated in the lyophilization step, par-ticularly if hawing occurs. The maximum dimer con-te nt observed with samples lyophilized from ammoni-um acetate as described in the Methods section hasbeen loo/,.

Ximplified Purification ProcedureThe first step in the purification of the principalNaja naja siamensis toxin was direct chromatographyof the crude venom on Bio-Rex 70 (or AmberliteIRC-50). A 3.2x20-25 cm bed of the ion exchangerwas adequate for a t least 1.5 g of venom, and if oneis not interested in separating the substances thatelute after fraction 3 in the gradient run, stepwiseelution can be used instead of th e gradient illustratedin Fig. 1.The sta rting conditions and init ial operationswere exactly a s described for the gradient procedurein the Methods section. Following application of thesample, the weably adsorbed constituents of thevenom were eluted with two bed volumes ( e . g .400 mlfor a 3.2x25 cm column) of 0.09 M ammonium ace-tate. The principal toxin emerged in a symmetrical

peak with a maximum at about 1.5 bed volumesafter the breakthrough of the 0.14 M acetate. Whenthree bed volumes of the 0.14 M solution had passedinto the column, the eluant was changed to 1.40Mammonium acetate to displace the remainder of thevenom proteins from the column. With regard tothe preparation of the principal neurotoxin, the step-wise elution method was completely equivalent tothe gradient procedure, and had the advantage th atspecial closed-bed columns and elaborate gradient-forming devices were not required. When the step-wise elution procedure was used, the presence of adead space above the resin bed did not affect theoutcome of the run if the dead volume liquid waswithdrawn and replaced with the new eluant a t eachstepwise change. If closed-bed columns were not used,a disk of thin filter paper was placed atop the resinbed to guard against disturbances of the surface layerduring the sample application or changing of theeluant.The second and final step, gel filtration on Sepha-dex G-50 in 0.20M ammonium acetate, was requiredonly to remove the minor neurotoxic contaminant Cand the small amounts of dimers and trimers generat-


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8 Naja m ja Neurotoxins Eur. J. Biochem.

Table 3. Amino acid composition of the principal Naja najasiamensis neurotoxinIn column 1, all values based on more than five determina-tions are given with standard deviations, otherwise theaverage deviation is given. The values in columns 2 and 3were obtained with 160 pg samples hydrolyzed n the absence(col.2) and presence (col.3) of lo/, henol (see text)Amount in toxin

Amino acid 1 2 3

TryptophanLysineHistidineAmide NH,ArginineAspartic acidThreonineSerineGlutamic acidProlineGlycineAlanineHalf-cystine

(as CM-cysteine)ValineMethionineIsoleucineLeucineTyrosinePhenylalanine

residues

5.01 5 0.070.96 f .055.17 f 0.504.99 f .069.02 5 0.108.96 & 0.152.98 f .141.00 0.026.04 f .254.00 f .032.99 & 0.03....

,/mole(1)a ...5 4.901 0.975 ...5 4.949 9.169 8.923 3.081 1.006 6.164 3.953 2.9610 8.06

10.00 f .12c3.96 f 0.016 4 3.870.00 0 0.004.96 & 0.02d 5 4.620.98 f .01 1 1.030.91 f .07e 1 0.712.96 f 0.10 3 2.92Total residues 71Formula weight =7820Molar absorptivity at 279 nm =8300 f 100(1 mg/ml)=1.06

...4.820.984.829.138.912.960.9s6.243.912.965.41

...

3.830.004.581.030.992.92

P From absorbance data.Three determinations.Two detorminations24 h hydrolysis.d Two determinations (7 2 h values only ).0 Five determinations (24 h values only).

ed in the lyophilization of the ion exchange fraction.A 3.2x 100 cm column operated at 20 mlxh-1handled the 340 mg of toxin (about 400 mg of lyo-philized material) obtained from 1.5 g of venom.Amino Acid Composition

With the exceptions indicated, the amino aciddata given in column 1 of Table 3 for the principalNaja naja siamensisneurotoxin are averages of valuesfrom a total of seven analyses (five 24 h hydrolysates,two of which were done with the reduced and X-car-boxymethylated derivative, and two 72 h hydro-lysates) done with samples from five different prep-arations of the toxin. The two 72 h hydrolyses weredone in parallel with 24 h hydrolyses of the same prep-arations, and the zero time values obtained forthreonine and serine by extrapolation, assumingzero- and first-order destruction kinetics, respectively,of the data from these pairs of runs are the basis of

the recovery factors (0.95 for threonine and 0.90 forserine) used for these amino acids with the other 24 hhydrolysates.The three samples on which the amide value isbased were gel filtered on a 1.9x20 cm column ofSephadex G-25 in loo/, acetic acid prior to analysis,and aliquots equivalent t o th e volume of the proteinfraction used for analysis were taken from the voidvolume effluent and carried through the entire ana-lysis procedure to serve as ammonia blanks.The value given a t the bottom of Table 3 for themolar absorptivity of the native neurotoxin a t neutralpH was obtained in connection with the amino acidanalyses as described in the Methods section, and isbased on five independent determinations. For neutralsolutions of the toxin,

The values given in column 1 of Table 3 for all theamino acids except tyrosine are very close to integers.The recovery of tyrosine isnot a simple function of thehydrolysis time and declines well below the 90level as the sample size is reduced below about 1 mg.Low recoveries of tyrosine are invariably observedwith ordinary hydrochloric acid hydrolysates ofcystine-rich proteins, and the effect is particularlypronounced with this toxin, where the ratio of half-cystine to tyrosine is 10: 1. Recently, we have inves-tigated the observation of Benisek et al . [23] that thedestructive effect of cystine on tyrosine could be pre-vented by performing the hydrolysis in the presenceof a small amount of phenol. The data given incolumns 2 and 3 of Table 3 were obtained with 24 hhydrolysates of 100 p1 aliquots of a stock solutioncontaining 1 .60mg toxin per ml, as determined byabsorbance measurements. The samples, each con-taining 160 pg or 20.5 nmole of the toxin, were hydro-lyzed with 2.0 ml portions of 6 N HCl, the one (co-lumn 2) lacking phenol, and the other (column 3)containing phenol (Merck, reagent grade) a t a con-centration of 10 mg/ml. The hydrolysates were ana-lyzed with the Bio-Gal BC-200 analyzer using the15 mm microcuvets. The values given in columns 2and 3 were calculated directly from the amino aciddata on the basis of the amount of sample (20.5nmole)taken for analysis ( i . e . , the dimensions are moleobserved/mole of toxin hydrolyzed), and only in th ecase of threonine and serine have corrections (thosementioned above) been applied. The excellent agree-ment between columns 2 and 3 indicate the analyticalprecision attainable in analyses done on the 160 pgscale, and the very close proximity t o integral valuesdemonstrates the mutual accuracy of the analyticalprocedure and the absorption coefficient used todetermine the initial sample concentration. The slight-ly low values for valine and isoleucine are due to in-complete liberation, and are typical for 24 h hydro-lysates of this toxin.The only amino acids affected by the inclusions ofphenol in the hydrolysis medium are cystine and tyro-

(1.0mg/ml) =1.06.


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Vol. 21, No . 1, 1971 E. KARLSSON,. ARNBERC, and D. EAKER 9

sine. In the absence of phenol, the recovery of tyro-sine a t this scale of analysis is only 710/,, and therecovery of half-cystine (determined as cystine) isabout 81/,. I n the presence of phenol the tyrosinerecovery is quantitative, while the recovery of cystineis depressed to about 54O/,. Since half-cystine is bestdetermined as S-carboxymethyl cysteine or cysteicacid anyway, the sacrifice of the cystine da ta is not aserious disadvantage in practice.

Analytical UltracentrifugationTwo equilibrium runs done a t 20410 rev./min bythe short-column method of Yphantis [24] a t initialprotein concentrations of 0.3 and 0.8O/, indicatedmolecular weight values of 8100 and 8000, respec-tively, using a par tial specific volume of 0.71 cm3/gcal-culated from the amino acid composition.

ImmunoelectrophoresisImmunoelectrophoresis of the principal toxinagainst horse anti-Naja naja serum (Pasteur nstitu te,Garches) gave a single sharp precipitin arc, corre-sponding to the F, antigen discussed by Boquet et al.

[10,25].Th e Reducedan d S -Carboxymethylated Derivative

The reduced and S-carboxymethylated derivativeof the principal toxin is inactive and contains 10.000.12 residues of CM-cysteine per molecule, as deter-mined with two different preparations. The molarabsorptivity of this derivative a t 279nm in 100/,acetic acid is 6800,which is very close to the sum (6680)of the molar absorptivities of free tyrosine (1170) andtryptophan (5510)under t he same conditions, as wasalso the case with the Naja nigricollis toxin OL describ-ed earlier [1]. The principal Naja naja siamensisneurotoxin thus contains one residue of tryptophan.

Amino -Termina l SequenceAs determined by identification of the phenyl-thiohydantoin derivatives of amino acids obtainedin 13 stages of degradation of 0.55 pmole of the re-duced and S-carboxymethylated derivative, theamino-terminal sequence of the principal toxin is:H-Ile-Arg-Cys-Phe-11e-Thr-Pro-Asp-Ile-Thr-Ser-Lys-Asp-. Positive identification of residues 1, 5, and 9as isoleucine was accomplished by amino acid ana-lysis after alkaline hydrolysis of the amino acid do-rivatives according t o Van Orden and Carpenter [26].Residues 3, 8, and 13 were checked in the same fa-shion, owing to the very similar behavior of aspar-tate and CM-cystine derivatives in the thin-layerchromatographic systems used. Alkaline hydrolysisof the aspar tate derivative gives aspartic acid ingood yield, whereas CM-cysteine is completely des-

troyed. The yield of isoleucine in the first stage, asdetermined spectrophotometrically, was 1O residuesper mole of the reduced and carboxymethylatedneurotoxin.Carboxyl-Term inal Sequence

Neither carboxypeptidase A nor carboxypepti-dase B liberated any free amino acids from eitherthe reduced and S-carboxymethylated, the reducedand S-carboxamidomethylated, or the per formic acidoxidize derivative of the toxin. However, prolinewas obtained in a yield of 0.8 residues per mole byhydrazinolysis of the reduced and carboxymethyl-ated neurotoxin. The presence of proline a t the C-terminus has been confirmed by the electrophoreticisolation from chymotryptic digests of the per formicacid oxidized toxin of an extremely basic tetrapeptidehaving the sequence:H-Arg-Lys-Arg-Pro-OH.Absence of Free Sul fhy dry l Groups

The native neurotoxin gave no reaction withEllman's reagent, and no trace of CM-cysteine wasdetected in an acid hydrolysate of a sample that hadbeen treated with iodoacetate for 24 h a t -+4 "Cin 8 M urea without prior reduction.MINOR NEUROTOXIC COMPONENTS

OF Naja naja siamensis VENOMThe first minor neurotoxin, which we shall call3C, elutes in fraction 3 of the gradient run (Fig.l),but appears as a separate peak behind the principaltoxin in the subsequent gel filtration step, as illus-

trated in Fig.2. The Kd-value of 0.765 obtained forneurotoxin 3C on the calibrated G-50 column (Tablel)indicates a molecular weight of 4600.Chromatography on the calibrated G-50 columnof the neurotoxic fraction 5 (Fig.1. and Table2)obtained in the gradient run gave a single symmetri-cal protein peak with a Kd of 0.739, indicating a

4

0 20Fraction no.Fig3 Gel filtration on the calibrated B-50 olumn of 1 O m glyophhilized material fr o m fraetion 7 of the gradient run shownin Fig. 1


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10 Naja naja Neurotoxins Eur. J.Biochem.Table 4.Amim acid compositions of min.orNaja naja siamensisneurotoxins

Molar ratios in toxin83 c 5 7 cAmino acid

TryptophanLysineHistidineAmide NH,ArginineAspartic acidThreonineSerineGlutamic acidProlineGlycineAlanineHalf-cystineValineMethionineIsoleucineLeucineTyrosinePhenylalanine

(as CM-cysteine)

(1)s2.96 3 4.931.93 2 1.948.3b 8 ....5.95 6 4.077.32 7 7.9310.0 10 6.423.01 3 3.968.25 8 6.061.79 2 2.847.01 7 4.930.00 0 0.48.... 8 7.188.01 ....1.00 1 1.95c0.00 0 0.000.00 0 1.9oc1.96 2 1.931.94 2 0.820.00 0 0.00

(2)=5 2.022 1.87- 8.9b4 6.988 8.04(6.5) 7.734 3.896 7.063 2.225 6.65(0.5) 0.008 ....7.792 0.940 0.002 1.882 1.001 1.97e0 0.00

47

120

Total residuesFormula weightM, (Sephadex)M, (centrifuge)AbsorptionmaximumMolarabsorptivity

62679346008200

279 nm89001.29150

61687550009600

280 nm13000

1.91100

62698542009500

279 nm90001.29150

From absorbance data .b One determination.C One determination(72 h value only).d One determination-phenol present.e Tw o determinations-phenol present.

molecular weight of 5000. The component obtainedby gel filtration of the gradient fraction 5 shall becalled neurotoxin 5.Chromatography on the calibrated G-50 columnof t he third neurotoxic fraction (no. 7) observed inthe gradient run (Fig.1 and Table 2) gave the patt ernshown in Fig.3. Peak A, which elutes with a K dof 0.544, indicating a molecular weight of 8700, hasnot been investigated. Peak B, which contains neuro-toxic activity, has K d of 0.646, indicating a molec-ular weight of 6600, and has not been studied fur-ther . The component c eluting with a K d of 0.796, in-dicating a molecular weight of 4200, shall be calledneurotoxin 7C.

Analytical UltracentrifugationSedimentation equilibrium analysis of the neuro-toxins 3C, 5, and 7C a t initial concentrations of0.08 to0.10/, was done by the long-column meniscus de-

pletion technique of Chervenka [27]. The centri-fugations were done for 20 h a t rotor speeds of 40000or 48000 rev./min. Linear plots of log c vs. r2 wereobtained with all three toxins. The molecular weightsgiven at the bottom of Table 4 were calculated usinga partial specific volume of 0.71 cm3/g for all threetoxins, as derived from the amino acid compositiondata.

Amino Acid CompositionThe amino acid compositions of the neurotoxins3C, 5, and 7C are given in Table 4. The values for thetoxin 3C are averages from three 24 h hydrolysates:two of the native toxin and one of the reduced andcarboxymethylated derivative. The values for thetoxin 5 are based on one 24 h and one 72 h hydro-lysate of a single preparation. The da ta for toxin 7Care derived from two 24 h hydrolysates of the nativetoxin and one 24 h and one 72 h hydrolysate of thederivative. The molar absorptivities given at t hebottom of the table were determined in connectionwith the amino acid analyses as described in theMethods section.The amino acid compositions of all three toxinsare compatible with molecular weights of about7000. In the case of toxin 3C, th e values for all theamino acids except proline are acceptably close tointegers, aspartic and glutamic acids being marginal.The low proline value is probably due to inaccurateintegration of the very small proline peak obtaineda t the scale on which the analyses of this proteinwere done.In the case of toxin 5, satisfactory values wereobtained for all amino acids except threonine and

alanine. The alanine value of 0.5 certainly cannot beascribed to analytical error, and unless the mole-cule contains 122 rather than 61 amino acids, whichseems improbable, the preparation is definitely in-homogeneous. However, since the threonine valueis very close to 6.5, the simplest explanation of thedata is that the Naja naja siamensis fraction 5 isan approximately equimolar mixture of moleculesdiffering by a discrete threoninelalanine substitution.No traces of either methionine or phenylalanine wereobserved in the analyses.The analytical data for the 7C toxin are satis-factory for all amino acids except glycine and pro-line. The value for glycine (6.65& 0.09) is signifi-cant ly less than 7, and appears to be complementedby a proline value of 2.2. I n the analyses of the re-duced and carboxymethylated derivative, whereinterference from cysteine is ruled out, the samplesize was chosen to give proline peaks very near insize to that obtained (25 nmoles) in the calibrationruns with the 15 mm cuvet system, and on the basisof large numbers of standard runs performed on thelatter scale we would not expect an analytical errorgreater than + 3 O / , . I n th is case, therefore, t he high


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Vol.21, No.1, 1971 E. KILRLSSON,. ARNBERG,nd D. EAKER 11

proline value is probably significant, and might in-dicate that the toxin 7C preparation is an approxim-ately 7:3 mixture of molecules Wering by a dis-crete Gly/Pro substitution.Am ino - and Carboxyl-Terminal Sequencesof Neurotoxin 7C

24 cycles of Edman degradation were done ona sample of the reduced and carboxymethylatedderivative corresponding to 3.0 mg total amino acidresidues. Interpretable results were obtained in thefirst 23 stages, indicating the sequence:10H-Leu-Glu-Cys-His-Asn-Gln-Gln-Ser-Ser-Gln-Thr-

Pro-Thr-Thr-Thr-Gly-Cys-Ser-Gly-Gly-Gln-Thr-Asn-Alkaline hydrolysis was not required to establishthe identity of the amino-terminal residue, since

amino acid analysis of the residual peptide after24 cycles of degradation gave only 0.04 residues ofleucine as compared t o values of 1.0, 2.0 an d 7 .0 forvaline, isoleucine, and arginine, respectively, noneof which had been encountered in the degradation.The yield of leucine in t he first stage was 0.38 pmole;which corresponds to 0.94 residues/mole for thereduced and carboxymethylated derivative (formulaweight 7457) of a molecule having the integralamino acid composition given for the toxin 7C inTable 4. Residue 24 appeared to be CM-cysteine, butowing to an appreciable carry-over of asparaginephenylthiohydantoin from the preceding asparagineresidue, the issue could not be settled by the (nega-tive alkaline hydrolysis approach.To determine the carboxy-terminal residue, thereduced and carboxymethylated derivative of toxin7C was digested with carboxypeptidase A as describ-ed in the Methods section. I n the analysis done withhalf of the digest in the lithium-citrate buffer system[15] asparagine was observed in an amount corres-ponding to 2.0 residues per 7000 molecular weight.Since a tiny peak was observed in the aspartic acidposition, the other half of the digest was analyzedin the ordinary sodium citrate system to distinguishbetween CM-cysteine and aspartic acid, which are

19 23

not resolved in the lithium buffers [ la] . CM-cysteinewas observed in an amount corresponding to 0.05residues per mole, indicating the carboxyl-terminalsequence -Cys-Asn-Asn-OH.Na ja naja naja VENOM

The gradient chromatographic separation of theNaja naja naja venom is illustrated in Fig.4. Thefractions 3 and 4 obtained from the run shown con-tained 100 and 110 mg of protein, respectively, andboth had an LD,,, of 100 pg/kg in the mouse assay.The crude Na ja naja naja venom used has an LD,,,of 300 pg/kg and is 78O/, protein. The fractions 3 and4 together account for 28O/, of the to tal venom proteinand 67O/, of the LD,,, units applied to the column.Fractions 1 , 2, 5, 7, and 8 also exhibit neurotoxicactivity, but have not been investigated further.The further purification of the principal neuro-toxins represented by the gradient fractions 3 and 4has involved two additional steps; namely, gel fil-trat ion on Sephadex-G-50 followed by re-chromatog-raphy on the ion exchange column.The gel filtration step is illustrated in Fig.5 fora run done with the lyophilized gradient fraction 3.The very low void volume peak was due to a slightturbidity and contained a negligible amount of pro-tein. The small peak extending from fraction 15 t o 17consists of dimer and trimer of the toxin, andaccounts for 9 Ilo of the protein applied to the column.The remaining 91O/, of the protein is represented bythe hatched peak. A similar gel filtration pattern wasobtained with the gradient fraction 4, except that atiny peak, probably corresponding to the gradientfraction 5 (see Fig.4), was observed behind the mainpeak. I n both cases, the gel filtration was done in0.09M ammonium acetate, from which the toxinscan be adsorbed directly t o the ion exchange columnused fo r the final purification step.The ion exchange chromatography of both toxinsis done on resin equilibrated with 0.20 M ammoniumacetate at p H 6.50. The pattern shown in Fig.6 wasobtained upon re-chromatography of the gel filteredfraction 3 as described in the legend. The hatchedpeak of Naja naja naja toxin 3 is preceded by atiny peak of unidentified material, and a smallshoulder of toxin 4 is observed on the trailing edge.

Effluent (ml)Fig.4. Gradient chromatography of 995 mg Naja naja naja venom on a 3 . 2 ~ 2 9 . 2m column of Bio-Rex 70 as described inMethods. Explanation of chart record given in legend to Fig.1


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12 Naja naja Neurotoxins Eur. J. Biochem.

t

5 15 25Fraction no .

Fig.5. Gel filtration on a 3.2~ 4 1 . 8m G-50 column of 70 mglyophilized Naja naja naja fraction 3 from run shown in Fig.4.Elution was with 0.09 M ammonium acetate at 30 mlxh-l

"0 200 400Effluent (ml)

Fig.6. Re-chromatography on 3.2x21.0 cm Bio-Rex 70 columnof gel-filtered Naja naja naja toxin 3 (hatched peak in Pig.5) .Resin equilibrated initially with 0.20 M ammonium acetateat pH 6.5. 40ml 0.09M ammonium acetate pumped intocolumn before and after application of sample (about 100 mgprotein) in 60 ml of same medium. Zero on the volume scaleindicates the start of elution with 0.18 M mmonium acetate,the breakthough of the latter in the effluent being indicatedby the conductivity (----) trace

The gel filtered fraction 4was re-chromatographedin the same fashion, except th at the elution was donewith 0.19 M ammonium acetate. The Naja naja najatoxin 4 eluted in a single peak with a small shoulderof toxin 3 on the leading edge.As was the case with the principal Naja najasiarnensis toxin described earlier above, the principalNaja naja naja toxins 3 and 4 are obtained in es-sentially pure form in the initial gradient chromatog-raphy of the crude venom. Gel filtration of the N a j anaja naja fraction 3 does little more than removethe dimers and trimers produced in the interveninglyophilization step, and the final re-chromatographyon Bio-Rex-70 is required only to remove traces oftoxin 4. Gel filtration of Naja na ja na ja fraction 4removes the dimers and trimers and a small amountof the overlapping fraction 5, which would, at anyrate, elute behind the toxin 4 peak in the final ionexchange step. The main function of the rechromatog-raphy is thus to eliminate the cross-contaminationof toxins 3 and 4,which are seen to overlap in the

initial gradient run. The overlap of peaks 3 and 4 sgreatly exaggerated by the logarithmic ordinate scalein Fig.4. he absorbance a t the minimum between thepeaks is only one-fourth that a t the two maxima.The toxins 3 and 4 re completely resolved fromeach other if the initial gradient run is done onresin equilibrated initially with 0.20 M ammoniumacetate a t p H 7.3, using a gradient of 0 .09M vs .1 . 4 0 M ammonium acetate with the same gradientcam (see Methods) as was used for th e pH 6.50 run.Under these conditions, however, component 5 elutessquarely between 3 and 4 and contaminates both.

Am in o Acid CompositionThe amino acid data given in Table 5 for the Na janaja naja toxins 3 and 4were obtained with samplesprepared by the complete three-step procedure de-scribed above. With the exceptions indicated, theaverage values given for for each toxin are based ona total of seven analyses (three 24 h and three 72 hhydrolysates of the native toxin and a 24 h hydro-lysate of the reduced and carboxymethylated de-rivative).

Table 5. Am ino acid compositions of principal Naja naja najaNeurotoxinsAmount in toxinsAmino acid 3 4

TryptophanLysineHistidineAmide NH,ArginineAspartic acidThreonineSerineGlutamic acidProlineGlycineAlanineHalf-cystineValineMethionineIsoleucineLeucineTyrosinePhenvlalanine

(as CM-cysteine)

....4.04f .010.92 f .045.35f .05b5.96 f .029.00f .068.99f .032.92 f .101.10-j=.036.03f .035.02 f .041.99&0.019.78d3.97&0.030.004.93f .131.00f .020.93f .02e3.01+0.05

(11%41569931652

10405113

....4.05f 0.40.93f .034.83fO.llC5.93f .058.97f .069.04 f .073.95f0.121.10 f .066.06 0.184.99 f .042.01 fO.019.91d3.93 f .040.004.03 f .040.99 f .030.93 f .04e2.93&0.05

( l ) a41569941652

10404113Total residues 71 71Formula weight 7834 7807Molar absorp-tivity at 279 nm 8500f 00 8500f 00

100LDlOO mouse) 100a From absorbance data.b Tw o determinations.0 Five determinations.d One determination.e Four determinations (34 h values only, no phenol).


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v01.21, No.1, 1971 E. KARLSSON,. ARNBERO,nd D. EAKER 13

The histidine values for the Na ja naja naja toxins3 and 4 are somewhat lower than the 9601, recoveryobserved with the other toxins described herein,but the difference is probably not signiiicant. Thedat a given in Table 5 are based on hydrolysates donein the absence of phenol, which accounts for the lowtyrosine value of 0.93. Recent analyses done withsamples hydrolyzed in the presence of l o / , phenolgave 1.00 residues of tyrosine. The amide values wereobtained with gel-filtered samples in the mannerdescribed for the principal Naja naja siamensistoxin, and in view of the small average deviationswe have no reason to doubt their reliability. Theglutamic acid value of 1.10 obtained for both toxinsis definitely significant and indicative of some typeof inhomogeneity, since a value of 1.00 was obtainedwith the nearly identical Naja naja siamensis toxin.The data for the other amino acids are very satis-factory.The molar absorptivities a t 279 nm of the reducedand carboxymethylated derivatives of toxins 3 and 4in loo/, acetic acid are 6600 and 6500, respectively,indicating that each molecule contains 1 residue oftryptophan in addition to the tyrosine residue ob-served in the amino acid analyses.Neither toxin gives any reaction with Ellmansreagent, indicating t ha t no free sulfhydryl groups arepresent.

Noleculaar WeightBoth toxins elute from the calibrated G-50column with a Ka of 0.587, indicating a molecularweight of 7700, in good agreement with the formulaweights derived from the amino acid composition

data.Am ino - and Garboxyl-TerminalSequences

As determined with the reduced and carboxy-methylated derivatives in the manner described forthe principal Naja naja siamensis toxin, the Najanaja naja toxins 3 and 4 both have the N-terminal se-quence :H-Ile-Arg-Cys-,and the C-terminal sequence:-Arg-Lys-Arg-Pro-OH.TOXICITY

The very specific and practically irreversiblecurare-like blocking action of the principal Najanuja siamensis toxin 3 at the motor end plates ofthe frog skeletal myoneural junction has been de-monstrated by Lester [28]. On the basis of theirsimilar actions in the mouse assay, we make thereasonable hypothesis that Naja naja nuja toxins3 and 4 and the minor Na ja naja siamensis toxins 3C,5 and 7C are also of the curariform type. At highdoses (e.g. ~ ~ X L D , , , )ll of these toxins produce ageneral flaccid paralysis. A t doses near the I~D,,,

level the conspicuous effect is protracted suffocationin all cases.With regard to the mouse assay, however, thesiamensis toxins 3C and 7C differ rather strikinglyfrom the others with respect to the following fourparameters :LD,,, Dose. For female albino mice weighing

20 g, the monomer forms of the 71 amino acid toxinssiamensis 3, naja 3, and naja 4 and the 61 aminoacid toxin siamensis 5 all show an LD,,, of 2.0 pg,as compared to values of 1.5- 1.8 pg for the Najanigricollis toxin (x [l] and the Hemachatus hemachatestoxins 3 and 5 [2] described earlier. The 62 aminoacid toxins siamensis 3C and 7C have an LD,,, of3 pg. 2 pg will no t kill a mouse and roughly half of theanimals survive 2.5 pg.Th e Latent Period. This is the time between thcinjection of doses a t or very near the LD,,, level andthe development of severe breathing difficulty, atwhich point the animal ceases to move about vol-untarily but will generally move in response to gentleprodding. For the toxins siamensis 3 and 5, naja 3an d 4, and the nigricollis ci and hemachates 3 and 5mentioned above the latent period is about 2 h.At doses of 2 xLD1,, (about 4 g) the latent periodfor these toxins is 30-40 min. For one LD,,, dose(3 pg) of the toxins siamensis 3C and 7C the latentperiod is 20-30 min.The Survival T im e at the LD,,, Dose Level. For allof the above-mentioned toxins except siamensis 3Cand 7C, most of the deaths occur between 2.5 and 6 hfollowing injection of one LD,,, dose, and injectionsof twice the LD,,, dose generally cause death in 50 to70 min, th e 61 and 71 amino acid toxins falling nearerthe lower and upper limits, respectively, of the in-dicated range. One LD,,, dose of toxin siamensis3C or 7C causes death in about 1 h. I n lots of assaysdone with the latter toxins at or slightly below theLD,,, dose level, no mouse has ever died later than70 min following the injection. A dose of 2xLD,,,(6 pg) kills in 25-30 min.The S ymptom Recession Tim e for Slightly Sub-lethal Doses. At dose levels of 0.5 LD,,, none ofthe toxins mentioned above cause any serious dis-tress. Doses in the range of 0.7-0.8 LD,,, cause res-piratory distress of varying severity and fatalitiesoccur ; but roughly half of the animals survive, andwith regard to the time course of the recoveryprocess the toxins siamensis 3C and 7C also differfrom the others. Mice given 2p.g of siamensis3C or 7C become lethargic and show moderate res-piratory distress after 20-30 min, but the symptomsrecede rapidly and the animals exhibit normal be-havior by I h. At doses of 2.5 pg half of the animalsdie, but if the mouse is not dead or very nearly soby 1 h the recovery process will have already begunand will be apparently complete with in anotherhour. I n the case of the other toxins, doses of 0.8


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14 Nuju naja Neurotoxins Eur. J. Biochem.

LD,,, will not produce serious respiratory distressbefore 2-3 h following the injection, and the out-come depends on the severity and duration of thesymptoms. If the symptoms progress to t he point ofimmobilization and severe dyspnea and do not beginto lighten noticably within the nex t hour, the animalusually dies, often several hours later, probably asa secondary result of the prolonged anoxia. Other-wise, considerable improvement is observed beforethe B t h hour and the animal will have recoveredcompletely 3-4 h later.The singularity of the toxins siamensis 3C and 7Cwith regard to the four assay parameters discussedabove clearly indicates that these toxins somehowact considerably faster and more reversibly than theothers.The mouse assay behavior of the siamensis 3dimer(s) (peak TI1 in Fig.2) is also interesting. TheLD1,, doses observed for different preparationshave varied from 8-12 pg for 20 g mice. The la tentperiod for the development of deep lethargy andlabored breathing can be as much as 12 h, and theintoxication may last for two days before the animalfinally dies. We do not know whether the delayedand prolonged action of the dimer preparations re-flects chronic poisoning by monomer arising byslow dissociation of the dimer(s) or whether thedimer(s) itself is toxic.

DISCUSSIONThe principal neurotoxins siamensis 3, naja 3 andnaja 4 f the Na ja naja siamensis an d Na ja naja najavenoms used in the present s tudy contain 71 aminoacids in a single peptide chain cross-linked by five

disulfide bridges. The complete amino acid sequenceof the toxin siamensis 3 has already been elucidatedand will be presented elsewhere when the compara-tive work on the Na ja naja naja toxins is complete.However, to simplify the discussion we shall statehere that the Naja naja naja toxins 3 an d 4 do, infact, differ from the Naja naja siamensis toxin 3 bythe discrete substitutions inferred in the simplestinterpretation of the amino acid composition datagiven in Tables 3 and 5 ; namely, arginine for lysine,glycine for alanine, and serine for isoleucine, whichoccur a t positions 49, 28, and 32, respectively in thelinear sequence. We have not yet encountered anyother differences among the three toxins, all of whichhave the same (five) amide content. The separationof the Naja naja naja toxins 3 and 4 thus appears tobe based only on the replacement of an isoleucineresidue by a residue of serine, which demonstratesthe very high selectivity of the chromatographicsystem used.The isolation procedures described herein aresimple, rapid, and very reproducible. The crudevenoms are chromatographed directly without priorextractions, precipitations, etc., which are in this case

a waste of time and material. The capacity of theion exchange columns is high. Although we havenever run more than 1.5 g of venom, we expect thata 3.2x25 cm column of the fine Bio-Rex 70 (or IRC-50) resin can handle a t least 2 g of the Naja najasiamensis venom, which means that 500mg of theprincipal toxin can be prepared a t one time in abouttwo days without scaling up the apparatus. I n con-nection with the simplified %on-gradient pro-cedure described for the isolation of the siamensis 3toxin, we might mention that the minor component3C elutes in a separate peak ahead of the principaltoxin if the ion exchange column is eluted with five,rather than two, bed volumes of the 0 .09M am-monium acetate before switching to the 0.14Meluant, which means that the principal toxin can beisolated in pure form directly from the crude venomin a single chromatographic operation. However,this nearly doubles the time required for the ion ex-change run, and since a second look a t importantpreparations is always advisable we prefer to use theshorter elution schedule and then separate off th e3C component in the subsequent gel filtration step.A very useful purity criterion for the toxinsiamensis 3 is tha t the ratio of glutamic acid to leu-cine should not be significantly greater than 1.0(e.g., not more than 1.03), the reason being that theminor component 3C is the only likely contaminant.Amino acid analysis of the material recovered fromfraction 3 of the gradient run shown in Fig.l givessatisfactory values for all amino acids except glut-amic acid, which runs a t about 1.14 residues. Thishigh value reflects the approximately 201, contamina-tion with the toxin 3C, which contains 8 residues ofglutamic acid per mole (Table 4). The Naja naja najatoxins 3 an d 4 both show unsatisfactory glutamicacid values of 1.10, but neither gel filtration throughI m columns of Sephadex G-50 nor ion exchangechromatography a t pH 6.5 or 7.3 has revealed any3C-like contaminant or improved the glutamicacid value. I n t he sequence work on these two toxinswe are looking for a 1001, substitution of glutamicacid for aspartic, or of glutamine for some otherneutral amino acid.

The amino acid composition data certainlysuggest a high degree of structural homology be-tween the three principal toxins described aboveand the 71 amino acid neurotoxins isolated recentlyfrom Naja nivea [4] and Naja haje [6] venoms.Although the principal neurotoxins of African cobrasare of the 61 amino acid type, a t least some of thesevenoms thus contain small amounts of 71 amino acidtype toxins that are very similar to the principalneurotoxins of the Naja naja subspecies that pre-dominate in India and continental Southeast Asia.The da ta given for the minor Na ja naja siamensiscomponent 5 indicate that the converse situationalso exists. Toxin 5 contains 61 amino acids and is


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V01.21, No.L,1971 E. KARLSSON,. ARNBERO,and D. EAKER 15

nearly identical in amino acid composition to theprincipal neurotoxin that we have isolated fromNaja naja oxiana venom of Iranian origin (unpub-lished results), which also contains two residues oftryptophan, and which is clearly homologous insequence to the African cobra toxins of the 61 aminoacid type.The minor Naja naja siamensis toxins 3C and 7C,on the other hand, contain 62 amino acids andappear to be more closely related to the cobrotoxinof Naja naja atra [7]. In fac t, the toxin 7C differsin amino acid composition from the cobrotoxin onlyin having one less residue of lysine and one more

of arginine. Furthermore, in the first 23 residues ofthe amino-terminal sequence, toxin 7C differs fromthe cobrotoxin[7] only with respect to residue 21,which is glutamine rather than glutamic acid.Certainly the two molecules are very similar, the onlyfurther difference perhaps being a simple Arg/Lysreplacement.The toxin 3C is always obtained in pure form asa by-product of the purification of the principaltoxin siamensis 3, whether the stepwise or th egradient approach is used. If t he gradient procedureis used, toxins 5 and 7C are also obtainable withoutmuch additional effort. We should also mentionthat all of the toxins siamensis 3, 3C, 5, and 7Coccur in the venom of a single snake.The latter point was established through thecourtesy of Mr. W. E. Haast of the Miami Serpen-tarium Laboratories, who, in connection with alabelling experiment in vivo that will be describedelsewhere, kindly sent us a t weekly intervals all ofthe venom produced by a single male cobra over a

period of 26 weeks. The reduced and carboxymethyl-ated derivative of 7C used for the determination ofthe N-terminal sequence was, in fact , prepared fromthe toxin obtained from the lat ter snake. We do notyet know, however, whether the Naja naja najatoxins 3 and 4 are both produced by a single indi-vidual, or whether they represent two dist inct geneticlines. Some indication that the latter might be thecase is the fact t ha t we have observed in some prep-arations of Naja naja naja venom collected fromsnakes in the Maharashtra region of India, and sentto us by Professor Deoras of the Haffkine Institutein Bombay, only a single principal toxin having theacid composition of naja 4.

I n the gel filtration experiments done on the cali-brated 6-50 column, the principal toxins of bothvenom fall nicely on the regression line determinedby chymotrypsin, ribonuclease A, and the Najanigricollis toxin a 113. The Hemaclmtatus hemachatestoxins 3 and 5 [2] behave exactly as toxin a. Theminor toxins siamensis 3C, 5, and 7C all elute a tpositions corresponding to molecular weights of5000 or less, as determined by the equation ofTable 1.

The 0 . 2 M ammonium acetate used as eluant inthe gel filtration runs seems adequate to eliminateelectrostatic interactions between these basic pro-teins and the small number of fixed negative chargespresent in the gel matrix. All of the toxins eluteslightly later in 0.1 M buffer and become moreretarded as the concentration is reduced further.However, increasing the eluant concentration from0.2 M to 0.5 M has no effect on the elution positions.The tardy elution of the toxin siamensis 5 is probablydue to the presence in the molecule of a secondresidue of tryptophan, which is known to adsorbrather strongly to Sephadex. This toxin acts like anordinary 61 amino acid toxin in the mouse assay.The toxins siamensis 3C and 7C elute even laterthan siamensis 5 on the 6-50 column, and theirmore rapid and reversible action in the mouse assaysuggested that they might really be small. Althoughthe latter possibility seemed to be ruled out by theamino acid data , the issue was settled unequivocallyby the partial sequence analysis of the toxin 7C.We doubt that the late elution of the 62 amino acidtoxins on Sephadex can be due only to the presenceof a second tyrosine residue in the molecules, butmight ra ther reflect a substantial difference in second-ary structure between these molecules and the neuro-toxins of the 61 amino acid type. At any rate, oneclearly should be cautious in the use of gel filtrationfor the estimation of molecular weights. I n the casemoleculesof the size discussed herein, accurate aminoacid da ta will generally answer a t once the questionsof homogeneity and minimum molecular weight.

The investigation was supported by the Swedish NaturalScience Research Council. To Drs. Paul Boquet and MichelJouannet of 1Institut Pasteur, Services de Recherches surles Venins, Garches, France, we are grateful for many yearsof stimulating cooperation. We also acknowledge the experttechnical assistance of Mr. Ragnar Thorzelius and Mr. SvenCentring in the amino acid analyses. To Prof. J. Porath weare grateful for ample laboratory space and for placing atour disposal the Beckman Spectrochrome Analyzer, whichwas purchased on a grant from the Wallenberg Foundation.

REFERENCES1. Karlsson, E., Eaker, D., and Porath, J., Biochim. Bio-2. Porath, J., Mem. In st . Butantan ( X a o Paulo) , 33 (1966)phys. Acta, 127 (1966) 505.379.3. Botes. D. P.. and Strvdom, D. J., J . Biol. Chem. 244.(1969) 4147.4. Botes, D. P., Strydom, A. J. C., and Strydom, D. J. ,2nd Int. Xymp. Animal and Plant Toxins, Tel Aviv1970.5. Eaker, D., and Porath, J., Ja p a n J . Microbiol. 11 (1967)353.6 . Miranda, F., Kupeyan, C., Rochat, H., Rochat, C., andLissitzky, S., Eur. J . Biochem. 17 (1970) 477.7. Yang, C. C., Yang, H. J., and Huang, J. S., Biochim.Biophys. Acta, 188 (1969) 65.8. Tamiya, N., and Arai, H., Biochern. J . 99 (1966) 624.9 . Tamiya, N., Arai, H., and Sato, S., in Animal Toxins(edited by F. E. Russell and P. R. Saunders), Per-gamon, Oxford 1967, p. 249.


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16 E. KARLSSON,. ARNRERC, nd D. EAKER:Va@naja Neurotoxins

10. Boquet, P., 2nd Int. Symp. Animal and Plant Toxins,Tel Aviv 1970.11. Porath, J., and Bennich, H., Arch. Biochem. Biophys.,Suppl. l(1962 ) 152.12. Bock,R. M., and Ling, N. S., Anal. Chem. 26 (1954)1545.13. Walsh, K. A., and Brown, J. R ., Biochim. Biophys. Acta,58 (1962) 596.14. Eaker, D., in Evaluation of Novel Protein Products,Pergamon, Oxford 1970,p. 171.15. Benson, J. V., Gordon, M. J., and Patterson, J. A., Anal.Biochem. 18 (1967) 228.16. HjertBn, S., J. Chromatog.50 (1970) 189.17. Ellman, G. L., Arch. Biochem. Biophys. 82 (1959)70.18. Iwanaga, S., WallBn, P., Grondahl,N. J., Henschen,A.,and Blomback, B., Eur. J . Biochem. 8 (1969) 189.19. Fraenkel-Coilrat, H., and Tsung, C. M., in Methods inEnzymology (edited by C. H. W. Hirs), AcademicPress, New Vorli 1967, Vol. 11, p. 151.

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Isolation of the Principal Neurotoxins of Two Naja Naja Subspecies - Karlsson - 2005 - European Journal of Biochemistry - Wiley Online Library