Snake venom toxins

Biochimica et Biophysica Acta, 359 (1974) 24~247

Y()Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 36784 SNAKE VENOM TOXINS T H E REACTIVITY OF T H E D I S U L P H I D E BONDS OF N A J A N I V E A TOXIN a

DAWIE P. BOTES National Chemical Research Laboratory, Councilfor Scientific and Industrial Research, P.O. Box 395, Pretoria (Republic of South Africa)

(Received February I lth, 1974)

SUMMARY The disulphide bonds of Naja nivea toxin ~I react stoichiometrically with dithioerythritol in the absence of denaturing agents. At an equimolar concentration of dithioerythritol to toxin, one disulphide is cleaved selectively. Alkylation leads to a derivative which retains toxicity and antigenicity. INTRODUCTION The unique conformation of a protein molecule, dictated by intramolecular forces arising from the primary structure [1 ], is stabilized by hydrogen bonding, hydrophobic interactions and, in certain cases, by intrachain disulphide bonds. Reduction of the disulphide bonds of several biologically active substances brings about complete loss of activity, antigenicity and immunogenicity [2-5]. However, not all disulphide bonds in a protein molecule are of equal importance in maintaining the tertiary structure. Thus, partial cleavage of disulphide bonds in several proteins, viz. immunoglobulins, ribonuclease [2, 7] and lysozyme [8] had no significant effect on the biological activity. Whereas selective cleavage could be effected under mild non-denaturing conditions, inclusion of 8 M urea led to essentially complete reduction of the proteins in the studies cited above. On the other hand, only one disulphide bond of papain could be reduced in 8 M urea and complete reduction could only be affected in 6 M guanidine hydrochloride [9]. The reactivity of disulphide bonds towards reducing agents varies considerably, not only from protein to protein but is also dependent on the reducing agent. Mercaptans such as mercaptoethanol, thioglycolic acid and mercaptoethanol amine are usually required in large excess. Dithiothreitol and its isomer dithioerythritol owe their increased effectiveness as reducing agents to the formation of a thermodynamically favoured, six-membered dithiane ring [10]. Using dithiothreitol, quantitative reduction of the two disulphide bonds of human growth hormone [11] and selective cleavage of one of the three disulphide bonds in bovine basic pancreatic trypsin inhibitor [12], in the absence of denaturing agents, have been achieved. Significantly, both the completely reduced growth hormone [11] and the partially reduced trypsin inhibitor [12], after alkylation retained full biological activity.

243 The primary structure as well as the positioning of the disulphide bonds of Naja nivea toxin a has been determined [13]. The present communication describes the reactivity of the disulphide bonds of toxin a towards dithioerythritol and the effect of selective reduction and alkylation on the toxicity. EXPERIMENTAL PROCEDURE N. nivea toxin a was isolated as described previously [14]. Carboxymethyl cellulose (Whatman CM-32) was converted to the ammonium form and equilibrated with starting buffer (0.05 M ammonium acetate at pH 5.5). The columns were developed by linear gradients of ammonium acetate, pH 5.5. 1,4-Dithioerythritol was obtained from Merck. lodo[2-14C]acetic acid, (The Radiochemical Centre, Amersham) was diluted with unlabelled carrier and recrystallized from petroleum ether. Toxin a was dissolved, to a concentration of 1/~mole/ml, in 0.2 M Tris buffer, pH 8.6, containing 1 mg EDTA/ml. To i-ml aliquots of this toxin solution were added graded quantities of dithioerythritol, in the range 1-5/~moles, and the mixtures allowed to react at 0 °C for 90 min. To the five solutions were then added iodoacetic acid, in 10~o excess over the total thiol. The iodoacetic acid solutions were made by dissolving the appropriate quantity in 1 ml of the above Tris buffer. Alkylation was allowed to proceed for 1 h at room temperature in the dark, after which the alkylated derivatives were desalted on Sephadex G-50 columns, equilibrated with 0.05 M NH4HCO3, and freeze-dried. Anaerobic conditions were maintained throughout the reduction and alkylation procedure. Toxicity of the different fractions were assayed subcutaneously in mice weighing 16-18 g. The death rate was recorded after 24 h, and the LDs0 and its 5 ~ fiducial limits calculated by the probit method according to Finney [15]. Gel diffusion was carried out on gels consisting of 1 ~o agarose in 0.9 ~'/NaCI. The alkylated fractions were compared with native toxin by diffusion against enzymepurified multivalent antivenom (courtesy Dr P. A. Christensen, S.A.I.M.R., Johannesburg). After 24 h diffusion the gels were washed in 0.9 ~ NaCI, dried in contact with Whatman No. 3 MM filter paper, and stained with 1 ~ Amido black 10 B solution in 3 ~ aqueous acetic acid. Background colour was removed by washing in 3 ~ aqueous acetic acid. The dialkyl- and tetraalkyl-derivatives were digested with thermolysin and the peptides separated by paper electrophoresis and chromatography essentially as described for the native toxin [13]. Aliquots of peptides were mixed with Bray's solution and counted in a Packard Model 3003 tri-carb scintillation spectrometer. Alkylated derivatives and S-carboxymethylcysteine-containing peptides were hydrolysed with redistilled, constant boiling HCI in evacuated sealed tubes for 24 h at 110 °C. Amino acid analyses were carried out on Beckman 120 B and 120 C automatic analysers. RESULTS Table I reports the degree of alkylation with increasing concentrations of dithioerythritol. The ratio of reducing agent to toxin was varied from equimolar to

244 TABLE I S-CARBOXYMETHYLCYSTEINE CONTENT OF DERIVITIZED TOXIN PREPARATIONS AFTER REDUCTION WITH VARIOUS DITHIOERYTHRITOL CONCENTRATIONS AND SUBSEQUENT ALKYLATION Ratio of dithioerythritol to toxin I:l 2:1 3:1 4:1 5:1

S-carboxymethylcysteine residues/molecule 1.94

3.89 5.81 7.93 9.77

5-fold molar excess. Dithioerythritol was quantitatively utilized in all cases, as judged from the corresponding a m o u n t of S-carboxymethylcysteine formed. The purification of a dialkyl-derivative on CM-cellulose is depicted in Fig. I. The elution profile of native toxin a, under identical chromatographic conditions, is indicated by the broken line. The dialkyl-derivative, being more acidic than the native toxin, is less strongly absorbed on the resin and is well separated from unreacted toxin. Table I[ relates the lethal activity obtained for alkylated derivatives to dithioerythritol concentration, lncluded in the table are the theoretical residual lethal activities, calculated from statistical considerations assuming a r a n d o m attack on the remaining four disulphides, after reduction of the first disulphide bond. An LD,~,0 value of 16 # g / m o u s e was obtained for the purified dialkyl-derivative as compared to the value of 14.7/zg/mouse, found for the unfractionated mixture at equimolar reactant ratio. This slight disparity can be explained in terms of residual native toxin present at equimilar ratio. The amino acid composition of the only carboxymethylcysteine-containing peptides, isolated from a thermolysin digest of the dialkylated-derivative is reported in Table II1. The amino acid compositions are consistent with the expected specificity o f ihermolysin on the known structure o f toxin a.

0.4

--" 0.25

A / i J J IL

t

o

0,2

c~ <~

020

I z

0'15 OqO

J

005 I

0

I000

2000

3000

4000

Eluote Volume (ml)

Fig. 1. Chromatography of the reduced (equimolar ratio of dithioerythritol:toxin) and alkylated derivative on CM-cellulose. Gradient elution from 0.05 M to 0.25 M ammonium acetate buffer at pH 5.5. -- - , elution profile of native toxin a.

245 TABLE II LETHAL ACTIVITY OF VARIOUS ALKYLATED TOXIN PREPARATIONS Dithioerythritol/toxin 1:1 1:1

(Purified dialkyl-derivative (Unfractionated)

2:1 3:1 4:1 5:1 *

LDs0

5 ~ limits

Calculated LDs0

16.0 itg 14.7/~g 50.5/tg 251/~g 3.69 mg N*

19.1-13.8 17.6-12.3 61.4-41.6 301.6-209.4 5.27-2.58

49.6/tg 256/~g 4.0 mg

Non-toxic at a dose of 10 mg.

TABLE Ill A M I N O ACID COMPOSITION OF S-CARBOXYMETHYLCYSTEINE-CONTAINING PEPTIDES OF DIALKYL-DERIVATIVE Amino Acid

Peptide I (residues/molecule)

Aspartic acid S-Carboxymet hylcysteine Glycine Phenylalanine Tryptophan

2.07 (2) 0.87 (1)

Peptide II (residues/molecule) 0.91 (1) 1.08 (1) 0.97 (1)

(1)*

* Ehrlich positive.

A similar digest of the tetraalkyl-derivative resulted in a multiplicity of 14Clabelled peptides indicating random cleavage of the remaining disulphide bonds. The immunodiffusion pattern is presented in Fig. 2. Complete coalescence is found between native toxin and the dialkyl derivative. The precipitin line fi)und for

Fig. 2. Comparison of native toxin a with various alkylated derivatives by immunodiffusion. The centre well contained polyvalent antiserum. The other wells contained toxin a and the derivatised fractions as indicated, a, native toxin a; 1:1, 2:1, 3:1 and 4:1, fractions derived from equimolar, 2:1 etc., ratios of dithioerythritol to toxin.

246 the preparation where dithioerythritol to toxin ratio was 2:1, is attributable to dialkyl derivative that had escaped reduction. DISCUSSION The dependence of lethality of snake venom toxins on the integrity of the disulphide bond is exemplified by the study of Yang [16, 17] on cobrotoxin, which was shown to lose activity when reduced with fl-mercaptoethanol or when oxidized with performic acid. A correlation was found between the rate of appearance of free sulphydryl groups and the decrease in toxicity. However, no attempt was made to show preferential reduction of any particular disulphide bond, or whether the remaining toxicity at any particular concentration of reducing agent was due to the corresponding percentage of unreacted toxin. In the present study it was shown that N. nivea toxin a reacted stoichiometrically with dithioerythritol in the absence of denaturing agents. Samples were alkylated in order to avoid questionable significance of toxicity tests, due to possible reoxidation either during manipulation or even in vivo. The high yield of a dialkyl derivative, obtained by CM-cellulose chromatography, suggested high reactivity of one specific disulphide bond at equimilar concentrations of toxin and dithioerythritol. The isolation of two S-carboxymethylcysteine-containing peptides, the only to be found in a thermolysin digest of the dialkyl-derivative, pin-pointed the reactive disulphide to the one linking half-cystine residues 26 and 30. The significance of this being that this particular disulphide bond comprises the additional loop in toxin ~ as compared to the shorter type of homologous toxin [13]. This finding of high reactivity of the additional disulphide bond seems to corroborate the suggestion that the function of this disulphide bond was to stabilize the extended size of the loop by forming an internal ring structure [13]. The finding that the dialkyl-derivative retains lethal activity with an LD,~0 value of 16 #g/mouse further substantiates the proposed function of this disulphide bond. The higher LD~0 value, as compared to the value of 1.37 #g/mouse for the native toxin [14], suggests a minor conformational change in the proximity of the active site, resulting in a somewhat less effective binding with the receptor. This change however, must be small enough for the derivatized toxin to be able to recognize the receptor site. A suggestion of disulphide interchange of toxin and receptor protein [18], is negated by the finding that the dialkyl-derivate retains toxicity, since the most reactive disulphide bond is the most likely to participate in such an interchange. Concentrations of dithioerythritol to toxin beyond the equimolar ratio leads to random cleavage of the remaining four disulphide bridges as manifested by the multiple and scattered distribution ofl4C-labelled S-carboxymethylcysteine-containing peptides. It appears, therefore, that once the rigidity of part of the molecule is perturbed, the remaining disulphide bonds exhibit no difference in reactivity. Lethal activity is removed completely only at a dithioerythritol to toxin ratio of 5:!, i.e. an equimolar ratio of toxin disulphide to reducing agent. Toxicity values in the range of dithioerythritol:toxin, exceeding a ratio of 1:1 do not depart significantly from the values, predicted from statistical considerations, for residual dialkylated toxin. Decreasing lethal activity must therefore be attributed to residual dialkylated toxin and not to decreasing toxicity of tetra-, hexa- and octa-alkyl derivatives. The di-S-carboxymethylated toxin, with the remaining disulphide bonds intact,

247 is i m m u n o c h e m i c a l l y indistinguishable f r o m the native toxin. N o t only is this region o f the molecule not c o n t r i b u t i n g t o w a r d s the antigenic m a k e - u p o f the toxin, but rupture a n d alkylation o f this disulphide b o n d has no drastic effect on the overall three-dimensional structure o f the molecule. This o b s e r v a t i o n could be exploited, by introducing heavy metal atoms, via two free sulphydryl groups, for X - r a y crystallography. ACKNOWLEDGEMENTS I wish to t h a n k M r D. Parris for the statistical analysis o f the toxicity data. REFERENCES 1 Epstein, C. J., Goldberger, R. F. and Anfinsen, C. B. (1963) Cold Spring Harbor Syrup. Quant. Biol. 28,439-449 2 Sela, M., White, Jr, F. H. and Anfinsen, C. B. (1957) Science 125, 691-692 3 Epstein, C. J. and Goldberger, R. F. (1963) J. Biol. Chem. 238, 1380-1383 4 Isemura, T., Takagi, T., Maeda, Y. and Imai, K. (1961) Biochem. Biophys. Res. Commun. 5, 373-377 5 Freedman, M. H. and Sela, M. (1966) J. Biol. Chem. 5225-5232 6 Palmer, J. k., Nisonoff, A. and Van Holde, K. E. (19631 Proc. Natl. Acad. Sci U.S.A. 5(1, 314-321 7 Neumann, H., Steinberg, I. Z., Brown, J. R., Goldberger, R. F. and Sela, M. (1967) Eur. J. Biochem. 3, 171-182 8 Azari, P. (1966) Arch. Biochem. Biophys. 115, 230-232 9 Shapira, E. and Arnon, R. (1969) J. Biol. Chem. 244, 1026-1032 10 Cleland, W. W. (1964) Biochemistry 3, 480-482 11 Bewley, T. A., Dixon, J. S. and Li, C. H. (1968) Biochim. Biophys. Acta 154, 420-422 12 Liu, W. K. and Meienhofer, J. (1968) Biochem. Biophys. Res. Commun. 31,467-473 13 Botes, D. P. (1971) J. Biol. Chem. 246, 7383-7391 14 Botes, D. P., Strydom, D. J., Anderson, C. G. and Christensen, P. A. (1971) J. Biol. Chem. 246, 3132-3139 15 Finney, D. J. (1971) Probit Analysis, 3rd edn., Cambridge University Press, Cambridge 16 Yang, C. C. (1967) Biochim. Biophys. Acta 133, 346-355 17 Yang, C. C. (1965) Toxicon 3, 19-23 18 Bartels, E. and Rosenberry, T. L. (1971) Science 174, 1236-1237