Proc. Indian Acad. Sci. (Chem. Sci.), Vol. 93', No. 7, September 1984, pp. 1117-1136. 9 Printedin .

Toxic proteins of snakes and

L K RAMACHANDRAN* § K E ACHYUTHAN § O P AGARWALt, L CHAUDHURYt, J R VEDASIROMANIt and D K GANGULIt § Department of Biochemistry, Osmania University, Hyderabad 500007, India t Indian Institute of Chemical Biology, Calcutta 700 032, India

Abstract. A chromatographic method utilising Sephadex C-25 as an ion exchanger and molecular sieve permits both small and large scale separation of the constituents of snake venom. Cross-contamination of peak materials is diminished when Triton X-100 is in- corporated in eluting buffers. Two long neurotoxins and two cardiotoxins isolated from Naja naja (cobra) venom differ from other such toxins, isolated earlier, in their amino acid composition. Inorganic pyrophosphatase activity is found in snake venoms, and the more basic of the two cardiotoxins (cardiotoxin II) of cobra venom possesses intrinsic enzyme activity. The properties of the latter toxin as an enzyme have been studied. Enzyme activity does not seem essential for the protein to display cardiotoxicity. Cardiotoxin II, as isolated, contains four magnesiumatoms per mole, which do not appear to be essential for its function. At least two atoms of magnesium per mole are required for the pyrophosphatase action. The venom of two Indian scorpions ( tamulus and bengalensis) are amenable to fractionation by chromatography on Sephadex C-25. Two fractions from Buthus tamulus elicit an initial hypotensive effect and then a hypertensiveeffect in rats. The former is cholinergic and the latter adrenergic in nature. Both fractions induce a biphasic contracture of the indirectly stimulated rat diaphragm. Of the two venoms, that of Buthus tamulus is more toxic to mice.

Keywords. Snakes; venoms; cobra; chromatography; Naja naja; neurotoxins; cardiotoxins; enzymes; Triton X-100; inorganic pyrophosphatase; magnesium; scorpions; Buthus tamulus; ; hypotensive and hypertensive effects.

1. Introduction

There have been numerous investigations on venoms from different varieties of snakes. A good summary of much of the work done so far is to be found in the book edited by Lee (1979). Conformational aspects of some of the toxins are dealt with in another review (Dufton and Hider 1980). We describe in this communication some of our observations on the toxins of the venom of the Indian cobra, Naja naja. Some of these findings have been recorded in literature (Achyuthan et al 1980; Achyuthan and Ramachandran 1981, 1983; Srinivasa et al 1982; Shashidharan and Ramachandran 1983). Progress in the study of toxic and other biologically active constituents of venoms of scorpions has also been striking in recent years (Bettini 1979; Fontella- Camps et al 1981; Ovchinnikov and Grishin 1982). A part of this paper is devoted to our observations on venoms of scorpions belonging to two different (Buthus tamulus and Heterometrus benoalensis ) found in India. The nature of enzymatic activities encountered in their venoms is dealt with elsewhere (Achyuthan et al 1982).

* To whom all correspondence should be addressed.

1117 1118 L K Ramachandran et al

2. Cobra venom

2.1 Fractionation of cobra venom A number of fractionation procedures applicable to snake venoms are reported in literature. Many of these procedures either involve repeated chromatography of the venom or fractions thereof or are not readily applicable to venoms other than the one for which they were designed. We have developed a separation procedure involving the use of CM-Sephadex C-25 which yields highly satisfactory results. Naja naja venom (Haffkine Institute) is readily separated into about seventeen fractions on such columns using phosphate buffers of varying molarity and pH for stepwise elution. Depending on column size, reproducible chromatographic profiles are obtained for even relatively large loads of crude venom (figures 1, 2 and 3). Proteins present in various fractions are recovered after desalting on Sephadex G-10 columns using 0.01 M acetic acid for elution. Ten of these fractions are lethal to mice at intraperitoneal dose levels of 12 micrograms or less. Four of the fractions appear to be homogeneous on disc gel electrophoresis. Two of the most basic venom proteins (fractions IX and X, figure 3) also appear to be homogeneous on rechromatography on CM-cellulose or Amberlite CG-50 columns. Two of the fractions (VA and VB, figure 3) appear to be the most toxic proteins of the lot and seem to correspond to the neurotoxins based on previously established LDso values for neurotoxins isolated from of cobra. Tables 1 and 2 provide information on yield and toxicity of various protein fractions derived from cobra venom (figure 3) and the LDs0 values of some of the fractions obtained from column chromatography. Fractions IX and X (figure 3) which account for 25-30 ~o of crude venom correspond qualitatively to two most basic proteins of cobra venom isolated by earlier workers and which go under labels such as cobramines, cardiotoxins, cytotoxins and direct lyric factors. The neurotoxic fractions account for less than 10~o of the weight of crude

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Figure 1. CM-Sephadex C-25 column chromatography of Naja naja venom (Haffkine Institute, venom batch no. 206), load, 99 mg in 1.0 ml of 0.02 M phosphate buffer, (pH 7.0); dimensions of column packing 0.8 x 70 cm, flow rate 20 ml/lar, fraction volume 2 ml; temperature, room temperature. Elution was carried out stepwise with phosphate buffers of molarities and pH's as indicated. The values in parentheses on the top of the figure refer, respectively, to the molarity of the eluent and its pH. Recovery, 82 % Toxic proteins of snakes and scorpions 1 119

1.5

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Figure 2. CM-Sephadex C-25 column chromatography of Naja ncja venom (Haffkine Institute, venom batch no. 204), load 862.4 mg/4-1 ml of 0.02 M phosphate buffer (pH 7.0); dimensions of column packing 2.5 • 74cm, flow rate 125 ml/hr, fraction volume, 10ml; temperature, room temperature. Elution was carried out stepwise with phosphate buffers of molarities and pH's as indicated. The values in parentheses on the top of the figure refer. respectively, to the molarity of the eluent and its pH. Recovery, 78 %.

( 0 02;7.0) (0.04;75) (0075;7.5) (0,1;7 5) I 0.10;8.0) (0.}5;8 o) to.z;8 o) [o25;80) (0.3;8,0) (0 4;8.0)

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Figure 3. CM-Sephadex C-25 column chromatography of Naja naja venom, Haffkine Institute, venom batch nos. 195-203; load 7.5 g/18 ml of 0-02 M phosphate buffer, pH 7.0; dimensions of column packing 4 x 110 cm; flow rate 340 ml/hr; fraction volume 40 ml; elution was carried out stepwise with phosphate buffers of motarities and pH's as indicated. The values in parentheses on top of the figure refer, respectively, to the molarity of the eluent and its pH. Recovery: 83 %.

venom and have LDsos of the order of 0.2-0.3 mg/](g mouse when administered intraperitoneally. Fractions IX and X which are similar to other cardiotoxins, display LDs0s in the range 2.6-3.5 mg/kg mouse when administered intraperitoneally. As many as three cardiotoxins have been recognized by other investigators in venom of Naja naja siamensis (Toxicon 1982). An even larger number is reportedly present in some other venoms.

2.2 Partial characterization of neurotoxins and cardiotoxins

The two neurotoxins examined are those isolated from fractions VA and VB (see figure 3) and possessing LDso values of 0.19 and 0.30 mg/kg mouse, respectively. The 1120 L K Ramachandran et al

Table 1. Yield and toxicity of fractions (see figure 3) from cobra venom.

Sample Yield Toxicity* peak no. (/o)o/ /~g/mouse

I 12 NT IA 2.4 10 IB 2.3 10 II 6.7 12 IliA 2-5 10 IIIB 2-4 11 IVA 1.8 10 IVB 1.8 NT VA 3.3 5 VB 3.2 5 VI 2.5 10 VII 5.3 NT VIIIA 0-9 NT VIlIB 5-0 9.3 IX 12.4 NT X 15'8 NT XI 2.4 NT

* Fractions that cause death of albino mice (18-20 gwt) at dose levels of 15 ~g or less/mouse are considered toxic; cobra venom was toxic at 15 pg/mouse. NT, non-toxic.

two cardiotoxins (I and II) analysed are those isolated from fractions IX and X, respectively (figure 3), and having LDso values of 3.5 and 2.6 mg/kg mouse. The compositions of the two neurotoxins are given in table 3. Comparisons of the compositions of the toxins isolated by us with those calculated from the structure of all long (type-II) neurotoxins so far isolated from all species of Naja (not only Nqia naja), as listed by Karlsson, leads to the following observations. Neurotoxin from fraction VA (70-5) shows maximum resemblance to toxin A or toxin 3 (71-5, N. naja naja, India) and toxin 3 (71-5, N. naja naja, Pakistan) with the difference of one residue less of arginine and lysine, respectively. On comparison with toxin B or toxin 4 (71-5, N. naja naja, India) the difference amounts to one residue less of serine and arginine and one residue more ofisoleucine, while with toxin C (71-5, N. naja naja, India) or toxin 3 (71-5, N. naja siamensis) there is one residue less ofalanine and lysine and one residue excess of glycine. All the toxins compared above have five disulphide bonds and lack methionine. Toxin VB (73-5) shows maximum resemblance to toxin B or toxin 4 (71-5, N. naja naja, India) and toxin A or toxin 3 (71-5, N. naja naja, India) with differences to the extent of six and eight residues respectively. There are five disulphide bonds in these toxins and methionine is absent. Comparisons of toxins VA and VB with other known toxins (type II) show differences ranging from 7 to 34 amino acid residues. The amino acid compositions of the two cardiotoxins are presented in table 4. When similar comparisons are made for cardiotoxins I and II (table 4) maximum resemblance (4-8 residue differences) is found with cytotoxins I, II and III from Naja naja and Toxic proteins of snakes and scorpions 1121

Table 2. LDso values of cobra venom and fractions thereof.

Reference LD~o Slope Potency to column Sample (mg/kg) function ratio run

Cobra venom 0.46 1.5 1.0 -- (0.58--0.37) Peak V 0.18 1.3 2.5 figure 1 (0.22-0.15) Peak IX 3-5 1.26 0-13 -do- (4.11-2.98) Peak X 2.5 1.36 0.18 -do- (3.0-2.1) Peak V 0.18 1.3 2-5 figure 2 (0. 22-0.15) Peak IX 3.6 1.4 0-13 -do- (4.5-2.7) Peak X 3.1 1.17 0.15 -do- (3.46-2.78) Peak VA 0.19 1.65 2-5 figure 3 (0.25-0.14) Peak VB 0-30 1.26 15 -do- (0.34-0.26) Peak IX 3"5 1.26 0.13 -do- {4.11-2.98) Peak X 2.6 1.36 0.18 -do- (3.09-2.18)

* Values in parentheses represent 95 ~o confidence limits. The potency ratio of the crude venom in mice is taken as I.

cytotoxin of N. naja atra (Formosa) which are all of the 60-4 type. With the other known cardiotoxins (including two which are of the 61-4 type) the differences are greater (10-21 residues). However, detailed resemblances and differences between the structures of the toxins reported now and those characterized earlier from various species of Naja can be worked out only after degradative structure analysis. A 1981 report (Charles et al 1981) recognised the presence of one neurotoxin of the 61-4 type containing 60 amino acid residues, 4 disulphide bonds and lacking alanine, methionine and phenylalanine. Three other toxins of unknown compositions but of similar toxicity also had been recognized in that report. It would seem that venoms of several Naja species contain neurotoxins of either short or long type; or both. Venoms of a single type of cobra are known to contain several cardiotoxins of varying toxicity.

3. Problem of aggregation of venom proteins and the use of Triton X-100 in column eluents.

Fraction VA, one of the neurotoxins (figure 3), on disc gel electrophoresis at pH 4.2, showed a multiplicity of bands (figure 4). Also when VA is rechromatographed, on a CM-Sephadex C-25 column, splitting into peaks (VAI and VA2, figure 5) could be

C-2 1122 L K Ramachandran et al

Table 3. Amino acid composition (residues/mole) of neurotoxins of venom of Naja naja.

Toxins isolated in Toxins from Naja species present study characterized earlier f

Amino acid VA VB I II III

Lysine 3.63 (4) 3.97 (4) 4 4 5 Histidine 1-01 (1) 1.03 (1) 1 1 1 Arginine 5.04 (5) 6'25 (6) 6 6 5 Aspartic acid 8.92 (9) 10.13 (10) 9 9 9 Threonine 8"78 (9) 7"97 (8) 9 9 9 Serine 2-90 (3) 3'92 (4) 3 4 3 Glutamic acid 1'27 (I) 2"75 (3) 1 1 1 Proline 5-52 (6) 5-35 (5) 6 6 6 Glycine 5-07 (5) 6'07 (6) 5 5 4 Alanine 2"07 (2) 1"75 (2) 2 2 3 Half-cystine* 9,75 (10) 9"88 (10) 10 l0 10 Valine 3-84 (4) 3'88 (4) 4 4 4 Methionine ..... Isoleucine 4'35 (5) 3-75 (4) 5 4 5 Leucine 1.00 (1) 1.21 (1) 1 1 1 Tyrosine (}89 (1) 1-00 (1) 1 1 1 Phenylalanine 2'89 (3) 2"98 (3) 3 3 3 Tryptophan 0.97 (1) 1"20 (1) 1 1 1 Total number of residues 70 73 71 71 71

In rounding values for the number of residues/mole of toxin, fractions greater than 0-5 have been considered as 1.0. Only in two instances, VA and cardiotoxin II (table 4), fractional yields in excess of 0-3 residues of isoleucine, which is occasionally known to be present in acid-resistant linkages in proteins, have been treated as equivalent to one residue. * Neither cardiotoxins nor neurotoxins revealed the presence of any thiol groups on assay with p-chloromercuribenzoate. Values for half-cystine were obtained by analysis of hydrolyzates of performic acid oxidized protein. f Yang (1978). observed. On the other hand, when fraction VA is subjected to SDS-disc gel electrophoresis only one band is observed at pH's 4.2 and 6-0 (figure 6). Also on scanning for eleven enzymatic activities in various fractions (table 5) obtained from crude venom considerable apparent cross contamination is observed. This relates to enzymatic activities noticed in early eluting fractions also appearing in later eluting fractions. Therefore, an effort was made to see whether the multiplicity in disc gel banding of fractions like neurotoxin of peak VA (figure 3) and cross contamination by enzymes in peaks could be avoided by running chromatographic separations of crude venom on CM-Sephadex C-25 with phosphate buffers containing 0 1 ~o Triton X-100. A chromatogram of that type for a 100 mg load of venom is shown in figure 7. That there is benefit derived in using this procedure in avoiding cross contamination in relation to specific enzymatic activities is shown in table 6. In regard to the nuisance of aggregation encountered with neurotoxin in peak VA (figure 3) which was commented on earlier, it should be observed that fraction V (figure 7) which does not show resolution of components VA and VB seen in bigger columns, behaves as if it is Toxic proteins of snakes and scorpions 1123

Table 4. Amino acid composition (residues/mole) of cardiotoxins of venom of Naja naja.

Toxins isolated in Toxins [cytotoxins) from Naja present study species characterized earliert Amino acid I II I II III

Lysine 9.54 (10) 8-65 (9) 9 9 9 Histidine ..... Arginine 1.82 (2) 1.94 (2) 2 2 2 Aspartic acid 7.18 (7) 6.65 (7) 8 6 8 Threonine 2-63 (3) 3.20 (3) 3 3 3 Serine 1.68 (2) 1.85 (2~ 2 2 2 Glutamic acid 1.00 (1) -- 1 -- Proline 4.21 (4) 5.24 (5} 4 5 4 Glycine 1.60 (2) 2-29 (2) 2 2 2 Alanine 1-82 (2) 2.20 (2) 2 2 2 Half-cystine 8.19 (8) 8-06 (8) 8 8 8 Valine 6-53 (7) 3.87 (4) 5 7 6 Methionine 1.67 (2) 1.70 (2) 2 2 2 Isoleucine 1-58 (2) 1.31 (2) 2 1 2 Leucine 6-09 (6) 7.08 (7) 6 6 6 Tyrosine 3-53 (4) 3.53 (4) 4 4 3 Phenylalanine --- 1.00 (1) 0 1 1 Tryptophan ..... Total number of residues 62 60 60 60 60

In rounding values for the number of residues/mole of toxin, fractions greater than 0.5 have been considered as 1.0. Only in two instances, cardiotoxin II and VA (table 1), fractional yields in excess of 0.3 residues of isoleucine, which is occasionally known to be present in resistant (acid) linkages in proteins, have been treated as equivalent to one residue. * Neither cardiotoxins nor neurotoxins revealed the presence of any thiolgroups on assay with p-chloromercuricbenzoate. Values of half-cystine were obtained by analysis of hydrolyzates of performic acid oxidized protein. t Yang 1978.

monodisperse at pH 4.2 and pH 6.0 (figure 6). Since this unexpected behaviour occurs in relation to fraction V (containing apparently two neurotoxins of venom) derived from Triton X-100 columns, conclusions made from disc gel electrophoresis as to individuality or homogeneity of various protein fractions derived from Triton X-100 columns should be treated with some reserve. The LDs0 values of neurotoxic peak fractions derived under various conditions is shown in table 7. As far as neuro-toxicity is concerned there does not appear to be any serious effect caused by the presence of contaminating Triton X-100 (see toxicity of peak V). However, Triton X-100 contaminating the protein fractions does seriously interfere with evaluating absorption due to aromatic amino acid residues present in the proteins isolated.

3.1 Inorganic pyrophosphatase activity in venoms It may be noted that in venom fractionations (figure 3) at least three fractions (VIII B, IX and X) display inorganic pyrophosphatase activity. On Triton X-100 chromato- grams, fractions VII, VIII, IX and X display such enzymatic activity. It is worth notiog

C-2A 1124 L K Ramachandran et al

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~Oq6 - Figure 5. Rechromatography of fraction VA ob- (o tained from column run (figure 2) on CM-Sephadex C-25 column; load: 43.1 mg fraction VA material in 00~OB - 0-5 ml of 0-05 M phosphate buffer, pH 6.7; dimen- sion of column packing 2-5 x 76cm; flow rate 100 ml/hr; fraction volume 5 ml; elution was carried o ~ 1 f 20 40 out at room temperature with 0-05 M phosphate FRACTION NUMBER buffer, pH 6.7.

that fractions X from both types of separations yield cardiotoxin II which possesses a nearly constant level of enzymatic activity. It is possible that this cardiotoxin possesses some intrinsic pyrophosphatase activity. That inorganic pyrophosphatase activity is to be found not only in Naja naja venom but others as well, is shown by the data given in table 8 for venoms from Crotalus, Bothrops, Agkistrodon, Trimeresurus, and Lapemis.

3.2 Properties of cardiotoxin II as a pyrophosphatase Cardiotoxin II shows a ten-fold higher inorganic pyrophosphatase activity (0-12 I.U.) relative to crude venom (0,012 I.U.). The protein is often obtained in an yield of about Toxic proteins of snakes and scorpions 1125

t~

Figure 6. SDS-electrophoresisat pH 4.2 (I) and pH 6.0 (2) respectively,of fraction VA obtained from column run shown in figure 3. Time of run: 1 hr.

15 ~. A twelve year old sample of Cobramine B originally isolated by Larsen and Wolff in 1967 still displayed half the activity observed in our cardiotoxin II preparations. The enzyme showed a broad pH optimum between 6-5 and 7.5. The enzyme shows a low Ks (70 #M) and the energy of activation is found to be about 10 kcal/mole. It tolerates a wide range of substrate concentrations as shown in figure 8. Table 9 presents the data on the sensitivity of the enzyme to various anions, cations and reducing agents. No thiol groups appear to be present. The kind of specificity observed for the pyrophosphatase activity is indicated by the data in table 10. The enzyme activity ofcardiotoxin is not as sturdy as the toxicity. The relative stability of the enzymatic activity under two different conditions is shown in figure 9. Whether the enzymatic activity of cardiotoxin is necessary for the exertion of its toxicity is difficult to say. But, it would appear it is not. 1126 L K Ramachandran et al

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Figure 7. CM-Sephadex C-25 column chromatography of Naja naja venom. Haffkine Institute, venom batch no. 200; load 100.80 mg in 1.0 ml of 0-02 M phosphate buffer, pH 7.0 containing 0.1% (v/v) Triton X-100; dimensions of column packing 0.9 x 70 cm; flow rate 20 ml/hr; fraction volume 2 ml; elution was carried out with phosphate buffers of molarities and pH's as indicated. All buffers contained 0"1% (v/v) Triton X- 100. The values in parentheses on top of the figure refer, respectively, to the molarity of the eluent and its pH. Recovery: 67 ~o.

4. The role of metal ions in the enzymatic activity and toxicity of cardiotoxin II

The role of metal ions in the enzymatic activity of cardiotoxin has been examined in detail. Whereas earlier the absence of any inhibition by EDTA at a level of 0.01 M had been commented on, experiments with EDXA at a higher level of 0"1 M could elicit lowering of the activity by 27 ~o, Whether divalent metal ions like Mg 2 § had any role in activity could be examined only after a deliberate effort to remove such metal ions from the protein. Such demetallation of the protein has been accomplished by repeated treatments with EDTA and recovery of protein after passage through Sephadex G-10. Three such treatments lowered the enzymatic activity to 15 ~ o, 7 ~ and 0 ~o of that originally displayed. The direct examination of crude venoms for magnesium content, by flame spectrophotometry, has given values in the range 0.45 to 0.9 ~o. Earlier reports in literature also show that crude venom contains appreciable amounts of magnesium. Analysis of cardiotoxin II as isolated (figure 3) showed a magnesium content of 1.47 ~ o. Demetallated protein which showed no enzymatic activity contained none. The content of magnesium in cardiotoxin II would amount to 4 atoms of Mg 2§ per 6000 g. Remetallation of demetallated cardiotoxin II at 37~ and purification by passage through a Sephadex G-10 column yielded a metallated derivative containing only 1.7 atoms per mole. The degree of restoration of enzymatic activity which is possible on reconstitution under different conditions is indicated by the data in table 11. It is interesting to note that the Mg(II)-EDTA complex also can restore nearly half the original enzymatic activity. The lower degree of metal incorporation into protein in reconstitution experiments may be because the cardiotoxin samples from which demetallated protein was derived may themselves contain loosely bound magnesium, which is not retained when remetallated protein is purified by gel chromatography on Sephadex G- 10. Having shown that cardiotoxin II is a magnesium containing metalloprotein and that Mg 2+ was essential for pyrophosphatase action, it was essential to ascertain whether the metal was important for the toxic action ofcardiotoxin II. Intraperitoneal injections of demetallated samples showed a LDso which was indistinguishable from 1128 L K Ramachandran et al

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Table 7. LDs0 values of cobra venom and fractions thereof.

LDs0 Slope Potency Refer to Sample (mg/kg) function ratiot column run

Cobra venom 0.46* 1.50 1.0 -- (0.58-0.37) Peak VA 0.19 1.65 2.5 figure 3 (0.25-0-14) Peak VB 0.30 1.26 1-5 figure 3 (0.34-0.26) Peak V 0.18 1-30 2.6 figure 7 (0.22-0.15) Peak VA~ 0.30 1.75 1.5 figure 5 (0.44-O.20) Peak VA 2 0.19 1.65 2-5 figure 5 (0-25-0-14)

* Values in parentheses represent 95 o~, confidence limits. t The potency ratio of crude venom in mice is taken as 1.

Table 8, Inorganic pyrophosphatase activity of some snake venoms.

Pyrophosphate (/~moles) hydrolyzed Specific Family and species 1 hr 2 hr activity (I.U.*)

Crotalidae: Crotalus viridis 0.06 0"10 4 • 10- 3 Crotalus atrox 0.06 0"12 4 x 10 -3 Crotalus t. terrificus 0.02 0-08 2.7 x 10-3t Bothrops atrox 0"03 0-04 2 x 10 -3 Aokistrodon rhodostoma 0.06 0-17 4 x 10 -~ Trimeresurus flavoviridis 0.06 O"12 4 • 1O- 3 Elapidae: Naja naja (Haffkine Inst) 0.16 0.28 1.1 x 10 -2 Naja naja (Astik Farms) 0.13 0.22 9 x 10 -3 Naja naja atra 0"10 0-22 7 x 10-3 Naja naja oxiana 0-12 0.20 8 • 10 -3 H ydrophiidae: Lapemis hardwickii 0.34 0-48 2'3 x 10 -2

* Specific activity is defined as pmoles of phosphate liberated/mg of protein/min. + Values obtained from the enzyme activity observed in 2 hrs of incubation. Specific activity for the rest were calculated from the enzyme activity in one hr.

that of the original protein (table 12). However, it is not easy to conclude that Mg 2+ is not essential in the lethal action in mice since possibilities do exist of the protein having got re-equilibrated with Mg 2 + present in the body pool before reaching the target sites. 1130 L K Ramachandran et al

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Figure 8. Effect of substrate concentration on enzyme activity. The enzyme (0-5 mg) was incubated with varying concentrations of sodium pyrophosphate (upto 15 pmol/ml). At the end of 15 min the inorganic phosphate liberated was estimated.

Table 9. Effect of metal ions, anions, tris, chelators, thiols and thiol reagents on Naja naja pyrophosphatase.

Concentration Inhibition* Ingredient (mM) (%)

Metal ions: Cu 2+ and Ag + 0"5 65-73 10 70-88 Ca 2 + 0-5 68 10 83 Zn 2+, Cd 2+ and Hg 2+ 0-5 70-75 10 77-90 Mn 2+ 0.5 57 10 69 Ni 2+ and Co 2+ .0"5 24-73 10 57-77 Anions: WoO42 - 10 17 MoO] - 10 24 Ox alate 10 10 F- 10 82 CN 10 50

Thiols (cysteine, reduced glutathione, and dithiothreitol) at 10 m M have no effect on activity at pH 7.0. N-ethylmaleimide and iodoacetate (0.01 to 10 mM) have no effect. Tris-HCl (pH 7.5), upto 0.8 M and the metal chelators, 8-hydroxy-quinoline, ct,~t'-bipyridyl and ethylene diamine- tetraacetate, at concentrations upto 10 mM, have no effect on enzyme activity. * Values for Zn 2 +, Cd 2 +, Cu 2 § and for WoO~ are corrected for protein precipitation; Mg z§ (1-10 mM) has no effect on activity. Toxic proteins of snakes and scorpions 1131

Table 10. Organic phosphates and pyrophosphates as substrates for Naja naja pyrophos- phatase.

Compound Effectiveness*

Pyrophosphate 100 Cytidylic acid (CMP-3') 25 Adenylic acid (AMP-5') 25 Uridylic acid (UMP-5') 22 Guanylic acid (GMP-5') 25 Glucose- 1-phosphate 13 Glucose-6-phosphate 13 Otherst 0

* The effectiveness of the enzyme on sodium pyro- phosphate under standard assay conditions is taken as 100. Under standard assay conditions, other sub- strates are scarcely attacked. The values entered in the table are obtained at ten-fold higher substrate concentration. t Several other organic phosphates (viz thymidylic acid (TMP-5') cyclic-AMP, adenylic acid (ATP-5'), cyti- dylic acid (CTP-5'), inosinic acid 0TP-5'), uridylic acid (UTP-5' and UDP-5'),cofactors (FMN, FAD, NADP, TPP), glycerophosphates (~- and fl-), fructose-l,6-diphos- phate and p-nitrophenyl phosphate) were not hydrolyzed.

I00

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20

0 0 20 40 60 80 TIME {rain)

Fig,.re 9. Effect of heat treatment at 60~ (- -) and 90~ (- -) on enzyme activity. The enzyme solution was heated in a water bath at the desired temperature (+ I~ for different durations. The solutions were taken out, cooled rapidly and assayed for pyrophosphatase activity.

C~3A 1132 L K Ramachandran et ai

Table II. Effect of metal ions, EDTA and metal chelates on enzyme activity.

Relative Concentration activity Sample Ingredient (raM) (%)

Cardiotoxin II (control) -- -- 100 Cardiotoxin II Na2-EDTA 100 73 Demetallated cardiotoxin A* -- -- 15 Demetallated cardiotoxin B* -- -- 7 Demetallated cardiotoxin C* -- -- 0 Dcmetallated cardiotoxin B Mn + + I ~

105 I 0 20 -do- Co + + 1 5 10 0 20 -do- Mg + + 20 75 -do- Mg + + 20 53 + EDTA 50 -do- Mg(II~EDTA 1 44 -do- Na2-EDTA 10 5 -do- Mg(II)-EDTA I ~ 17 + Naz-EDTA 9

* A, B and C refer to cardiotoxin II recovered after one, two and three cycles of demetallation.

Table 12. Toxicity of cardiotoxin II and demetallated cardiotoxin II.

Sample LD~'o (mg/kg) Slope function

Cardiotoxin-II 2'6 (3.09-2.18) 1"36 tDcmetallated cardiotoxin-II 2.8 (3'33-2.35) 1.28

* Values in parentheses represent 19/20 confidence limits. t This sample was obtained after two successive cycles of EDTA treatment and gel filtration and contained ~55 atoms of magnesium per mole of protein.

Whether the toxic and enzymatic actions in cardiotoxin II are resident in one and the same protein could be answered clearly if both activities showed the same sensitivity to heat inactivation. Table 13 provides data for the rate of heat inactivation ofcardiotoxic activity at 60~ Figure 10 which depicts the decay curves for both activities does show that the two activities do not decay in a parallel manner. As there has been no evidence to clearly show that the cardiotoxin II samples, whether obtained with or without the use of Triton X-100 by gel chromatography, contain contaminating proteins, one might speculate that cardiotoxin II entrains two apparently different active sites of which one Toxic proteins of snakes and scorpions 1133

Table 13. LDso values of cardiotoxin-II after heating at 60~ for different durations.

Heating Time Slope Potencyt (min.) LD*0 (mg/kg) function ratio

0 2-6 (3"09-2"18) 1"36 1-00 40 2'2 (4"16-2"46) 1"50 0"80 60 3.4 (4-08-2-83j 1"34 0"77 80 4'0 [4-96-3"23) 1-47 0"65 100 5"0 (6-75-3-76) 1"56 0"52

* The values in parentheses represent 19/20 confidence limits. i The potency ratio of unheated cardiotoxin II is taken as 1.0.

• Figure 10. Effect of heat (60~ treatment on 40 -- toxicity and pyrophosphatase activity of cardiotoxin If. The protein (native) solution was heated in a water bath at the desired temperature (_ I~ for different durations. The solutions were taken out, cooled rapidly and assayed for enzyme activity and o [ ] I I toxicity (LD~o). The lines ('best fit') have been drawn o 20 4o 6o 8o ~oo according to calculations based on regression

TIME(ram) analysis.

p I.IOSPHAT[ aue F (Rr

oo~ oo~ oo7~ o, o, ol o1~ oqa MOLAmTV 6~ 700 ~0 7.0 7~ aO aO eODH 0

0 J ;o

o

o , z o

tn ~o vii I~ vAv Vl v ix x "tl ii nl

,io ao ,~o leo 200 240 FRACTION NUMB[R

Figure 11. Chromatographic fraction of Buthus tamulus venom on CM-Sephadex C-25 column (1.8 x 88 cm); load 218 mg (207 rag/protein); flow rate 50-60 ml per hour; the stepwise schedule ofelution with phosphate buffers of known pH and molarity is indicated in the figure, Run at room temperature. Ch4 1134 L K Ramachandran et al

PHOSmHATE aUFfEgt I OU~ 00~ 002 004 OOrS 0~0 OlO 0~5 020 0~ 030 035 040 ~OPO,,,~" 6 / 70j 15 Z5 Z5 7~ ao eo ac, ~, s, ao a,~ ~H

~= 1 J , 1 i j i 2-

z

or r 5c if o 16o poo 250 ~oo 350 40(, iI TUBE NUMB[R Figure 12. Chromatographic fractionation of Heterometrus ben qalensis venom. Load 190 rag, other details as in figure 11.

(a) (b) (c) Figure 13. Effect of fraction V (PV) on rat blood pressure. (a) dose dependence, (b) blockade hypotensive effect on atropine treatment, (c) blockade of the hypertensive effect on dibenzyline (DBZ) treatment,

Figure 14. Kymograph tracing showing development of tachyphylaxis to the hypertensive effect of fraction V (PV) on rat blood pressure. Numbers within brackets indicate dose number. Toxic proteins of snakes and scorpions 1135 is involved in pyrophosphatase action and the other in manifestations of toxicity. Recent experiments (Shashidharan 1984, unpublished data) indicate that N- bromosuccinimide oxidation affecting concomitantly one residue of tyrosine and one disulphide bond in cardiotoxin II destroys toxicity. Further, one more type of membrane (or cell wall related) activity ofcardiotoxin II has been recognized. The toxin antagonizes the lyric action of lysozyme on killed cells of Micrococcus lysodeikticus.

Figure 15. Kymographtracing showingeffects of(a) fractionV (PV) (b) fractionIX (PIX) on response of isolated rat diaphragm preparation to phrenic nerve stimulation. W-wash. 1136 L K Ramachandran et al

5. venom constituents

So far there has been only one attempt, and that too recently, aimed at the separation of the active constituents in Buthus tamulus venom (Chatwal and Hubermann 1981). Three toxins, one histamine releasing factor and a trypsin inhibitor have been recognized. Fractionation of constituents present in Heterometrus bengalensis venom has not been done so far. We find that the chromatographic procedure we have successfully exploited in the study of cobra venom, is also eminently applicable in the study of scorpion venoms. Figures 11 and 12 depict the results obtained. Recovery of protein applied to the column is high. While disc gel electrophoresis of the first venom yields as many as 17 bands at pH 8.7, fewer peaks can be discerned in the column elution profile. Both fractions V and IX from B. tamulus venom elicit in the rat an initial hypotensive effect and then an hypertensive effect. The former effect (cholinergic) is antagonized by atropine and the latter effect (adrenergic) is abolished by dibenzyline (figures 13 and 14). Tachyphylaxis develops to the hypertensive effect of both fractions. Both these fractions induce a biphasic contracture of the indirectly stimulated rat diaphragm. The first contracture appeared immediately, and the second 5~i minutes later to persist till the agent was washed off. It took about 3-4 hr for normal twitch responses to return after washing. Of the two venoms B. tamulus is more toxic to mice (LDso 3.6 mg/kg mouse, figure 15) and the other very much less so. Many fractions from both venoms have shown interesting activities in preliminary studies, and these studies will be extended after careful purification of constituents (more than one) which may be present in individual fractions.

Acknowledgements

These investigations were supported by us PL-480 grants (01-040-I and 01-040-N) and by the Council of Scientific and Industrial Research (Indian Institute of Chemical Biology, Calcutta). The authors are grateful to Professors Anthony T Tu, Chen-Yuan Lee, Akhira Ohoaka and to Dr V I Tsetlin for gifts of some snake venom samples.

References

Achyuthan K E and Ramachandran L K 1980 Proc. Indian Natl. Sci. Acad. B46 603 Achyuthan K E and Ramachandran L K 1981 J. Biosci. 3 149 Achyuthan K E and Ramachandran L K 1983 J. Biosci. 5 1 Achyuthan K E, Agarwal O P and Ramachandran L K 1982 Indian J. Bioehem. Biophys. 19 356 Achyuthan K E, Ranganatha Rao K and Ramachandran L K 1980 Indian J. Biochem. Biophys. 17 228 Bettini S (ed.) 1979 Handbook of Experimental Pharmacology Vol 48 Venoms (Berlin: Springer- Verlag) p. 277-418 Charles A K, Gangal S V and Joshi A P 1981 Toxicon 19 295 Chatwal G S and Habermann E 1981 Toxicon 19 807 Dufton M J and Hider R C 1980 Trends in Biochemical Sciences 5 53 Fonteila-Camps J C, Almassy R J, Eslick S E, Suddath F L, Watt D D, Feldmann R J and Buggs C E 1981 Trends in Biochemical Sciences 6 291 Lee C Y (ed.) 1979 Hand Book of Experimental Pharmacology Vol 52 Snake Venoms (Berlin: Springer-Verlag) Ovchinnikov Yu A and Grishin E V 1982 Trends in Biochemical Sciences 7 26 Shashidharan P and Ramachandran L K 1983 Indian J. Biochem. Biophys. 20 132 Srinivasa B R, Achyuthan K E and Ramachandran L K 1982 Indian J. Biochem. Biophys. 19 52 Yang C C 1978 in Rosenberg (ed.) Toxins: , Plant and Microbial (Oxford: Pergamon Press) p. 261