Proc. Indian Acad. Sci. (Chem. Sci.), Vol. 93', No. 7, September 1984, pp. 1117-1136. 9 Printedin India. Toxic proteins of snakes and scorpions 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 (Buthus tamulus and Heterometrus 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; Heterometrus bengalensis; 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 genus (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 species 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 <OOZlZO)(O-05;~5~ (0.075;?.5~ (o.~;r 5) (o qeo) (o.15;8o) (o 2o~8 o) (o.3o;e o) (o,1o8o) I.i Ix I,o 0 I 0 0.8 F. x ~06 vii ~ 0.4 02 III VIII IV VIA s 4'o 5'0 8% ,6o &o ~;o ,go ,~o zbo z~o z,;o FRACTION NUMBER " 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 (0.02 7'0)(0-05; 7"5 ) (0-075;7.5) (0-I;?.5) (0.I;8-0) (O'LSj8.O) (O.ZO;8"O) (0.25;8-0) (0.30; 8.0) (0-40,8 O) l IX 0 II X ~0.5 0 tO VII X1 0 0 40 80 120 160 200 240 28 0 320 FRACTION NUMBER "~--~" 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) o < 0"5 B VII AIB VA V a: I IlIA IIIB I VA I'VB VI XI < 0 80 ~60 240 320 400 480 560 640 FRACTION NUMBER---~ 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.