Purification and characterization of a myotoxic phospholipase A2 from Indian cobra (Naja naja naja) venom

Toxlrorr Vol . 27, No . 8, pp . 861-873 . 1989. Printcd in Great Britain .

0041-0101 J89 53 .00+ .00 ~ 1989 Maxwell I~mon Maanillan plc

PURIFICATION AND CHARACTERIZATION OF A MYOTOXIC PHOSPHOLIPA5E A2 FROM INDIAN COBRA (NAJA NAJA NAJA) VENOM Mnxo~ KuMnx BxnT and T. VF.ERABJ~SAPPA Gowns*

Department of Studies in Biochealstry, Manasagangothri, University of Mysore, Mysore-570006, India (Accepted for publication 9 February 1989) M. K. BRAT and T. V. Gowns. Purification and characterization of a myotoxic phospholipase A2 from Indian cobra (Naja naja naja) venom. Toxicon 27, 861-873, 1989 .-A major phospholipase A2 (NN-XIII-PLA2) which constitutes 20% of the whole Naja naja naja venom was purified to homogeneity on CM-Sephadex C-25 column chromatography . NN-XIIIPLA2 is a basic protein with a mol. wt of 11,200 by SDS-PAGE . This enzyme has low enzymatic activity but is more toxic to mice than the whole venom. The l,n~ value (i .p .) of NN-XIII-PLA2 is 2.4 mg/kg body weight (whole venoms Ln~ is 2.8 mg/kg body weight). It induces neurotoxic-like signs in experimental animals. It induces myotoxicity when injected i.m . into the thigh muscle of mice and edema when injected into the foot pads of mice . This enzyme has a fluorescence maxima between 310-316 nm which is typical of tyrosine residues . INTRODUCTION PHOSPHOLIPASE A2 (PLA2; E.C .3 .1 .1 .4) is ubiquitous in nature, rich sources being venoms of snakes, scorpions and bees . Venom phospholipase A2 contributes to digestion of the prey (Tu, 1977) and induces pathological symptoms (FRANKEL-CONRAT, 1982-83), such as neurotoxicity, cardiotoxicity (FI,Erc1~R et al., 1981), myotoxocity (GOPALAKRISHNAKONE et al., 1984), hemolysis (CONDRIsA et al., 1980, 1981a), anticoagulability (CONDREA et al ., 1981b, 1982), internal hemorrhage and edema-inducing activity (VISHWANATH et al., 1987). Thus, PLA2 seems to be involved in many of the pharmacological effects induced by snake venom poisoning. Snake venoms contain multiple forms of PLA2. Indian cobra (Naja naja naja) venom has been reported to contain as many as 14 isoenzymes of PLA 2 (SHi[.OAH et al., 1973). The presence of several isoenzyme forms of PLA2 in cobra venom is not due to subspecies polymorphism but exists in a venom sample collected from a single snake (IIAZLETT and DExNts, 1985a) . Several workers attempted to purify different forms of PLA2 from cobra venom using multistep procedures involving acid treatment (BRAGANCA et al., 1969 ; D>~MS and DElvxrs, 1975), heat treatment followed by gel filtration and electrofocussing (SALACI-I et al., 1968, 1971), multiple ion-exchange chromatography (Srul.onx et al., 1973), 'To whom correspondence should be addressed. 861

862

M . K . BHAT ~wn T. V. GOWDA

electrophoresis (.TAYANTHI and GOWDA, 1983) and affinity chromatography (HAZLETT and DsxNls, 1985b) . KINI and GOWDA (1983) reported the presence of eight PLAZ positive peaks upon fractionation of Indian cobra venom on CM-Sephadex C-25 column by the modified method of AcxYUTHAN et al. (1980) . Further modification of this procedure resulted in the identification of ten peaks with PLA Z activity . Peak NN-XIII, which corresponds to peak IX Of ACHYUTHAN and RAMACHANDRAN (1980), showed PLA Z activity instead of pyrophosphatase, deoxyribonuclease and f-nucleotidase activities which they reported . In this paper we report the purification and characterization of a basic phospholipase A Z from cobra venom which induces myotoxicity. MATERIALS AND METHODS Lyophilized whole venom of Naja raja raja (Batch No . C) was purchased from hula Snake Catchers Industrial Co-opcraüve Society Ltd, Madras, India . CM-Sephadex C-25 (4.5 meq/g) and Sephadex G-50 were purchased from Pharmacia Fine Chemicals, Uppsala, Sweden. Linoleic acid, Freund's complete and incomplete adjuvants were from Sigma Chemical Company, St Louis, U .S.A . 1-Nitroso-2-naphthol was prepared according to the method of FURNt34 et al. (1978) . Phosphaüdylcholine (PC) was prepared from hen's egg yolk according to the method of Stxat .irroN et al. (1965). All other chemicals and solvents were of analytical grade . The solvents were redistilled before use . Swiss Wistar male mice weighing 20-22 g were used for all pharmacological studies . Phospholipase A, assay method Phospholipase A, activity was assayed by a new colorimetric procedure based on the Nov~x (1965) free fatty acid estimation method. The reaction was initiated by adding 0.01 ml (25 pg) of phospholipase or crude venom, to a reaction mixture containing 1000 nmoles of egg PC, 0 .3 ml of ether, 0.5 ml of 0.05 M Tris-HCl buffer, pH 7 .5, and 40 ptnoles of Ca=+ . The reaction mixture was thoroughly mixed and incubated at 37°C for I hr . The reaction was terminated by adding 0.5 ml of modified Dole's extraction mixture (isopropyl alcohol, petroleum ether, 1 N H 2S0 40:10:1, v/v/v), mixing and adding 1 ml of petroleum ether . The contents of the tube were thoroughly mixed for 1 min and centrifuged at 500 x d for 5 min at room temperature. All lipids were extracted into the upper organic phase . An aliquot (0 .8 ml) of the upper organic phase was transferred into a test tube containing 0 .8 ml of chloroform: petroleum ether (5 :1, v/v) followed by the addition of 0.5 ml of freshly prepared cobalt reagent . The reagent consists of 1 .35 ml of triethanolamine, made up to 10 ml with solution A [6 g of Co(NO,h6HZ0+0.8 ml glacial acetic acid, made up to 100 ml with saturated K zSO, solution], plus 7 ml of a saturated solution of Na,SO,. The contents of the tube were thoroughly mixed for 1 min and centrifuged for 10 min at 1200 x g at room temperature . Cobalt selectively forms a soap with the fatty acid which is extracted into the upper organic phase . This phase (0.6 ml) was carefully transferred to a tube containing 0 .75 ml of Initroso-2-naphthol, indicator solution (0 .4% of 1-nitroso-2-naphthol in 96% ethanol) . Cobalt gives a coloured reaction with 1-nitroso-2-naphthol, the intensity of which is direcüy proportional to the amount of cobalt present. After 30 min, the contents were diluted to 3 .35 ml by adding 2 ml of 96% ethanol and the absorbance was read at 500 nm in a Bausch and Lomb Spectronic-20 . Linoleic acid was used as the standard fatty acid . PLA, activity is expressed in terms of amount of fatty acid, in nmoles, released per min per mg of protein . The phospholipase assay of Mno~ and Txostrsox (1960) was carried out as described by VISHWANAYfi et al. (1987) . The radioactive method of phospholipase assay was carried out according to the method of FRANSON et al. (1974) using ["cl-labelled oleate, autoclaved E. coli cells . Radioactivity was measured in an LKB 1211 Rackbeta liquid scintillation counter . Phospholipase assay was carved out on fraction NN-I-PLA (Jever~rrtn and Gowne, 1983) by the three independent methods (MAGF~ and THO~SON, 1960 ; NOVAK, 1965; Fw~xsox et al., 1974) under idenücal experimental conditions . The relative sensitivities of these three methods are tabulated in Table l . It is evident that the sensitivity of the assay developed on the basis of estimation of free fatty acids by Nov~x (1965) is very close to the radioactive method of Fte~rtsoty et al. (1974) which is the most sensitive of the three methods . With this method, amounts as low as 25 nmoles of fatty acid liberated can be estimated (Fig . 1) . Positions! specificity The positions! specificity of the purified phospholipase was determined using PC labelled at the Sn-2 position with a fluorescent 11 (9-anthroyl) udecanoic acid according to the method of VLSHWANATH et al. (1987) and by the method of FwUVSON et al. (1974) using ["C]-labelled cleats autoclaved E. coli cells . Purification of NN-X717 phospholipase Naja naja naja venom (650 mg) was dissolved in 4.0 ml of 0 .02 M phosphate buffer (pH 7 .0) and applied to a CM-Sephadex C-25 column (1 .4 x 120 cm) which was eluted stepwise, using phosphate buffers of various

Naja naja naja Phospholipase A,

863

TABLE I . COMPARATIVE STUDY OF THREE PHOSPHOLIPASE ASSAY PROCEDURES

Methods Ct al. (1974)

FRANSON

Methods Fatty acids liberated (nmoles) Percentage recovery of liberated fatty acid

427 ± 7 (7) 100

NOVAK

MAGEE and THOMPSON

363 t 20 (6)

230 t 15 (6)

(1965)

85

(1960)

54

Values are expressed as mean f S .D. The numbers in parentheses are the numbers of independent experiments .

molarities and pH values, as described in Fig.2 . Fractionation was carried out at 20°C . The flow rate was adjusted to 40 ml/hr and 4 ml fractions were collected by Frac-100 automatic fraction collector . Protein elution was monitored at 280 nm using a Bausch and Lomb Spectronic 2000. The enzyme elution was studied by assaying 0 .1 ml aliquots from alternate tubes Cor PLA, activity . Individual fractions of the enzyme peak were pooled, desalted, lyophilized and stored at -IS°C . Fraction NN-XIII showed PLA2 activity and is one of the major peaks wntaining 20% of the venom protein rewvered from CM~ephadex C-25 column and was named NN-XIII-PLA,. Thirty milligrams of NN-XIIIPLA= in 2 ml of 0 .1 M NaCI was loaded on a Sephadex G-50 column (1 .4 x 92 cm) . The protein was eluted with 0.1 M NaCI with a flow rate of 30 ml/hr at 20°C and 2 .5 nil fraction were collected. The protein elution was monitored at 280 nm . Assay ojdirect and indirect hemolytic activity Direct and indirect hemolytic activity was assayed as described by VISHWANATH et al. (1987) . Human red blood cells separated from freshly collected blood from healthy donors were used for assay.

E e Ô a+ ô d u c 0 a 0 N a

0"4

0"3 0 "2

a 0"1

Fatty ocid concentration (nmoles) FIG. I . FATTY ACID CALIBRATION CURVE HASFD ON THE FREE FATTY ACID ESTIMATION PROCEDURE OF NOVAK.

Linoleic acid was used as the standard fatty acid . The bars represent standard deviations of the mean of five independent determinations .

gG4

M . K . BHAT ~xu T . V . GOWDA

Naja naja naja Phospholipase AZ

865

Electrophoresis, mol. wt determination and inrmLnological studies Polyacrylamide gel electrophoresis (PAGE) was carried out by loading 250 pg of crude venom and 25 pg of NN-XIII-PLA, protein on 7 .5% polyacryhimide gels using ß-alanine-acetic acid buffer (pH 4.3) according to the method of Dwts (1964) . The gels were stained with amido black 10 B. The mol. wt of NN-XIII-PLA, was determined by gel filtration on Sephadex G-50 (1 .4 x 92 cm) according to the method of AxnxEws (1964) . Hacitracin (1486), clupeine (4000), sahnine (7000), lysozyme (14,400), trypsin (23,000), egg albumin (45,000) and bovine serum albumin (66,000) were used as standard protein markers. SDSPAGE was carried out on 15% acrylanvde gel slab (13 x 13.5 x 0 .17 cm) containing 0. l % SDS according to the method of L enKe~~ (1970) . Cytochrome C (12,400), myoglobin (16,800), carbonic anhydrase (29,000), egg albumin (45,400) and bovine serum albumin (66,000), were used as standard protein markers . The gels were stained with 0.25% Coomassie billiant blue R-250. Mice were immunized by injecting i .p. half c,n,° doses of the protein NN-XIII-PLA, in O .I ml of saline mixed with an equal volume of Freund's complete adjuvant . Three booster doses were administered at weekly intervals at the same concentration of protein . After resting the animals for 7 days, blood was drawn by retroorbital puncture and antiserum was separated after allowing the blood to coagulate for 4 hr . Ouchterlony immunodiffusion technique was carried out as described by wILLtAM3 and Chose (1971) . Edema-inducing activity The method of Yeautuw~ et al. (1976) was followed . Groups of four to six mice (20-22 g) were injected in the right foot pads of the hind limbs with different doses of protein in 20 pl of saline. The left foot pads received 20 pl of saline which served as control . After 45 min the mice were sacrificed by cervical dislocation and both legs were cut at the ankle joints and weighed individually . The increase in weight due to edema was calculated as the edema ratio which equals the weight of edematous leg x 100/weight of normal leg. Minimum edema dose is defined as the l+g of protein causing an edema ratio of 120% . [ .D determination The procedure of MEten and Tt~KSroN (1986) was followed . The enzyme dissolved in 0.3 ml of saline, was injected i .p. into mice with doses ranging from 0-12 mg/kg body weight . Survival times of each animal were recorded. r.n m was calculated by comparison of the doses injected with the observed survival times of the experimental animals according to the mathematical scheme described by Mitt and T~ttsrox (1986). Histopathological studies The purified NN-XIII-PLA, was injected i .p . at half t,n doses into a group of five mice . After 4 hr the animals were killed by cervical dislocation and autopsy was carried out immediately. Brain, pituitary gland, heart, lung, liver, pancreas, spleen, kidney and testes were removed, fixed in Bouin's solution and subjected to dehydration by processing the tissues through different grades of alcohol, and chloroform: alcohol mixtures . The processed tissues were embedded in paraffin and 5letn thick sections were taken using a Spencer `800' miaotome and were then stained with hematoxylin~sin stains. Myotoxic activity . Five micrograms of NN-XIII-PLA, in 0.1 ml of saline was injected i .m. into the thigh muscle of five mice . Saline (0 .1 nil) was injected into the other thigh which served as a control . All the animals were killed by cervical dislocation 4 hr after injection and the muscle around the site of injection was removed, fixed in Bouin's solution and processed as described in histopathological studies. Fluorescence and protein determinations Fluorescence emission of 200 nmole of NN-XIII-PLA, in 2 ml of 0.9% NaCI solution was recorded after exciting at 280 nm in an Amines Bowman Spectrofiuorometer. Protein was estimated by the method of Lowxv et a1. (1951) . RESULTS

Purhcation ofNN-XIII-PLA z Naja raja raja venom was fractionated on a CM-Sephadex C-25 column by modifying

the method of ICiNt and GownA (1983). The enzyme elution profile showed the presence of phospholipase A~ activity in ten peaks (Fig. 2). Recovery of total PLA2 activity from the CM-Sephadex C-25 column was 47% and the protein recovery was 83% . NN-XIIIPLAZ accounted for 20% of the total protein recovered and 6 .6% of the total PLAZ

866

M. K. BHAT

FIG .

AND T . V.

GOWDA

z

3.

OUCHTERLONY IMMUNODIFFUSION OF ANTIGEN NN-XIII-PLA owGIN) To MYOTOxIN NN-XIII-PLA,.

AND ANTISERUM (MOUSE

Immunodiffusion test was carried out in a 1 % agarose in 0.05 M Tris-HCl buffer (pH 7 .5). The gels were run at 20°C for 36 hr. The central well contained antiserum (50 pl). The peripheral wells contained 50 pl of antigen NN-XIII-PLAz solution in 0.05 M Tris-HCl buffer (pH 7.5) at the following concentrations: Wells 1 and 2 contain 50 hg and wells 3 and 4 contain 100 ug of NNXIII-PLA, antigen, respectively .

activity recovered (Table 2). The contaminating protein found along with NN-XIII-PLAz ( < 2%) was revmoved by subjecting it to gel filtration on a Sephadex G-50 column . Characterization of NN-XIII-PLA 1

Purified NN-XIII-PLAz eluted as a single symmetric peak on Sephadex G-50 cohunn. The antisera raised against this phospholipase reacted with the NN-XIII-PLAz antigen giving a single precipitin line on agarose plate (Fig. 3). This fraction gave a single Amido TAe1.E

Fraction no . Crude venom NN-I-PLA NN-I ;PLA NN-II-PLA NN-III-PLA NN-III;PLA NN-IV-PLA NN-V-PLA NN-VI-PLA NN-XI-PLA NN-XIII-PLA

2.

FRACI7GNATIGN OF Naja naja naja VENOM ON CM-SEPHADEX

C-25

COLUMN

Total protein (mg)

Total activity' (nmoles of FA/min)

Specific activity (nmoles of FA/min/mg of protein)

Yieldt

650 88 .7 19 .6 1.4 1.9 2.4 31 .0 17 .7 18 .2 35.6 I10

69,550 18,290 2510 187 236 350 4731 1513 2064 1029 2178

107 206 128 104 124 l46 153 86 113 29 20

100.0 55 .3 7.6 0.57 0.71 1.1 14 .3 4.6 6.2 3.2 6.6

Total protein recovered from the column was 83% (which includes non-PLA, fractions) and total enzyme activity recovered from the column was 47 .6%. 'Total enzyme activity was estimated in the pooled fractions. tPercent contribution to the total enryme activity recovered. Values are presented as means of two experiments.

Naja naja naja

PhosphoGpase A=

867

FIG. 4 . POLYACRYLAMIDE GEL ELECTROPHORESIS (PAGE) OF CRUDE Naja raja Raja VENOM AND PURIFn~ NN-XIII-PLA, .

Samples containing 250 pg of crude venom and 25 kg of purified NN-XIII-PLA, were loaded on to 7.5% polyacrylamide gels and run at pH 4.3 using ß-alanine acetic acid butïer . An electric current of 3 mA per tube was applied for a period of 4 hr. Methyl green was used as a tracking dye and the protein in the gels was stained with Amido black 10 B. solution. (A) NN-XIII-PLA, ; (B) Naja raja naja venom.

black positive band at pH 4.3 on PAGE (Fig. 4). It also gave a single sharp Coomassie brilliant blue band on SDS-PAGE under both reduced and non-reduced conditions. The bands migrated to the same distance, indicating the presence of only one peptide chain in the enzyme. The mol. wt estimated for NN-XIII-PLAZ was 10,200 by gel filtration and 11,200 by SDS-PAGE (Fig. 5). The positional specificity of NN-XIII-PLAZ was confirmed by two independent methods: (1) PC labelled at Sn-2 position with fluorescent 11 (9-anthroyl) udeca.noic acid; (2) autoclaved E. coli cells with phospholipids labelled at the Sn-2 position with [14C]-oleate. In both cases NN-XIII-PLAZ released fluorescent/radioactive fatty acid and the lysophosphatides did not contain fluorescence/radioactivity . NNXIII-PLAZ exhibited low direct hemolytic activity of 5.4% hemolytis on washed intact human erythrocytes when 100 ~g of enzyme were incubated with the assay mixture for 10 min. It also showed indirect hemolytic activity, causing 50% hemolytis when 100 Fig of enzyme were incubated with the assay mixture for 10 min. The optimum pH and temperature at which NN-XIII-PLAZ showed maximum activity were 7-7.5 and 37-~5°C,

M. K. BHAT ~rm T. V. GOWDA

868

8 "0

c v~ 3 0

u d ô E o~ 0

5~0

Myglobin CytochromeC N-XIII-PLAZ

4~0

T 0

0ß

0" 4

0 "8

O~B

1~0

Relative mobility

FIa. 5. Mol. Wt DETERAiINAT10N OA THE PURIFIED NN-XIII-PLAz ev SDS-PAGE Ei .ecraOPHOx~s . The standard proteins used were : bovine serum albumin (66,000), egg albumin (45,000), carbonic anhydrase (29,000), myoglobin (16,800) and cytochrome C (12,400) . The mol. wt of NN-XIIIPLA Z was estimated to be 11,200 .

respectively . The Km with PC as substrate was 1 .178 x 10 - a M. The Vm ~ of the enzyme was 24 nmoles of FA/min/mg of protein. Lethal potency and biological activities

NN-XIII-PLAZ is slightly more toxic to mice than crude venom with an LD Sp of 2.4 mg/ kg body weight (i.p.), as compared to the LDso of 2.8 mg/kg body weight for the whole venom. The animals injected with NN-XIII-PLAZ showed neurotoxic-like signs such as respiratory distress, paralysis of hind limbs and lacrimation 1-2 hr following injection, with the survival time being 2-6 hr. The mice moved with difficulty and were completely immobilized at the time of death. Post mortem examination did not show any visible damage to the organs . NN-XIII-PLAZ induced edema without hemorrhage when injected into the foot pads of mice with a minimum edema dose of 0 .35 Fig. Edema ratio increased linearly with increase in protein concentration (0.5-3.0 pg). NN-XIII-PLAZ affects the lung but did not affect other organs . It is myotoxic, causing degeneration of muscle fibres and muscle necrosis around the site of injection, as revealed by histological studies (Fig. 6). Fluorescence emission spectra

NN-XIII-PLAZ has a fluorescence emission maximum at 310-316 nm when excited at 280 nm. Tyrosine also exhibited a similar fluorescence emission maximum (Fig. 7).

Naja naja naja Phospholipase A2

FIG . 6 . LIGHT MICROGRAPH OF LONGITUDINAL SECTION OF THIGH MUSCIE OF A MOUSE.

Four hours after i.m . injection of NN-XIII-PLA, (5 hg) in 0. I ml of saline, showing widespread distribution of necrotic muscle fibres and disorganised nuclei . (H) Four hours after i.m . injection of O. l ml of saline showing normal muscle fibres with intact nucleus. Bars 12 plrt . (A)

869

2eo

310 340 EMSSION WAVELENGTH

Flc . 7. FLUORESCENCE EMISSION SPECTRA OF 200 nmole of NN-XIII-PLA, . Inset: fluorescence emission spectra of 20 nmole of tyrosine. Solution was prepared in 0.9% NaCI . The samples were excited at 280 nm. DISCUSSION BRAGANCA and SAMBRAY (1967) reported the presence of a basic protein in the acid precipitate of Naja naja naja venom which had the capacity to lyre erythrocytes embedded in an agar gel plate. SA.LACH et al. (1971) isolated eight PLAZ isoenzymes from Naja naja naja venom; all of them possessed pI values between 4.63 and 5.66. KINI and GOWDA (1983) fractionated Naja naja raja venom on CM-Sephadex C-25 column and reported the presence of eight peaks with PLAZ activity, which included three to four basic phospholipase peaks. NN-XIII-PLAZ, the most basic PLAZ purified from Naja naja naja venom, is similar to other basic PLAIS isolated from snake venoms (NISHIDA et al., 1985 ; VlsxwnxATx et al ., 1987) with respect to mol, wt (10,200-11,200), optimum temperature (375°C) and optimum pH (7-7.5). However, NN-XIII-PLAZ is unique with respect to the following properties: it has low enzymatic activity and has a fluorescence emission maxima at 310316 nm. Basic phospholipases from Trimeresurus jiavoviridis and Vipera russelli exhibited a fluorescence maximum between 340-360 nm (VISIiwnNATx et al., 1987 ; Jayanthi, Ph.D . thesis, 1987). Fluorescence maximum exhibited by NN-XIII-PLAZ is typical of tyrosine residues, indicating the presence of exposed tyrosine residues in the molecule. NN-XIIIPLAZ is toxic (LDsp 2.4 mg/kg body weight of mice) but possesses low enzymatic activity (20 nmoles FA/min/mg) as compared to the phospholipase isolated by BRAGAIVCA et al. (1969) from Naja naja raja venom which has an I,D~ > 19.2 mg/kg body weight and a high specific enzymatic activity . NN-XIII-PLAZ is the first myotoxic phospholipase AZ isolated from the Indian cobra Naja naja naja venom, though there are reports of purification of myotoxic phospholi-

Naja naja naja Phospholipase A,

87 1

pries from Australian elapid venoms (MEHS and .SAMPJIMA, 1980) and from the venoms of Naja nigricollis, Naja haje haje and Naja nivea (MEBS, 1986). Myotoxicity is induced by two different groups of PLAZ enzymes: those which are primarily presynaptically neurotoxic (HARRIS et al., 1975 ; HARRiS arid MACDONNEL, 1981 ; HARRIS and MALTIN, 1982; HAW('~OOD, 1982; GOPALAKRISHNAKONE et al., 1984) and those which are non-neurotoxic (FOHLMAN and ERKER, 1977; MEBS and SAMEJIMA, 1980). Some of the non-neurotoxic PLAZ enzymes induce myoglobinuria, whereas the presynaptically active neurotoxic phospholipases do not induce myoglobinuria, probably due to their high neurotoxicity (MEHS, 1980). ICINI and IWANAGA (1986) reported that neurotoxic phospholipases which are also myotoxic have a distinct cationic site (around residues 79-87), which is available for interaction with membranes. This site is in addition to the neurotoxic site present in the presynaptic neurotoxic phospholipases, which are also myotoxic . NN-XIII-PLAZ may belong to the first group as it induces signs such as respiratory distress, hind limb paralysis and lacrimation in addition to myotoxicity in experimental mice . NN-XIII-PLAZ induces edema in the foot pads of mice without causing hemorrhage . Edema ratio reached a maximum 45 min after injection and was maintained for at least 4 hr . This property is similar to the other basic PLAI S from V. russelli and T.flavoviridis venoms (VISHWANATH et al., 1987). On the other hand, edema induced by a polypeptide from Agkisirodon piscivorus venom is of a transient type and declines after 30 min (BHARGAVA et al ., 1972). The edema inducing property of phospholipase AZ enzymes may be due to the release of arachidonic acid from membrane phospholipids which is in turn transformed into prostaglandins and leukotrienes, resulting in increased vascular permeability (DAMERAU et al., 1975) or due to the release/formation of pharmacologically highly active autacoids such as histamine, 5-hydroxytryptamine, slow reacting substances, prostaglandins and kinins (OHSAICA, 1979 ; Hunxc, 1984). Thus, the edema induced by NNXIII-PLAZ may be mediated through one or more of the above mechanisms . Acknowledgements-The authors thank Dr M . S . THAKUR for his help. M . K . B. thanks the University Grants Commission, New Delhi, India, for financial assistance . REFERENCES AcxvuTxex K. E. and Resiwctterrnww, L. K . (1980) Separation of protein and enzyme components of venom

of the Indian Cobra (Naja naja ) . Proc . Ind. natn. Sci . .lead. B46, 603-b14 .

AeFtYtrrx~N, K . E ., RANGANAI'HA Reo, K . and RAMACHANDRAN, L . K . (1980) A single-step separation

procedure for several protein constituents of venom of the Indian Cobra (Naja naja ). Ind. J. Biochem. Biophys . 17, 228-232 . AxuxEws, P. (1964) Estimation of the molecular weights of proteins by Sephadex gel filtration . Biochem . J. 91, 222-233 . BHARGAVA, N., V~tecnt~ttc, B. B., Vos, C. J ., DE BOMA, I . L . and Tus, T. (1972) Dissociation of oedema provoking factor of Agkistrodom piscivores venom from kininogenase . In : Vasopeptides Chemistry, Pharmacology and Pathophysiology, pp. 141-148 (Beck, N. and StetrreRi, L, Eds). London: Plenum Press . HRAGANCA, B. M . and Sestaxev, Y. M . (1967) Multiple forms of cobra venom phospholipase A . Nature 216, 1210-1211 . BRAGANCA, B. M., Sestaww, Y . M . and GHADIALLY, R . C. (1969) Simple method for purification of phospholipase A from cobra venom . Toxicon 7, 151-157. Corroxae, E., Yerro, C. C. and RosexaERG, P. (1980) Comparison of a relatively toxic phospholipase A, from Naja nigricollis snake venom with that of a relatively non-toxic phospholipase A, from Hemachatus haemachatus venom-I. Enzymatic activity on free and membrane bound substrates. Biochem . Pharmac . 29, 1555-1563 . CONDREA, E., Ft .Erct~R, J . E., RAPUANO, B. E., YANG, C . C . and Ros~asxc, P. (I981a) Dissociation of enzymatic activity from lethality and pharmacological properties by carbamylation by lysines in Naja nigricollis and Naja naja atra snake venom phospholipase A, . Toxicon 19, 705-720.

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M . K . BHAT wxn T . V. GOWDA

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