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COMPARATIVE CHARACTERIZATION OF TWO TOXIC PHOSPHOLIPASES AZ FROM INDIAN COBRA (NAJA NAJA NAJA) VENOM B.

S. BASAVARAJAPPw and T. VT~RABwSAPPA Gownw"

Department of Studies in Biochemistry, Manasagangothri, University of Mysore, Mysoro-570 006, India (Received 20 February 1992 ; accepted 22 Apri! 1992)

S. Bwswvwlwwrrw and T. V. Gownw. Comparative characterization of two toxic phospholipases AZ from Indian cobra (Naja raja raja) venom. Toxicon 30, 1227-1238, 1992.-Indian cobra venom contains many phospholipase AZ (PLA 2) toxins . In the present study two toxic PLA2s have been purified from the Indian cobra (Naja naja raja) venom by column chromatography . The NN-XI;and NN-XIb-PLA 2s have mol. wts between 10,700 and 15,000 . The NN-XIàPLA2 induces myotoxic effects, oedema and neurotoxicity in mice and has an i.p. Ln,o of 8.5 mg/kg body weight . The NN-XI;PLA2 is also cytotoxic to Ehrlich ascites tumour cells. The other PLA2, NN-XIb, in contrast has an i.p. I,n~ of 0.22 mg/kg body weight, and it induces acute neurotoxicity. The NN-XIb-PLA2 is devoid of the other biological activities which are exhibited by NN-XI,-PLA2. B.

IIVTRODUCTION

A2 (EC 3.1 .1 .4; PLA2) enzymes are major constituents of snake venoms. Venom PLA2 enzymes differ in enzymatic and pharmacological properties but show a remarkable degree of structural homology (Iwwxwaw and Suzuxl, 1979; RosaIVBi~tG, 1979, 198 . Snake venom PLA2s show a wide variety of toxicities and induce several pathological symptoms (RINI and Evwxs, 1989) in addition to their contribution to digestion of prey . Lethal potency of basic PLA2s was apparently inversely related to their in vitro enzymatic activity (ROSSxB~tc et al., 1983; FLSrcItTat et al., 1980; CONDRBw et al., 1981). Snake venoms contain multiple forms of PLA2 and have been isolated from almost all snake venoms. In most cases, the purification involves a number of steps (BxwGwxcw et al., 1%9; SwL.wcx et al., 1%8; SFmowH et al., 1973; JAYAN~ and GoWDw, 1983 ; HwzL.gl-r and Di~xts, 198 . Indian cobra (Naja naja naja) venom has been reported to contain as many as 14 isoenzymes of PLA2 (Slm owx et al., 1973). BxwT and GOWDA (1989) identified the presence of ten peaks with PLA2 activity by a modified method of Kn~n and Gownw (1983) . Three basic PLA2s have been purified to homogeneity, from this venom. They are characterized as a myotoxic (NN-XIII-PLA2) and neurotoxic (NN-Vb-PLA2s NN-IVb,-PLA2) (BxwT and GownA, 1988, 1991 ; BxwT et al., 1991). In the present PHOBPHOi;.IPASE

' Author to whom correspondence should be addressed. 1227

1228

B. S. BASAVARAJAPPA and T. V. GOWDA

investigations we report the purification and characterization of two more basic toxic phospholipases A2 which differ in biochemical and pharmacological properties from each other . MATERIALS AND METHODS

Lyophilized whole venom of Naja n4ja naja (Batch No . K-~ was purchased from ISCICS Ltd (Madras, India) . Swiss wrstar male mice weighing 20-22 g, used for pharmacological studies, were obtained from the Mysore University Animal House (Manaaagangothri, Myeore, India). CM-Sephadex G25 (4 .5 meq/g), Sephadex G-50 and mol. wt markers were purchased from Pharmacia Fme Chemicals, Uppaala, Sweden. Linoleic acid, 1-nitroso-2-naphthol, Freund's complete and inoompleis adjuvants were from Sigma Chemical Company (St. Louis, U.S .A.) . Phosphatidylcholine (PC) was pt+epared from hen's egg yolk according to the method of SnvarErnx et al. (i%~. All other chemicals and solvents were of analytical grade. The solvents were rediatilkd beforo use . Purifecatian ojphospholipate A~ enzymes Nq%a ngja naja venom was fractionated on a CM-Sephadex G25 column as described earlier (BrrAT and Gowns, 1989), lyophilized whole venom of Naja nqja naja (1 ~ was dissolved in 8 ml of 0.02 M phosphau buffer

(pH7.0) and applied to a CM-Sephadex G25 column (1 .4 x 120cm) which was eluted stepwise, using phoaphais buffers of various molarities and pH values at 20°C . The Bow rais was adjusted to 40 ml/hr and 10 ml fractions were collected by a Frao-100 automatic fraction collector. Protein elution was monitored at 280 nm using a Bausch and Lomb spectroaio-2000. The enzyme elution was studied by assaying 0.1 ml aliquots from alternais tubes for PLAs activity . Individual fractions of the enzyme peak were pooled, desalted, lyophilized separaisly and stored at -18°C. Rechromatography ojNN-XI-PLA fraction on CM-Sephadex C-25 colwrm

Fraction NN-XI is the protein fraction containing 6% of the total venom protein recovered and 3% of the PLA activity from CM-Sephadex C-25 column and was named NN-XI-PLA. Sixteen milligrams of NN-XI-PLA was dissolved in 3 ml of 0.1 M phosphau buffer (pH 8.0) and loaded on a CM-Sephadex G25 column (1 .6 x 30 cm). The protein was eluted stepwise, using phosphais buffers of molaritiea and pH values (0 .1 ; 8.0, 0.15; 8.0) with a flow rais of 30 ml/hr at 20°C, and 2.5 ml fractions were collected by Frac-100 automatic fraction collector. Protein elution was monitored at 280 nm using a Haunch end Lomb Spectronio-2000. Pkospholipare A~ assay

Phospholipase A~ sassy was done as described in Bier ~ad Gowns (1989) by using 1000nmoles of egg phosphatidylcholine (PC) as a substrau. Linoleic acid wsa used as the ataadard fatty acid . PLA2 activity is expressed as nmoles of frce fatty acid released/min/mg of protein. Positional specj~rcity

The positional apedficity of purified phoapholipases was determined (Faetaort et ol., 1974) using autoclaved E. cola a1>8 with phospholipid labelled at the Sn-2 position with (~~ oleais . Electroplroresdr and molecular weight dettrmination

Polyacrylamide gel electrophoresis (PAGE) was carried out for PLAT enzymes oa 7.5% polyacrylamide gels using ß-alanine-acetic acid buffer (pH 4.3) according to the method of Devis (1964) . The gels wero stained with amido black 10 B. The molecular weights were estimated by gel filtration on Sephadea G-50 (1 .4 x 92 cm) (Armresws, 1964). Bacitracin (1 .486), sahnine (7000), trypsin (23,000) and bovine serum albumin (66,000) were used as standard protein markers. SDS-PAGE was carried out on 1SX acrylamide gel slab containing 0.1 % SDS (L~o~aa, 1970). Low mol. wt markers were used from Pharmacia. The gels wet+e stained with 0.25% Coomassie brilliant blue R-250. Detox{frcatiar of NN-XI, PL .Is and preparation ojantt-NN-XI,PLAs antibody

NN-XI,-PLAs was detoxified by formalin treatment, according to the method of Koxno et al. (1970). NN-XI,-PLAz toxoid (0.5 mg) in phoephais buffered saline was mixed with equal volumes of Freund'a oompleis

1229 Cobra Venom i?hoapholipases A2 adjuvant and injected into male rabbit intradermally at aeweral spots. Four booster doses were administered at weekly intervals at the same concentration, but with equal volumes of Freund's incomplete adjuvant. After resting the animal for 10 days, blood was drawn from the marginal ear vein and antisera was separated after allowing the blood to coagulate for 24 hr.

Preparation ojq-8lobulin and detection ojantibodies Gamma-globulin (anti-NN-XI ;PLAZ Ig) was prepared from male rabbit antiserum injected with NN-XI,-PLA2 toxoid, by precipitation with ammonium sulphate as descxibed by Ham$ and Sc~nvtcc (1973) . The ouchterlony agar gel double diffusion technique was used to detect precipitating antibodies as described by Wn.~ r~uc and Cttw~ (1971).

Fluorescence and protein deterrr6rations Fluorescence emission of phoepholipase Az in 2 ml of saline (pH 7.4) solution was recorded after exciting at 280nm. The protein was estimated by the method of LowRY et al. (1951).

Oedemaforming activity The method of Ywaswxwww et a1. (19717 was followed . Different amounts of PLAT (dissolved in 20 pl of saline solution) were injected subcutaneously in the right foot pad of groups of four mice (20-22 g) . The left foot pad was injected with 20 pl of saline solution. After 45 min the mix were eacriSced bY cervical dislocation and both legs wen cut off at the ankle and weighed individually. The increase in weight due to oedema was calculated as oedema ratio by the following equation. Oedema ratio equals the weight of the oedematous kg x 100/weight of normal kg . Minimum oedema dose is defined as the amount of protein causing an oedema ratio of 120°/..

Myotoxicity dettrmirratiore PLA2s (emu dose, dissolved on 0.3 ml of phosphate bufferod saline solution, pH 7.4) were injected (i.p .) into groups of mice (six animals each). After 2 hr, blood samples were collected by retroorbital puncture. Creative phosphokinase and lactate dehydrogenase levels were determined in the serum using span diagnostic kits 23903 and 25939, respectively. Control experiments were performed by injecting saline solution under the same conditions.

Cytotoxicity Cell viability was assayed as described in G~wBrtot+t+ et al. (1989), using Edith satires tumour cells (EAT ills) grown in the peritoneal cavity of Swiss albino mix, suspended in Tyrode's ring buffer and incubated with various oonoentrationa of PLA= (10-501rg/10° ills in 2ml) at 37°C for 30 min. Ono-hundtnd microGtrea of 1% Trypan blue saline solution was then added, and stained (dead) and unstained (viable) ails were independently counted using a haemocytometer .

Dettrrrlnation of direct acrd indirect Jrocmolytk activity Direct and indirect haemolytic activity was assayed as described by VI~iwANA1H er al. (1987). Human red blood ills separated from freshly collected blood from healthy donors were used for assay.

i~~ determination The enzyme, dissolved in 0.3 ml saline, was injected i.p. into mice with doses ranging from 1 to 13 mg (NN-XI,-PLAN and 50 to 8001rg (NN-XIb-PLA~/kg body weight . Survival time of each animal was reoot+ded. t~~ was calculated by comparison of the doses injected with the observed survival time of the experimental animals according to the mathematical scheme dexribed by Mgt and TFa;AâB'I'ON (198 . The values given in all the experiments, t S.E ., were based on six independent determinations.

1230

B. S. BASAVARAJAPPA and T. V. GOWDA

L `~CL O

Ô m ii ~

E c

Fraction number Ftc . 1. CM-St~tun~c C-25 cot..ue~t xectoeoawroax~ttY of NN-XI-PLA meoM Naja naja naja vwox. The column (1 .6 x 30 cm), equilibrated with O.l M phosphate buffer (pH 8.0), was loaded with 16 mg of NN-XI-PLA dissolved in 3.0 ml of the same buffer. Elution was carried out stepwise with phosphate buffer of molarities aad pH as indicated, at 211°C. Fractions of 2.5 ml were collected at a Bow rate of 30 ml/hr. RESULTS

Purgation ofPLA 2 enzymes Naja naja raja venom was fractioned on a CM-Sephadex C-25 column (BHAT and GownA, 1989). The enzyme elution profile indicated the presence of 10 fractions with PLAz activity. NN-XI-PLA fraction, which accounted for 6% of the total venom protein recovered and 3.2% of the total PLA2 activity recovered, was refractionated on CM-Sephadex C-25 column. The enzyme elution profile showod the presence of PLA2 activity in two peaks (NN-XI, and NN-XI~J (Fig. 1). Recovery of total PLA2 activity and protein from the CM-Sephadex C-25 column was 93 .3% and 75%, respxtively (Table 1). The contaminating protein found along with NN-XI, aad NN-XIb PLA2s ( < 1 %) was removed on a Scphadex G-50 gel filtration column . Homogeneity and characterization of PLAz enzymes The purified enzymes moved as a single band in both polyacrylamide (Fig. 2) and SDS-polyacrylamide gel electrophoresis under both reduced and non-reduced conditions and yielded a single, symmetrical peak on Sephadex G-50 column, indicating that they are homogeneous. The mol. wt determined by Sephadex G-50 for NN-XI, and NN-XIb-PLA2 was 11,200 . However, by SDS-PAGE they were found to be 15,000 and 10,700, respec-

1231 Cobra Venom Phospholipasea A2 T~aLe 1. Stnot~tr oe rujtnnc~nox or PLAz t~vzrra+s t7aoY Nr~a rraJa rwja vBtvoY Total protein (mg)

Total' activity (nmoka of FA released/min)

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

Yieldt

Purification

CM-Sephadex G25 Whole venom NN-XI-PI;.A

1000 35 .6

107,000 1553

107.0 43 .6

100.0 3.3

1.0 -

Rechromatography on CM-Sephadex C-25 NN-XI-PLA NN-XI,-PLA NN-XIb-PL.A

16.0 6.4 5.6

697.92 376.44 274.40

43 .6 58 .8 49 .0

100.0 57 .8 42 .2

1.0 1.4 1 .2

Step

Total protein recovered by rechromatography of NN-XI friction on CM-Sephadex G25 was 75% and total enzyme activity tecoverd from the column was 93 .3% (not shown) . 'Total activity of the enzyme was estimated from the elution profile . $Per ant contribution to the total activity recovered values are presented as means of five acperimenta.

tea. 2. Pa.r~cxr~s ~. ~.ecrxot~oseeats (PAGE) aF drone Naja raja nqM (A), NN-XI-PLA

(B), Ptntn+~ NN-XI, (C), rum NN-XIe (D) PLA~e. Samples containing 230 Pg of crude venom, 100beg of NN-XI-Pï,.A, 60 Pg of purified NN-XI, and NN-XIb-PLA3s were loaded on to 7 .5% polyacrylanvde gds and ru~ et pH 4.3 using ß-alanine antic acid buffer. An elecUric 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 lOB soiution .

1232

B. S. BASAVA1tAJAPPA and T. V. GOWDA 5.0 4.8 r 'm

3 4.8 ro

m 4 .4 ô E ae 0 4 .2 4 .0 0

0

0.2

0 .4

0.8

0.8

1 .0

Relative mobility FYo . 3. Moucuux w~axr nsta~uwenox oF rux~ NN-XI, nrm NN-XIb-PLAza HY SDS-PAGE ~c,-ixor .

tively (Fig. 3). When anti-NN-XI ;PLAZ Ig was tested on ouchterlony immtmodiffusion plates against the protein samples of NN-XI;PLAZ tozoid, NN-XI, and NN-XIb-PLAZ gave only one precipitin line in all cases (Fig . 4). In contrast, preimmune serum did not give any precipitin line on an agar gel plate with any of these antigens . The positional specificity of phospholipases was confirmed using autoclaved E. coli cells with phospholipid labelled at the sn-2 position with ('~ oleate . Both the enzymes releasod radiolabelled fatty acid and non-radiolabelled lysophosphatides. Both NN-XI, and NN-XIb-PLA Z exhibited indirect haemolytic activity causing 28% and 23% haemolysis, respectively, whereas NN-XIb-PLA Z showed low direct haemolytic activity of 2.4% haemolytis, when 100 Rg of PLAZs were incubated with the assay mixture on washed intact human erythrocytes for 10 min. The optimum pH and temperature at which NN-XI, and NN-XIb-PLAZ showed maximum activity were 7.3 to 7.8, 7.0 to 7.5 and 45 to 50°C, respectively . The ~ for NN-XI, and NN-XIb-PLAZ with PC as a substrate was 9.43 x 10-5 M and 2 x 10_s M, respectively .

Lethal potencies and biological activities Of the two PLAZ enzymes, NN-XIb-PLAZ was most toxic, with an >~m value of 0.22 mg/kg body weight (i.p.) in mice. The animals injected with this protein showed acute neurotozic symptoms such as respiratory distress and paralysis of hindlimbs, 1-2 hr following injection. The mice moved with di~culty and were completely immobilized at the time of death. i ns value for NN-XI;PLAZ in mice was 8.5 mg/kg body weight . Postmortem examination for NN-XI;PLAZ and NN-XIb-PLAZ showed no internal bleeding in the peritoneal cavity of the mice. The other biological properties of phospholipases are given in Table 2.

Cobra Venom Phospholipases Az

1233

FyO. 4 . OUCH~RLONY IMMUDK)DLFFIJSION OF ANTIOENa AOA1NSr ANr®ODY; I!®liJNODIFFUSION WAS cAxte~ our IN 1 % AaAaose aß. . The central well contained anti-NN-XI, Ig (100 pg). (a) NN-XI;toxoid (50 Rg); (b) NN-XI,-PLAz (lug) ; (c) NN-XIb-PL.A: (100pB) .

Changes in serum levels of creative phosphokinase and lactate dehydrogenase

A drastic increase in serum creative phosphokinase and lactate dehydrogenase levels was observed for NN-XI;PLAv whereas only the creative phosphokinase level was high for NN-XIb-PLAZ, after 2 hr of PLAZ injection (Fig. 5).

TABt.a 2 . PttArtMwwLOGtcAi. FttoF~rms oF NN-XI, Arro NN-XIb-PLAz eNZr~s Properties mss° (mg/~g ~Y weight) Survival time ( t 5 sec) (min) Neurotoxicity Myotoxicity Cytotoxicity Oedema inducing activity (a) Minimum oedema dose (~ (b) Oedema ratio (5 Pg)

NN-XI,-PIA~

NN-XIb-PLA2

8 .5 t 0.2 95 .0 t 1 .0 Mild Yes Yea Yes 1 .710.1 140.0 t 5.5

0.22 f 0.003 77 .00 t 1 .000 Acute Nil Nil Nil Nil Nil

Values of oedema ratio are expressed as mean+S .D. (n = ~

1234

H. S. HASAVARAJAPPA and T. V. 130WDA

FYo. 5. C~~xo>s ~ s»elnt cleanrn~m rxas>~POSw~ w~ ucxere n~lrnAOO>~v~ csve~s nv rrox~ut (A), NN-XI. (H), and NN-XIb (CJ PLAz injected (iP.) mice.

Fluorescence emission spectra NN-XI;PLA z has a fluorescence emission maximum at 310-320 nm whereas NN-XI b-PLA z has one at 350-365 nm (tryptophan specific).

(tyrosine specific),

DISCUSSION

Two basic PLAZ enzymes have been purified from Indian cobra venom. The fraction NN-XI-PLA (BRAT and GownA, 1989) was refractionated on CM-Sephadex C-25 followed by gel filtration on Sephadex G-50 column, and resulted in the purification of

Wavelength (nm)

Fya. 6. F co pox sraclxe oa 25 kg oa NN-XI, nivn NN-Jüb-PLA z. Solution was prepared in saline (pH 7.4) and excited at 280 mn . NN-XI~PLAN ("--") and NN-XIb-PLA= (O-O)~

Cobra Venom Phosphoflpases A2

1235

NN-XI, and NN-XIb-PLAZ enzymes. They were confirmed as PLAZ as they released radioactive fatty acid from autoclaved E. coli cells with phospholipids labelled at the sn-2 position with ('~ oleate . They were homogeneous as judged by gel filtration, PAGE and SDS-PAGE . The mol. wt of both the PLAIS was found to be 11,200 by Sephadex G-50 column chromatography . However, by SDS-PAGE it was found to be 15,000 for NN-XI, and 10,700 for NN-XIb. Formation of a single precipitin line against NN-XI~PLAZ indicates the presence of specific antibodies. Anti-NN-XI Q-PLA Z Ig gave a single identical precipitin line against NN-XIb-PLA Z, suggesting that anti-NN-XI;PLAZ Ig cross-reacts with the other neurotoxic PLAI S from Naja raja raja venom. The pH and temperature optimum for NN-XI, and NN-XIbPLAZ was consistent with the values reported for PLAIS from the same venom (BxwT and Gownw, 1989, 1991 ; BxwT et al., 1991) and other snake venoms (N~w et al., 1985; Vrsxwwxw~rfz et al., 1987, 1988 ; Kws~ and Gownw, 1989; Twxwswxc et al., 1990). The NN-XIb-PLAZ has a specific activity of 58.8 nmoles (nmoles of FA released/min/mg of protein) and 1~ value 9.43 x 10- s M, whereas NN-XIb-PLA Z has 49 .0 nmoles and ~ value 2.0 x 10- s M . These values are in contrast to the enzyme activity of 20 nmoles and ~ value 1.2 x 10 -s M for NN-XIII-PLAZ (BxwT and Gownw, 1989) but are nearer to the specific activity of 101 nmoles, 1~ value 2.5 x 10- s M for NN-Vb-PLAZ (BxwT et al., 1991), from the same venom. Thus NN-XI, and NN-XIb PLAIS are more active compared to NN-XIII-PLAZ. The NN-XI;PLAZ has an Ln~ value of 8.5 mg/kg body weight belonging to the low-toxicity enzymes (mow > 1 mg/kg body weight) . In contrast NN-XIb-PLA Z with an LDsp Value Of 0.22 mg/kg body weight belongs to the high toxicity enzymes (LDs p > 0.1 mg/kg but less than 1 mg/kg body weight) (ROSHNBSRG, 1988). This PLAZ analogous to NN-Vb-PLA Z with an t n~ value of 0.27 mg/kg body weight and reported to induce neurotoxic symptoms (BxwT et al., 1991). Two other PLAZ enzymes have been purified from this venom, NN-XIII basic PLAZ, r..n~ 2.4 mg/kg body weight (BxwT and Gownw, 1989), and an acidic PLAZ purified by BxwGwxcw et al. (1969) had > 19.5 mg/kg body weight belong to low toxicity enzymes. The lethal potency of NN-XI;PLAZ is less when compared to crude venom whose r..n~ is 3.6 mg/kg body weight; this may be due to synergistically acting components present or due to the presence of other highly toxic components in the venom. These results are in contrast to the report of EAxat (1978) ; Fr.Erc~ et al. (1980) that `generally basic PLAZ enzymes are highly toxic followed by acidic and neutral ones', but in agreement with the finding that pharmacological effects of PLAIS cannot be directly correlated with their iscelectric points (Coxnxsw et al ., 1983). The NN-XI;PLAZ, being a less toxic enzyme, induces mild oedema in the foot pads of mice without causing haemorrhage. Oedema ratio reached a maximum in 45 min after injection, oedeomatic condition maintained for at least 4 hr. This property is similar to the NN-XIII-PLAZ from the same venom (BxwT and Gownw, 1989) and other snake venoms (VL4HWwNwTH et al., 1987 ; Cfma et al., 1989; SBi.ISTRE et al ., 1990). The NN-XI;PLAZ exhibited myotoacicity as shown by an increased level of cytoplasmic markers, CPK and LDH level in the serum. Similar increase in serum CPK and LDH levels was reported for myotoxin from Bothrops venom (Gv~xBZ et al ., 1989; LoMOxz~s et al., 1990). Myotoxicity of NN-XIII-PLAZ from Naja raja raja venom was confirmed by histopathology of muscle (BxwT and Gownw, 1989) as well as measuring serum LDH level (BxwT, Ph.D. Thesis, 1991), Generally, myotoxic PLAZ enzymes are classified into two groups; those that are primarily presynaptically neurotoxic (HARxis and MwLrnv, 1983 ; GOPALAKRISHNAKONS et al., 1984) and those that are non-neurotoxic (Foxi.Mwx and

H. S. IiASAVA1tATAPPA and T. V. GOWDA

1236

100

w H Q W Ô a

80 80

40

W

20

0

0

10

20

90

40

50

Protein (Rg/assay)

FIa.

~. EFPSGT OF INCRBAStPIO CONCENTRA170N OF

NN-XI;PLAz ON VIAHILCfY

OF

EAT CHLL4.

and SAMFJIMA, 1980) . The NN-XI,-PLAZ belongs to the first group as it exhibited neurotoxic symptoms such as respiratory distress, hind limb paralysis and lacrimation in addition to myotoxicity in experimental mice. The NN-XI;PLAZ exhibited cytotoxicity (Fig. 7) on EAT cells and this effect was proportional to the concentration of NN-XI; PLAZ enzyme (within the tested concentration limits). Phospholipase A Z enzymes from Naja raja raja and Viper~ russelli also had cytotoxic properties (BABU and GOWDA, 1991) . In contrast to NN-XIII-PLA Z which exhibits both direct and indirect haemolytic activity, NN-XI;PLAZ showed only indirect haemolytic activity. The NN-XI b-PLAZ, in contrast to NN-XI, and NN-XIII-PLA Z, is highly tonic, exhibiting acute neurotoxic symptoms, and it lacks other pharmacological properties. In this respect it is similar to NN-Vb-PLA Z, but NN-XIb-PLAZ exhibited both direct and indirect haemolytic activity, whereas NN-Vb-PLAZ exhibits only indirect haemolytic activity . The fluorescence emission maxima of NN-XI;PLAZ (310-320 nm) is analogous to NN-XIII-PLAZ (310-316 nm) and contrast with NN-XIb and NN-Vb-PLAZ (340-360 nm) from the same venom and from other snake venoms (VI3IiWANATH et al., 1987 ; JAYAxTt-u et al., 1989). This suggests the presence of three types of PLA Z enzymes in snake venom based on the fluorescence emission pattern, i.e. those that are (1) tyrosine specific, less toxic, (2) tryptophan specific, and highly toxic, and (3) both tyrosine and tryptophan specific, and moderately toxic. The NN-XI,-PLAZ belongs to enzymes of low toxicity with multiple pharmacological properties, whereas NN-XIb-PLAZ is highly toxic with a single effect . The multiple pharmacological properties exhibited by NN-XI;PLAZ may be due to the presence of multiple pharmacological sites (Knvi and EvAxs, 1989) on this molecule. These results are in agt~eement with the earlier reports for the cobra venom PLA IS (BI3AT et aL, 1991), that the PLA IS which have a single pharmacological site are more toxic than the PLAI S that have multiple pharmacological sites. In other words, highly toxic PLAIS induce fewer EAx>~t, 1977; MEas

Cobra Venom Phosphoüpaaes A~

1237

toxic effects, and low toxic PLAZs induce several toxic effects and hence dilution of the specificity. Acknowledgement-B.S .B. thanks the Lady Tata Memorial Trust, Bombay, for their financial assistance. REFERENCES Aivnxews, P. (1964) Estimation of the molecular weights of proteins by sephadex gel filtration. Blochem. J. 91, 222-233. Beau, A. S. and GOWDA, T. V. (1991) Efforts ofchemical modification on enzymatic and toxicological properties of phospholipase A= from Naja nsja ngja and Vipers russelli snake venom. Toxicon 29, 1251-1262. Blur, M. K. and Gowne, T. V. (1989) Purification and characterization of a myotoxic phoapholipase A2 from Indian cobra (Ngjs raja raja) venom. Toxicon 27, 861,873. Bier, M. K. and Gowne, T. V. (1991) Isolation and characterization of a lethal phospholipase AZ (NN-IV b-PLAN from the Indian cobra (Naja raja raja) venom. Biochem . Int. 25, 1023-1034. Blur, M. K., Pxeseu, H. N. and Gowne, T. V. (1991) Purification and nt,e*,~rr'*;~ tjon of neurotoxic phoapholipase Az from Indian cobra (Naja rraja raja) venom. Toxicon 29, 1345-1349 . Bxeaexce, B. M., Swamtuv, Y. M. and Gaeuur.LY, R. C. (1%9) Simple method for purification of phospholipase A from cobra venom. Toxicon 7, 151-157. Cxuv, H. C., Cr~tv, I. J. and Tmva, C. M. (1989) Edema formation and degranulation of mast cells by a basic phospholipase A2 purified from Trimeresurus mucrosquamatus snake venom. Toxicon 27, I15-125. CHwsrzo>=e, S., TsuvwsAwe, S., Serww~+e, F. and Mt~z, A. (1989) Nigexine, a phoapholipase A, from cobra venom with cytotoxic properties not rslated to esterase activity. Purification, amino acid sequence and biological properties . J. biol. Chem . 264, 13,289-13,297. Coxnaee, E., Fr trrc~x, J. E., RAPUANO, B. E., Yexc, C. C. and Ito~>imea, P. (1981) Effect of modification of one histidine residue on the enzymatic and pharmacological properties of a toxic phospholipase A from Hemachatus keamacltatus and Naja najs ngja snake venoms. Toxicon 19, 61-71 . Coxnaee, E., Reruexo, H. E., FiErcrn=a, J. E., YeNa, C. C. and Ro~xaeAa, P. (1983) Ethoxy formylation and guanidination of snake venom phwpholipase Ab effect on enzymatic activity, lethality and some pharmacological properties. Toxicon 21, 209-218. Dwts, B. J. (1964) Disc electrophoresis-II . Method and application to human serum proteins . Ann. N.Y. Acad. Sci. 121, 404-d27. Ewt~, D. (1978) Studies of presynaptically neurotoxic and myotoxic phospholipeses Ar. In : Versatility of Proteins. pp. 41431 (Ly C. H., Ed.) . New York: Academic Press. FLSrc~t, J. E., RePUetvo, B. E., CorronFa, E., Yexa, C. C., RYAN, M. and RosexaEaa, P. (1980) Comparison of a relatively toxic phospholipase A2 from Naja nigricollis snake venom with that of a relatively non-toxic phospholipase A2 from Hemachtw haemachatus snake venom. II . Pharmacological properties in relationship to enzymatic activity . Biochem. Pharmac. 29, 1565-1574. Fofn.~seN, J. and EAt~e, D. (1977) Isolation and characterization of a lethal myotoxic phospholipase A from the venom of the common sea snake Enhydrirra schistosa causing myoglobinuria in mice . Toxicon 1S, 385-394. FxANSON, R., PArauxce, P. and Ersaecx, P. (1974) PhosphoGpid metabolism by phagocytic cells. Phospholipase A= associated with rabbit polymorphonuclear leukocyte granules. J. Lipid Res. 15, 380-388. GorAr.AgxmtANAxoNE, P., D~IP31Ea, D. W., Hewcoon, B. J. and Ecn~t, H. Y. (1984) Cellular and mitochondrial changes induced in the structure of marine muscle by crotoxin, a neurotoxic phospholipase A2 complex. Toxtcon 22, 85-98. GurmaAt:z, J. M., Cxev®, F., G», J. A., LOMONTE, B., CeerecHO, Z. and SCÜ08rN3KY, K. (1989) Myonecrosis induced in mice by a basic myotoxin isolated from the venom of the snake Bothrops monmifer (jumping viper) from Costa Rica . Toxicon 27, 735-745. HASws, J. H. and MAr.rav, C. A. (1982) Myotoxic activity of crude venom and the principal neurotoxin, taipoxin of the Australian taipan, Oxyuramis seutellatus. Br. J. Phsrmac. 76, 61-75. HAZr.err, T. L. and DeNNrs, E. A. (1985) A)finity chromatography of phoapholipase A~ from Naja nsja naja (Indian cobra) venom. Toxicon 23, 45966. Hene, K. and Scxwrcx, H. G. (1973) Preparation of globulin. In : Handbook of Experimental Immunology, Vol. 1, pp . 61-64 (W>rrn, D. M., Ed.). New York : Academic Press. IweNAae, S. and Suzuu, T. (1979) Enzymes in snake venom. In : Handbook of Experimental Pharnwcology, Vol. 52, pp . 61-144 (L~, C. Y., Ed .) . Berlin: Springer . JAYANTrn, G. P. and GOWDA, T. V. (1983) Purification of acidic phospholipaees from Indian cobra (Naja raja naja) venom. J. Ckromat. 281, 393-3% . IAYANTrû, G. P., KASwxr, S. and GownA, T. V. (1989) Dissociation of catalytic activity and neurotoxicity of a basic phospholipase A2 from Russell's viper (Vipers russelh) venom. Toxicon 27, 875-885.

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Comparative characterization of two toxic phospholipases A2 from Indian cobra (Naja naja naja) venom.

Indian cobra venom contains many phospholipase A2 (PLA2) toxins. In the present study two toxic PLA2s have been purified from the Indian cobra (Naja n...
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