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Simple method for purification of phospholipase A from cobra venom
Taxiton, 1969, Vol. 7, pp. lS1-157. PerQamon Preae. Printed in Great Britain
SIMPLE METHOD FOR PURIFICATION OF PHOSPHOLIPASE A FROM COBRA VENOM BEATRIZ M. BRAGANCA, Y. M. SAMBRAY
and
ROSHAN C. GHADIALLY
Biochemistry Division, Cancer Research Institute, Tata Memorial Centre, Bombay, India (Accepted for pr~bllcation 25 Naueroher 1968j Abstract-A simple method is described for the purification of phospholipase A . The enryme preparation obtained was purified 20-fold, and was homogeneous on starch gel electrophoresis . The yield was 5 to 6 per cent with a recovery of 60 to 7l) per cent of the total units present in the starting material .
INTRODUCTION
A (phosphatide acyl-hydrolase EC 3.1 .1 .4) catalyses the formation of a lysophosphatide by removing one fatty acid residue from the phospholipid molecule . Several partially purified preparations of this enzyme from the venoms of different snakes have been reported . DE (1944) obtained a `haemolysin' from the venom of Naja tripudians which was relatively non-toxic and had a molecular weight of 32,000. Suzula et a/. (1958) prepared crystalline phospholipase A from Naja naja atra venom which was markedly toxic. The crystalline `Crotoxin' from the venom of Crotalus terrificus terrificus had both haemolytic and neurotoxic activities (FRAENKEL-CONRAT and FRAENKEL-CONRAT, 1950) . SALTO and HANAHAN (1962) showed the presence of two forms of phospholipase A in Crotalus adamenteus venom. Recent reports from this laboratory (BRAGANCA and SAMBRAY, 1967) have demonstrated the occurrence of 7 different forms of phospholipase A in the venom ofthe cobra Naja raja. The present paper describes a simple method for the preparation of the major form of phospholipase A in cobra venom, free from the lethal factors and other known constituents ofthis venom, in a high state ofpurity and with a good yield. PHOSPHOLIPASE
MATERIALS AND METHODS
Naja naja venom was a lyophilised preparation, purchased from the Haffkine Institute, Bombay . CM~ellulose fine mesh, capacity of 0~7 m-equiv per g; Sephadex G-50 (100--300 was obtained from Pharmacia (G.B.) Ltd . ; Tris (hydroxymethyl amino-methane), lecithin (egg yolk), phosphatidyl ethanolamine (brain), and phosphatidyl serine (brain) from Koch Light Lab . Ltd . ; lysolecithin (egg yolk) from V.P. Chest institute, Delhi ; oleic acid, used for the standard fatty acid spot, from Albright and Wilson Ltd . All other chemicals were of analytical grade . Protein determination Protein was estimated by the absorbance method described by 151
KALCKAR
(1947) using
15 2
BEATRIZ M. BRAGANCA, Y. M. SAMBRAY and ROSHAN C. GHADIALLY
the following formulation : protein mg per ml- I ~45 A;,~ m~-074 A~~ m~. where 280 m~ and 260 mtc were the absorbante of the solution at wave lengths 280 and 260 mt~. Toxicity determinations Swiss mice (25-30 g) were injected s.c. with various doses of the material contained in 025 ml of distilled water. The LD P was expressed in mg protein per kg body weight, with deaths recorded at 24 hr. Determination ofphospholipase A activity by haemolytic method Phospholipase A was assayed essentially as described by BoMnx and KALETi'A (1957) . In the assay method, blanks were run with and without addition of egg yolk which eliminate the effect of the direct lytic factor (CONDREA et al., 1964) . The haemolysis was estimated by the increase in absorbance at 540 m~ after incubation of the system for 90 min at 37°. The unit of activity was the amount of enzyme protein required to produce haemolysis corresponding to an optical density of 0~5 at 540 m~. Assay. for the direct lyticfactor The presence of the direct lytic factor (CONDREA et al., 1964) was estimated by the direct count of erythrocytes in a haemocytometer after incubation with the material for 30 min at 37° . For this purpose blood was collected in citrate . The packed cells were washed 3 times with Tyrode solution and suitably diluted to a concentration of 2~5 x l0e cells per ml . Assayfor the cytotoxin preferentially cytotoxic to Yoshida sarcoma cells This was determined according to the method Of BRAGANCA et al . (1967) . Cytotoxicity was defined as the concentration, expressed as tag protein per ml, ofthe active material which caused 50 per cent of the cells to become permeable to lissamine green on incubation for 30 min at 32° . RES U LTS
Isolation procedures An aqueous solution of 1 g lyophilised cobra venom (20 mg per ml) was centrifuged to remove the insoluble material. lit was then treated with 4 vol. of 6 per cent HClOa at 4° with constant stirring. The mixture was allowed to stand for 7 hr at 5°. The precipitate was separated by centrifuging at 80008 for 30 min and dissolved in a minimum amount of distilled water . The pH was adjusted to 6'S to 7~0 with 1 M KOH . Precipitation with HCIOs (Table l, step 2) resulted in 83 per cent recovery of the phospholipase A units in the precipitate. The specific activity was doubled . As previous experiments had shown (BRAGANCA et al., 1967), chromatography on CM-cellulose and elution with acetate buffer (pH 6~0) of molarities ranging from 0005 to 044 M was effective in separating the toxin and many other active proteins from cobra venom, this step was included in the present procedure . The results noted in Fig. l demonstrate that the component having phospholipase A activity eluted at 0005 M buffer, indicating that under these experimental conditions the enzyme was not bound to the ion exchanger and probably eluted at the break-through volume of the column along with the other anionic proteins. As expected, the removal of other venom proteins increased the specific activity from 1~9 to 4~7 (Table l, steps 2 and 3). However, at this stage there was a consistent loss of the total number ofactive units of phospholipase A, which decreased from 83 to 49 per cent.
Simple Method for Purification of Phospholipase A from Cobra Venom
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BEATRIZ M. BRAGANCA, Y. M. SAMBRAY and ROSHAN C. GHADTALLY
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(HCIO, ppt.) ON CM-cELLULOSF Experimental conditions : Column : 1 x25 cm 10 ml (120-140 mg protein) of the material loaded . Elution with acetate buffer (pH 6"0), 3 ml fractions. FIG . I .
CHROMATOGRAPHY OF COBRA VENOM
The fraction having phospholipase A activity was precipitated with (NH 4 )9S04 saturated to 80 per cent. The precipitate (25 mg per ml) was dissolved and dialysed against distilled water for 6 hr at 0°. As cobra venom phospholipase A is heat stable (BRAGANCA and QUASTEL, 1952) further purification was achieved by heating the active peak at 100' for 10 min at pH 5"0. The results (Table l, step 4) show that heating leads to a slight recovery in the total number of units. The specific activity of the products was also higher . The heat treated solution was next passed through 5ephadex G-50 and eluted with 0"05 M Tris buffer (pH 7~5). This resulted in the separation of a major peak which was active and a minor one which was inactive by the haemolytic assay method . The active fraction was dialysed against 3 l. ofdistilled water for 6 hr. It is clear from Table 1, step 5 that Sephadex filtration gave a preparation which was purified 20-fold . Also, the part of the active units lost in the CM-cellulose step were recovered, and the percentage of the total number of units increased from 49 to 658 per cent (fable l, step 5). The I_Dsp was ~ 19"2 mg per kg body weight as compared to 0"7 mg per kg body weight found for crude venom . Electrophoresis on starch gel (Fig. 2) showed a single band at pH 8~6 and at pH 7~0 migrating to the anode. In recent years, CONDREA e! al. (1964) have described the existence of a direct lytic TABLE 2 . THE ABSENCE OF CYTOTOXIC FACTOR (P,) AND DLF A~f DIFFERENT 9TAOE3 OF PURIFICATION OF PHOSPHOLIPASE A
Material 1 . Crudecobra venom 2. HC10, precipitate 3. Final preparation of phospholipase A
CytOLOXlC1Iy * Yoshida sarcoma Washed human cells erythrocytes (tag protein/ml) (Wg protein/m!) 0"71 t "25 > 80 "00
125 > 500 -
" N.g of protein required to produce 50 per cent ttll lysis in 30 min.
FIG . 2. STARCH GEL ELECTROPHORETIC PATTERN OF (A) COBRA VENOM (HCIO, (B) PURIFIED PHOSPHOLIPASE A (i) pH 8~6 (ii) pH 7" 0.
ppt.)
AND
Technique according to POULIK (195 in discontinuous buffer system using 8"0 M urea . pH 8" 6, V 200, temperature 20° and time 7 hr. Protein bands visualised with amido black.
Tox. f.p . 154
FIG .3 .
DEGRADATION OF LECITHIN BY PHOSPHOLIPASE A PREPARATION .
Incubation mixture : 0~2 moles of lecithin, 75 tsg of protein fraction in 40 ~l of NaHC08 buffer pH 7~1 with 8 .moles of Ca'= . Total vol : 100 ~.I made with distilled water, time 1 hr, temperature 37° . The product formed extracted with CHCIs-MeOH (2 :1) and I/5 vol. of H,O. Thin-layer chromatography of the lower phase. Solvent system : (a) Lysoderivative : chloroform-methanohacetic acid-water (75 :23 :7 :3 v;v) (b) Fatty acid : hexane~thyl ether-acetic acid 75 : 23 : 2 v/v). Detection: exposure to I, vapours. 1. Lecithin+Ph . A preparation . 2. Lecithin . 3 . Lysolecithin . 4. Olei c acid .
Simple Method for Purification of Phospholipase A from Cobra Venom
155
factor which brings about haemolysis of erythrocytes in the absence of an external source of phospholipids . Cobra venom had also beets shown to contain a cytolytic factor which preferentially lyses certain tumour cells (BRAGANCA et al., 1967) . The direct lytic factor, tested by lyses of red blood cells, described by CONDI2EA et al. was absent from the preparation after precipitation with HCIO,. The purified phospholipase A was also free of the cytolytic factor as tested in a standard system using Yoshida sarcoma cells (Table 2). Thin-layer chromatography (Fig. 3) of the products formed when pure lecithin was incubated with the purified enzyme demonstrated the presence of lysolecithin and a fatty acid. The specificity of purified phospholipase A preparation for various phospholipids was detet7llined by following the decrease in the aryl ester bonds by the hydroxamic acid method essentially as described by STERN and SHAPIRO (1953) . The results given in Table 3 show that the enzyme attacks only one ester bond per mole of lecithin or phosphatidyl ethanolamine, as expected of phospholipase A. Lysolecithin was not attacked, showing the absence of phospholipase B in the preparation . Phosphatidyl serine, which in thinlayer chromatography appeared to be pure with only a slight contamination of the lyso derivative, was also not attacked by the cobra venom phospholipase A. TABLE 3.
SUBSTRATE SPECIFICITY OF PURIFIED PH08PHOLIPASE A
Substrate (mole) 1 . Lecithin Lecithin 2. Lysolecithin Lysolecithin 3. Phosphatidyl ethanolamine Phosphatidylethanolamine 4. Phosphatidyl serine Phosphatidyl serine
Phospholipase A (Eag protein) -1-F -i-f-
Total styl groups present/Wmole of phospholipid
No. of styl groups liberated
200
-
I"35 116
0"19
1 ~OS
2" 00
1"08 200 1 ~76
095 092
0"24
(1)~ (0) (1)
(0)
incubation mixture: 2 tamoles phospholipid in 0~1 ml ether, 0"2 ml NaHCOs buffer (pH 7"1), 25 l+g protein phospholipase A, 8-0 moles Ca'+ . Total volume 1"0ml, temperature 37° and time 120 min. 'Number in brackets=whole number of acyl groups liberated. DISCUSSION
The presence of phospholipase A was first demonstrated in cobra venom by DELEZENNE and LBUFBT in 1911 . This venom is a rich source of phospholipase A and has been employed to prepare lysolecithin from lecithin. Although considerable evidence (BRAGAIVCA and PATEL, 1965) has demonstrated that phospholipase A is relatively non-toxic, many of the purified preparations of this enzyme (Suzuxr et al., 1958 ; FRAENICEL-CONRAT and FRAENKELCONRAT, 1950) have been shown to carry the toxic components of cobra venom. Suzuki's crystalline phospholipase A (Suzuxt et al., 1958), on passage through CM-cellulose, had 4 protein fractions, only 2 of which had phospholipase A activity . The isolation procedure described here for the preparation of phospholipase A gives a good yield of the enzyme in a minimal number of operations . The phospholipase A activity
156
BEATRIZ M. BRAGANCA, Y. M. SAMBRAY and ROSHAN C. GHADIALLY
was determined by the haemolytic assay method which measures only the haemolysis resulting in the presence of an external source of phospholipids (egg yolk), and does not include the haemolysis produced by such factors as the direct lytic factor (CONDRF.A et al., 1964). This procedure is simple and convenient for screening the various fractions during isolation of the enzyme . Treatment of cobra venom with HCLO, under cold conditions led to the precipitation of more than 80 per cent of the total units of enzyme activity . While the toxins, as well as many other proteins including the cytotoxic factor (BRAGANCA et al., 1967) which make up more than 60 per cent of the total protein of the venom, remained in the supernatant . The lethality of the preparation at this stage increased by a factor of 9. Chromatography on CM-cellulose with acetate buffer of varying concentrations gave further purification of the enzyme, especially from the lethal factors. It was observed however, that treatment with CM-cellulose led to a loss of the total number of phospholipase A units, which decreased by 50 per cent. These could be recovered to a small extent when the preparation was heated to 100°. A considerable increase in the recovery of the total number of phospholipase A units was obtained when the heated product was subjected to gel filtration . Experiments to be described elsewhere have shown that the enzyme is partially bound to an inhibitor when it is passed through CM-cellulose . The inhibitor separates out on heating and can be removed from the enzyme mixture by gel filtration . Studies (BRAGANCA and SAMBRAY, 1967) have shown that cobra venom contains at least 7 different forms of phospholipase A, all of which are precipitatable by HC104, but which are eluted from the cationic exchanger at different buffer concentrations . It has also been found that the particular kind of enzyme described here is present in much greater proportion and eluted out at the break-through volume under the conditions employed. Nevertheless, this step helps to separate this kind from the rest. Thus, the loss of activity units found after CM-cellulose chromatography is probably due to the presence of the inhibitor which complexes with the enzyme, and also to the elimination of other forms of phospholipase A . The specificity of the enzyme for various phospholipids was studied by a method which measures the decrease in acyl ester bonds produced on incubating the substrate with the enzyme . The enzyme preparation degraded lecithin and phosphatidyl ethanolamine but not phosphatidyl serine. Lecithin was found to be more actively degraded. Thin-layer chromatography of the product formed when the preparation was incubated with a sample of hydrogenated lecithin produced the corresponding lyso derivative and a fatty acid. It was also free of phospholipase B described in some snake venoms (DOERY and PEARSON, 1964) . Many of the other known enzymes present in cobra venom could also be eliminated, as they are unstable to heat (BRAGANCA and QUASTEL, 1952) . The final enzyme was purified 20 .fold, and the yield was 5 to 6 per cent with a recovery of 60 to 70 per cent. The protein was homogeneous on starch gel electrophoresis, with an isoelectric point below pH 7~0 . Acknowledgement-We thank the Wellcome Trust for ßnancial support. REFERENCES H. G. and KA>.e~rrA, U . (1957) Chromatography of rattlesnake venom : A separation of three phosphodiesterases . Bloch/m. Blophys. Acta 24, 619. BRAGAtvcA, B. M., PATBL, N. T. and BADRINATH, P. G. (1967) Isolation and properties of a cobra venom factor selectively cytotoxicto Yoshida sarcoma cells. Blochim. Blophys. Acta 136, 508. BRAGANCA, B. M, and PAS. N. T. (1965) Glycoproteins as components of the lethal factors in cobra venom Nqja ngja. Can.J. Biochem. 43, 915. BRAGANCA, B. M. and Qvastec, J. H. (1952) Action of snake venom on acetylcholine synthesis in brain. Nature, Lortd.169, 695. BOtKAN,
Simple Method for Purification of Phospholipase A from Cobra Venom
157
Bx .~aexce, B. M. 8IId SAMBRAY, Y. M. (1967) Multiple forms of cobra venom phospholipase A. Natare, Lord. 216,1210. Corrox$a, E., ns Vrtrtnis, A. and Meaga, J. (1964) Henolysis and splitting of human erythrocyte phospholipid by snakevenons . Blochim. Biophys. dcta 84,60. DE, S. S. (1944) Physico-chemical studies on haemolysin-I . Crystalline haemolysin (Lecithinase). dnn. Blochen. exp.Med. 4, 45 . Dg~zexnE, C. and L.~~r, S. (1911) Action du venin de cobra sur le serum de cheval . Ses rapports avec l'hémolyse . C. r. SEanc, dead. Sd. Parts 152, 790. Doexv, H. M. and Pe,~xsoN, J. E. (1964) Phospholipase B in snake venons and bce venom. Blochen. J. 92,599. Fw~xxarrCoxxwr, H. and Fa~ax~ar.-Cox~r, J. (1950) Inactivation of crotoxin by group-specific magenta. Blochim. Btophys. Acte 5, 98 . K~+t.ceax, H. M. (1947) Differential spectrophotometry of purine compounds by means of specific enzymes. J, blot. Chem .167, 461 . Povr uc, M. D. (1957) Starch gel electrophoresis in a discontinuoussystem of buffers. Natwe, Loxd.180,1477. S~ua~o, K. and H~v~x, D. J. (1962) A study of the purification and properties of the phospholipase A of Crotalrrsadamanteus venom. BlochemLstry 1, 521 . Sreßrr, I. and Sxnrmo, B. (1953) A rapid aad simple method for the determination of esterified fatty acids and for total fatty acids in blood.J. Clin . Path . 6,158. Suzuxi, T., Iwnx~ae, S. and Kww~c~, S. (1958) Isolation of crystalline lecithinase A (haemolysin) from Formosan cobravenom (Najanq/a atra Cantor). J. Pharm. Soc. (Japan)78, 568.
SIMPLE METHOD FOR PURIFICATION OF PHOSPHOLIPASE A FROM COBRA VENOM BEATRIZ M. BRAGANCA, Y. M. SAMBRAY
and
ROSHAN C. GHADIALLY
Biochemistry Division, Cancer Research Institute, Tata Memorial Centre, Bombay, India (Accepted for pr~bllcation 25 Naueroher 1968j Abstract-A simple method is described for the purification of phospholipase A . The enryme preparation obtained was purified 20-fold, and was homogeneous on starch gel electrophoresis . The yield was 5 to 6 per cent with a recovery of 60 to 7l) per cent of the total units present in the starting material .
INTRODUCTION
A (phosphatide acyl-hydrolase EC 3.1 .1 .4) catalyses the formation of a lysophosphatide by removing one fatty acid residue from the phospholipid molecule . Several partially purified preparations of this enzyme from the venoms of different snakes have been reported . DE (1944) obtained a `haemolysin' from the venom of Naja tripudians which was relatively non-toxic and had a molecular weight of 32,000. Suzula et a/. (1958) prepared crystalline phospholipase A from Naja naja atra venom which was markedly toxic. The crystalline `Crotoxin' from the venom of Crotalus terrificus terrificus had both haemolytic and neurotoxic activities (FRAENKEL-CONRAT and FRAENKEL-CONRAT, 1950) . SALTO and HANAHAN (1962) showed the presence of two forms of phospholipase A in Crotalus adamenteus venom. Recent reports from this laboratory (BRAGANCA and SAMBRAY, 1967) have demonstrated the occurrence of 7 different forms of phospholipase A in the venom ofthe cobra Naja raja. The present paper describes a simple method for the preparation of the major form of phospholipase A in cobra venom, free from the lethal factors and other known constituents ofthis venom, in a high state ofpurity and with a good yield. PHOSPHOLIPASE
MATERIALS AND METHODS
Naja naja venom was a lyophilised preparation, purchased from the Haffkine Institute, Bombay . CM~ellulose fine mesh, capacity of 0~7 m-equiv per g; Sephadex G-50 (100--300 was obtained from Pharmacia (G.B.) Ltd . ; Tris (hydroxymethyl amino-methane), lecithin (egg yolk), phosphatidyl ethanolamine (brain), and phosphatidyl serine (brain) from Koch Light Lab . Ltd . ; lysolecithin (egg yolk) from V.P. Chest institute, Delhi ; oleic acid, used for the standard fatty acid spot, from Albright and Wilson Ltd . All other chemicals were of analytical grade . Protein determination Protein was estimated by the absorbance method described by 151
KALCKAR
(1947) using
15 2
BEATRIZ M. BRAGANCA, Y. M. SAMBRAY and ROSHAN C. GHADIALLY
the following formulation : protein mg per ml- I ~45 A;,~ m~-074 A~~ m~. where 280 m~ and 260 mtc were the absorbante of the solution at wave lengths 280 and 260 mt~. Toxicity determinations Swiss mice (25-30 g) were injected s.c. with various doses of the material contained in 025 ml of distilled water. The LD P was expressed in mg protein per kg body weight, with deaths recorded at 24 hr. Determination ofphospholipase A activity by haemolytic method Phospholipase A was assayed essentially as described by BoMnx and KALETi'A (1957) . In the assay method, blanks were run with and without addition of egg yolk which eliminate the effect of the direct lytic factor (CONDREA et al., 1964) . The haemolysis was estimated by the increase in absorbance at 540 m~ after incubation of the system for 90 min at 37°. The unit of activity was the amount of enzyme protein required to produce haemolysis corresponding to an optical density of 0~5 at 540 m~. Assay. for the direct lyticfactor The presence of the direct lytic factor (CONDREA et al., 1964) was estimated by the direct count of erythrocytes in a haemocytometer after incubation with the material for 30 min at 37° . For this purpose blood was collected in citrate . The packed cells were washed 3 times with Tyrode solution and suitably diluted to a concentration of 2~5 x l0e cells per ml . Assayfor the cytotoxin preferentially cytotoxic to Yoshida sarcoma cells This was determined according to the method Of BRAGANCA et al . (1967) . Cytotoxicity was defined as the concentration, expressed as tag protein per ml, ofthe active material which caused 50 per cent of the cells to become permeable to lissamine green on incubation for 30 min at 32° . RES U LTS
Isolation procedures An aqueous solution of 1 g lyophilised cobra venom (20 mg per ml) was centrifuged to remove the insoluble material. lit was then treated with 4 vol. of 6 per cent HClOa at 4° with constant stirring. The mixture was allowed to stand for 7 hr at 5°. The precipitate was separated by centrifuging at 80008 for 30 min and dissolved in a minimum amount of distilled water . The pH was adjusted to 6'S to 7~0 with 1 M KOH . Precipitation with HCIOs (Table l, step 2) resulted in 83 per cent recovery of the phospholipase A units in the precipitate. The specific activity was doubled . As previous experiments had shown (BRAGANCA et al., 1967), chromatography on CM-cellulose and elution with acetate buffer (pH 6~0) of molarities ranging from 0005 to 044 M was effective in separating the toxin and many other active proteins from cobra venom, this step was included in the present procedure . The results noted in Fig. l demonstrate that the component having phospholipase A activity eluted at 0005 M buffer, indicating that under these experimental conditions the enzyme was not bound to the ion exchanger and probably eluted at the break-through volume of the column along with the other anionic proteins. As expected, the removal of other venom proteins increased the specific activity from 1~9 to 4~7 (Table l, steps 2 and 3). However, at this stage there was a consistent loss of the total number ofactive units of phospholipase A, which decreased from 83 to 49 per cent.
Simple Method for Purification of Phospholipase A from Cobra Venom
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BEATRIZ M. BRAGANCA, Y. M. SAMBRAY and ROSHAN C. GHADTALLY
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(HCIO, ppt.) ON CM-cELLULOSF Experimental conditions : Column : 1 x25 cm 10 ml (120-140 mg protein) of the material loaded . Elution with acetate buffer (pH 6"0), 3 ml fractions. FIG . I .
CHROMATOGRAPHY OF COBRA VENOM
The fraction having phospholipase A activity was precipitated with (NH 4 )9S04 saturated to 80 per cent. The precipitate (25 mg per ml) was dissolved and dialysed against distilled water for 6 hr at 0°. As cobra venom phospholipase A is heat stable (BRAGANCA and QUASTEL, 1952) further purification was achieved by heating the active peak at 100' for 10 min at pH 5"0. The results (Table l, step 4) show that heating leads to a slight recovery in the total number of units. The specific activity of the products was also higher . The heat treated solution was next passed through 5ephadex G-50 and eluted with 0"05 M Tris buffer (pH 7~5). This resulted in the separation of a major peak which was active and a minor one which was inactive by the haemolytic assay method . The active fraction was dialysed against 3 l. ofdistilled water for 6 hr. It is clear from Table 1, step 5 that Sephadex filtration gave a preparation which was purified 20-fold . Also, the part of the active units lost in the CM-cellulose step were recovered, and the percentage of the total number of units increased from 49 to 658 per cent (fable l, step 5). The I_Dsp was ~ 19"2 mg per kg body weight as compared to 0"7 mg per kg body weight found for crude venom . Electrophoresis on starch gel (Fig. 2) showed a single band at pH 8~6 and at pH 7~0 migrating to the anode. In recent years, CONDREA e! al. (1964) have described the existence of a direct lytic TABLE 2 . THE ABSENCE OF CYTOTOXIC FACTOR (P,) AND DLF A~f DIFFERENT 9TAOE3 OF PURIFICATION OF PHOSPHOLIPASE A
Material 1 . Crudecobra venom 2. HC10, precipitate 3. Final preparation of phospholipase A
CytOLOXlC1Iy * Yoshida sarcoma Washed human cells erythrocytes (tag protein/ml) (Wg protein/m!) 0"71 t "25 > 80 "00
125 > 500 -
" N.g of protein required to produce 50 per cent ttll lysis in 30 min.
FIG . 2. STARCH GEL ELECTROPHORETIC PATTERN OF (A) COBRA VENOM (HCIO, (B) PURIFIED PHOSPHOLIPASE A (i) pH 8~6 (ii) pH 7" 0.
ppt.)
AND
Technique according to POULIK (195 in discontinuous buffer system using 8"0 M urea . pH 8" 6, V 200, temperature 20° and time 7 hr. Protein bands visualised with amido black.
Tox. f.p . 154
FIG .3 .
DEGRADATION OF LECITHIN BY PHOSPHOLIPASE A PREPARATION .
Incubation mixture : 0~2 moles of lecithin, 75 tsg of protein fraction in 40 ~l of NaHC08 buffer pH 7~1 with 8 .moles of Ca'= . Total vol : 100 ~.I made with distilled water, time 1 hr, temperature 37° . The product formed extracted with CHCIs-MeOH (2 :1) and I/5 vol. of H,O. Thin-layer chromatography of the lower phase. Solvent system : (a) Lysoderivative : chloroform-methanohacetic acid-water (75 :23 :7 :3 v;v) (b) Fatty acid : hexane~thyl ether-acetic acid 75 : 23 : 2 v/v). Detection: exposure to I, vapours. 1. Lecithin+Ph . A preparation . 2. Lecithin . 3 . Lysolecithin . 4. Olei c acid .
Simple Method for Purification of Phospholipase A from Cobra Venom
155
factor which brings about haemolysis of erythrocytes in the absence of an external source of phospholipids . Cobra venom had also beets shown to contain a cytolytic factor which preferentially lyses certain tumour cells (BRAGANCA et al., 1967) . The direct lytic factor, tested by lyses of red blood cells, described by CONDI2EA et al. was absent from the preparation after precipitation with HCIO,. The purified phospholipase A was also free of the cytolytic factor as tested in a standard system using Yoshida sarcoma cells (Table 2). Thin-layer chromatography (Fig. 3) of the products formed when pure lecithin was incubated with the purified enzyme demonstrated the presence of lysolecithin and a fatty acid. The specificity of purified phospholipase A preparation for various phospholipids was detet7llined by following the decrease in the aryl ester bonds by the hydroxamic acid method essentially as described by STERN and SHAPIRO (1953) . The results given in Table 3 show that the enzyme attacks only one ester bond per mole of lecithin or phosphatidyl ethanolamine, as expected of phospholipase A. Lysolecithin was not attacked, showing the absence of phospholipase B in the preparation . Phosphatidyl serine, which in thinlayer chromatography appeared to be pure with only a slight contamination of the lyso derivative, was also not attacked by the cobra venom phospholipase A. TABLE 3.
SUBSTRATE SPECIFICITY OF PURIFIED PH08PHOLIPASE A
Substrate (mole) 1 . Lecithin Lecithin 2. Lysolecithin Lysolecithin 3. Phosphatidyl ethanolamine Phosphatidylethanolamine 4. Phosphatidyl serine Phosphatidyl serine
Phospholipase A (Eag protein) -1-F -i-f-
Total styl groups present/Wmole of phospholipid
No. of styl groups liberated
200
-
I"35 116
0"19
1 ~OS
2" 00
1"08 200 1 ~76
095 092
0"24
(1)~ (0) (1)
(0)
incubation mixture: 2 tamoles phospholipid in 0~1 ml ether, 0"2 ml NaHCOs buffer (pH 7"1), 25 l+g protein phospholipase A, 8-0 moles Ca'+ . Total volume 1"0ml, temperature 37° and time 120 min. 'Number in brackets=whole number of acyl groups liberated. DISCUSSION
The presence of phospholipase A was first demonstrated in cobra venom by DELEZENNE and LBUFBT in 1911 . This venom is a rich source of phospholipase A and has been employed to prepare lysolecithin from lecithin. Although considerable evidence (BRAGAIVCA and PATEL, 1965) has demonstrated that phospholipase A is relatively non-toxic, many of the purified preparations of this enzyme (Suzuxr et al., 1958 ; FRAENICEL-CONRAT and FRAENKELCONRAT, 1950) have been shown to carry the toxic components of cobra venom. Suzuki's crystalline phospholipase A (Suzuxt et al., 1958), on passage through CM-cellulose, had 4 protein fractions, only 2 of which had phospholipase A activity . The isolation procedure described here for the preparation of phospholipase A gives a good yield of the enzyme in a minimal number of operations . The phospholipase A activity
156
BEATRIZ M. BRAGANCA, Y. M. SAMBRAY and ROSHAN C. GHADIALLY
was determined by the haemolytic assay method which measures only the haemolysis resulting in the presence of an external source of phospholipids (egg yolk), and does not include the haemolysis produced by such factors as the direct lytic factor (CONDRF.A et al., 1964). This procedure is simple and convenient for screening the various fractions during isolation of the enzyme . Treatment of cobra venom with HCLO, under cold conditions led to the precipitation of more than 80 per cent of the total units of enzyme activity . While the toxins, as well as many other proteins including the cytotoxic factor (BRAGANCA et al., 1967) which make up more than 60 per cent of the total protein of the venom, remained in the supernatant . The lethality of the preparation at this stage increased by a factor of 9. Chromatography on CM-cellulose with acetate buffer of varying concentrations gave further purification of the enzyme, especially from the lethal factors. It was observed however, that treatment with CM-cellulose led to a loss of the total number of phospholipase A units, which decreased by 50 per cent. These could be recovered to a small extent when the preparation was heated to 100°. A considerable increase in the recovery of the total number of phospholipase A units was obtained when the heated product was subjected to gel filtration . Experiments to be described elsewhere have shown that the enzyme is partially bound to an inhibitor when it is passed through CM-cellulose . The inhibitor separates out on heating and can be removed from the enzyme mixture by gel filtration . Studies (BRAGANCA and SAMBRAY, 1967) have shown that cobra venom contains at least 7 different forms of phospholipase A, all of which are precipitatable by HC104, but which are eluted from the cationic exchanger at different buffer concentrations . It has also been found that the particular kind of enzyme described here is present in much greater proportion and eluted out at the break-through volume under the conditions employed. Nevertheless, this step helps to separate this kind from the rest. Thus, the loss of activity units found after CM-cellulose chromatography is probably due to the presence of the inhibitor which complexes with the enzyme, and also to the elimination of other forms of phospholipase A . The specificity of the enzyme for various phospholipids was studied by a method which measures the decrease in acyl ester bonds produced on incubating the substrate with the enzyme . The enzyme preparation degraded lecithin and phosphatidyl ethanolamine but not phosphatidyl serine. Lecithin was found to be more actively degraded. Thin-layer chromatography of the product formed when the preparation was incubated with a sample of hydrogenated lecithin produced the corresponding lyso derivative and a fatty acid. It was also free of phospholipase B described in some snake venoms (DOERY and PEARSON, 1964) . Many of the other known enzymes present in cobra venom could also be eliminated, as they are unstable to heat (BRAGANCA and QUASTEL, 1952) . The final enzyme was purified 20 .fold, and the yield was 5 to 6 per cent with a recovery of 60 to 70 per cent. The protein was homogeneous on starch gel electrophoresis, with an isoelectric point below pH 7~0 . Acknowledgement-We thank the Wellcome Trust for ßnancial support. REFERENCES H. G. and KA>.e~rrA, U . (1957) Chromatography of rattlesnake venom : A separation of three phosphodiesterases . Bloch/m. Blophys. Acta 24, 619. BRAGAtvcA, B. M., PATBL, N. T. and BADRINATH, P. G. (1967) Isolation and properties of a cobra venom factor selectively cytotoxicto Yoshida sarcoma cells. Blochim. Blophys. Acta 136, 508. BRAGANCA, B. M, and PAS. N. T. (1965) Glycoproteins as components of the lethal factors in cobra venom Nqja ngja. Can.J. Biochem. 43, 915. BRAGANCA, B. M. and Qvastec, J. H. (1952) Action of snake venom on acetylcholine synthesis in brain. Nature, Lortd.169, 695. BOtKAN,
Simple Method for Purification of Phospholipase A from Cobra Venom
157
Bx .~aexce, B. M. 8IId SAMBRAY, Y. M. (1967) Multiple forms of cobra venom phospholipase A. Natare, Lord. 216,1210. Corrox$a, E., ns Vrtrtnis, A. and Meaga, J. (1964) Henolysis and splitting of human erythrocyte phospholipid by snakevenons . Blochim. Biophys. dcta 84,60. DE, S. S. (1944) Physico-chemical studies on haemolysin-I . Crystalline haemolysin (Lecithinase). dnn. Blochen. exp.Med. 4, 45 . Dg~zexnE, C. and L.~~r, S. (1911) Action du venin de cobra sur le serum de cheval . Ses rapports avec l'hémolyse . C. r. SEanc, dead. Sd. Parts 152, 790. Doexv, H. M. and Pe,~xsoN, J. E. (1964) Phospholipase B in snake venons and bce venom. Blochen. J. 92,599. Fw~xxarrCoxxwr, H. and Fa~ax~ar.-Cox~r, J. (1950) Inactivation of crotoxin by group-specific magenta. Blochim. Btophys. Acte 5, 98 . K~+t.ceax, H. M. (1947) Differential spectrophotometry of purine compounds by means of specific enzymes. J, blot. Chem .167, 461 . Povr uc, M. D. (1957) Starch gel electrophoresis in a discontinuoussystem of buffers. Natwe, Loxd.180,1477. S~ua~o, K. and H~v~x, D. J. (1962) A study of the purification and properties of the phospholipase A of Crotalrrsadamanteus venom. BlochemLstry 1, 521 . Sreßrr, I. and Sxnrmo, B. (1953) A rapid aad simple method for the determination of esterified fatty acids and for total fatty acids in blood.J. Clin . Path . 6,158. Suzuxi, T., Iwnx~ae, S. and Kww~c~, S. (1958) Isolation of crystalline lecithinase A (haemolysin) from Formosan cobravenom (Najanq/a atra Cantor). J. Pharm. Soc. (Japan)78, 568.