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Phospholipase isoenzymes from Naja naja venom —I. Purification and partial characterization
Toricon. 1973 . Vol. 11, pp . 481
90. Per¢amon Prcss. Printed in Grcat Britain.
PHOSPHOLIPASE ISOENZYMES FROM NAJA NAJA VENOM -I. PURIFICATION AND PARTIAL CHARACTERIZATION J. SHILOAH, * CHAYA KLIBANSKY 8nd A. DE Vx1Es The RogofFWellcome Medical Research Institute, Tel-Aviv University, 13eilinson Medical Center, Petah Tilcva, Israel (Accepted jor pubi'icalton 14 May 1973)
AbstraM-Six phospholipase A~ontaining fractions were isolated from cobra (Nglo agja) venom by ion~atchange and gel filtration chromatography. Each fraction was homogeneous by immunological criteria and comprised more than 1 phospholipaae isoenzyme, their toW number amounting to 14 . The molecular weights of the isoenzymes ranged from 11000 to 24000. None of them contained free SH groups . The six fractions exh]bited similar specific activities. INTRODUCTION
years, cobra (Naja naja) venom has been shown to contain several phospholipase iscenzymes. BRAGANCA and SAMBRAY (1967) reported the presence of 7 phospholipase iscenzymes in Naja naja venom and obtained 1 phospholipase fraction which appeared homogeneous on starch gel electrophoresis . CURRIE et al. (1968) crystallized from this venom a phospholipase A fraction which was further separated into two phospholipase A components, but no additional information was given concerning their purity . SALACH et al. (1971), applying iscelectric focusing to a Naja naja phospholipase preparation obtained after heat-inactivation and gel filtration, concluded that there existed 9-11 phospholipase iscenzymes in the venom. Only one of these was purified to give a single band on disc electrophoresis . Jn the course of our studies on the specificity of snake venom phospholipases A, Naja naja phospholipase iscenzymes were obtained in 6 fractions. Their purification and molecular properties are described in the present communication. IN RECENT
EXPERIMENTAL PROCEDURE
Naja naja venom was purchased from L. Light and Co. Ltd., Colnbrook, England. Chromatographic techniques All procedures for purification of the enzyme were conducted at 4°. CM-cellulose (Whatman) was washed with alkali and acid, and equilibrated with ammonium acetate buffer, pH 5~8, at the appropriate ionic strength . DEAF-cellulose (Whatman) was washed with acid and alkali, and equilibrated with ammonium bicarbonate-ammonium carbonate buffer, pH 8~1, at the appropriate ionic strength . Sephadex G-50 (Pharmacia, Uppsala, Sweden) was allowed to swell in water for 24 hr, packed into a column under a pressure "In partial fulfilment of therequirements for a Ph .D. degree of the Feinberg Graduate School, the Weiunann Institute of Science, Rehovot, Israel. TOXICON 1973 Vol. Il
481
482
J. SHILOAH, CHAYA KLIBANSKY and A. DE VRIES
head of 50 cm water, and equilibrated overnight with O~l M ammonium bicarbonate pH 7~1 at a flow rate of 10 ml/hr. Protein concentrations were estimated by the method of LOWRY et al. (1951) using bovine serum albumin as a standard ; absorbance was read at 750 nm. Electrophoresis
Disc electrophoresis in polyacrylamide gel (Eastman Organic Chemicals, Rochester, N.Y.) was performed using the method of Dwls (1964) . The separation was carried out in a Shandon Disc Electrophoresis Apparatus (Shandon Scientific Co. Ltd., London, England) in 5 x 5 mm gels (15 per cent acrylamide in Tris-glycine buffer, pH 8~3). The amount of protein applied was 20-100 pg in 100 pl buffer solution containing one drop of glycerol. Proteins were stained with 1 per cent Amido Black in 7 per cent acetic acid for 30 min, and washed thereafter with the latter acid containing the anion exchange resin Dowex AG 1-x 8 (220-400 mesh Bio-Rad) . Immunization procedure
The antiserum against Naja raja venom was prepared using white New Zealand rabbits of about 3 kg weight . The animals were injected intradermally in several locations on the back and in each foot pad with 1 ml emulsion prepared from equal volumes of whole venom in saline (6 mg/ml) and complete Freund's adjuvant. Ten days later an antigen booster was given intramuscularly (1 mg of whole venom in complete Freund's adjuvant). The booster injection was repeated twice, at 4-week intervals. One week after each booster injection the animals were bled by cardiac puncture and sera were tested for precipitating antibodies. The antibody content of the immune sera was estimated by absorbance measurements at 280 nm after dissolving the washed specific precipitates in 0~1 N NaOH . Immunological techniques
Immunodi$'usion experiments were performed as described by Oucx~i.ox~t (1948) with 1 per tent Difco agar in 0025 M veronal buffer pH 8~2. The peripheral wells were filled with 001 ml of each phospholipase fraction (0~5 mg/ml) and the central well with 0025 ml rabbit antiserum. Diffusion was allowed to proceed for 2-3 days at 4° in a moist chamber. After extensive washing with saline for 3 days, the plates were covered with Whatman paper and dried at 37° for several hr. The paper was removed and the plates were placed for 5 min in a solution of 1 per cent Amido Black in 7 per cent acetic acid, and then rinsed in 3 changes of the latter solvent. For immunoelectrophoresis, glass slides (76 x 2~ were coated with 3~5 ml of 1 "5 per cent agar solution in 0"025 M veronal buffer pH 9"0. Whole venom and purified phospholipase fractions were placed in the holes and electrophoresis was performed for 120 min at 140 V and 4°. The concentration of the tray buffer was 0"OS M. After completion of the electrophoresis, anti-whole Naja naja serum was placed in the main trough and the slides were developed, stained and washed as described above for immunodiffusion. Molecular weight determination and Thiol-group estimation
The molecular weight determination of the phospholipases were tamed out by dodecyl sulfate polyacrylamide gel electrophoresis as described by WESEIt and OSHOxrt (1969), using RNase, lysozyme, performec acid-oxidized hemoglobin, trypsin, chymotrypsinogen A, pepsin and ovalbumin as molecular weight standards. TOXICON 1973 Yol. 11
483
Naja naja phospholipase isoenzymcs
Free thiol groups of purified phospholipases in 1 per cent SDS were determined with 5,5-dithio-bis (2-nitrobenzoic acid) according to the method of ELLMAN (1959) . Assay of phospholipase A activity For specific activity determinations at the various purification stages of the enzyme, the pH-stet method (TTA s Radiometer autotitrator type SBR2C/SBU 1) was employed, using dilute egg yolk (1 :20 with saline) as substrate. Liberation of acid was measured at pH 8"0 and 37° by titration with 0"005 N NaOH under a constant stream of argon. One unit of activity was defined as the amount of enzyme which liberates one prllole of free fatty acid per min under the above conditions . This was permissible since the activity of a given sample was found to be proportional to its concentration in the assay mixture, indicating that the substrate was present in large excess. , RESULTS
Pur~cation procedure Crude lyophilized Naja naja venom (2"16 g) was dissolved in 5 ml of 0005 M ammonium acetate buffer pH 5"8 and dialyzed at 4° overnight against 2 I . of the same buffer. The material was then subjected to serial ion-exchange and gel filtration chromatography, as summarized in Table 1 and illustrated by Figs. 1-9. The phospholipase containing fractions TARIE 1 . SUMMARY OF THE PURIFICATION OF PHOSPHOLIPAaBa FROM
Fraction
Protein (mg)
Lyophilized venom CM-cellulose Erst column (Fl-I-F2-}F3) " (zone I, Fig. 2) Sephadea G-SOt (Fl ~-F2~-F3) DF.AFrcelluloae first column (Fl+F2) (zone I, Fig. 3) F3 (zone II, Figl . 3) DEAE-cellulose second column Fl (zone I, Fig. 4) F2 (zone II, Fig. 4) DBAP-cellulose third column F3 (zone I, Fig. S) CM~ellulose first wlumn (F4+FS) (zone II, Fig. 2) CM-cellulose second column (F4) (zone I, Fig. 6) (FS) (zone II, Fig. 6) Sephadex G-50, F4 (zone I, Fig. 7) Sephadea G-S0, FS (zone I, Fig. 8)
CM-cellulose first column (F6) (zone III. Fig. 2) CM-oeiluloae third column (F6) (zone I, Fig. 9) Sephade~c G-50f, F6
2160
Specific activity (snits/mg protein) 831 600 385 Total activity (units)
Naja naja
Yield (%~ 100
VENOM
Ratio to specific activity of whole venom 1
43 "5
139 635
3210
16"8
8" 34
41~0
129 970
3170
15"6
8" 23
12 "9 23 "9
40 377 76122
3130 3185
4"9 9"2
8"13 8"27
4"2 5"0
l3 230 16000
3150 3200
1"59 l "92
8"18 8 "31
18 "5
58 460
3160
7"03
8"21
49ß
69 020
1400
8"3
3"64
12"6 19~6 3"5 9"0
14 918 35 672 l0 920 27 630
1184 1820 3120 3070
1 "8 4"3 1 "31 3"32
3"O8 4~73 8"10 7"97
85 "7
83 986
980
20 "1 16 "3
60 903 50 856
3030 3120
10~1 7"3 6~11
2"55 7"87 8"]0
"Fs in parenthesis represent eluates containing fractions separated in subsequent steps. tSephadal G-50 column which did not result in purification of materials applied on and one single peak was obtained. 71DXlCON 1973 Vol. !!
484
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FIß. I. PtntISIC,~~noN sue. Abbreviations : S-Sephadex ; CMC-carboxymethyl xllulose ; DEAEC-diethylamincethyl cellulose; serial numbers indicate appropriate columns (see text). The elution buffers for SG-S0, DEAEC and CMC columns wen 0"1 M ammonium bicarbonate pH 7~1, ammonium bicarbonate-amawnium carbonate pH 8~1, ammonium acetate pH S"8, respectively. The elution gradients of the two latter buffers are given in the legends to Figs. 3-9.
3000
o.sa 26 22
IA
~â2
_I ae 0 N aa d
ale als 14 .1E 0
IOOOâ
Q4
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000
1600
2400 ~200 Elation Velonlelml)
4000
4000
.10 Q06 Q08
0
FIß. 2. CM~.LULOSe CIIJtOMA1nCiRAPHY, wear COLiJI~I . Purification of 2"16 g crude venom on a 2"2 x 90-cm column in a 4"2 L linear gradient of 0"OOS M vs 0"S M ammonium acetate buffer, pH S'8, at 112 ml hr -1 . 7~OXICON J973 Yo(. 11
Naja ngja phospholipase iscenzymes
485
~-0.30 i
-oao t0 . _ e¬0 ä E
-a10 ~~ É E
Elusion Volume (ml)
Fta. 3. DEAB~-cs.ctrcosE etneo~roan.~rt>r, ~nes"r coiv~nv. Puri&cation of zone I (Fig. 2), after gel filtration through Sephadex G-S0, on a 1 x 20-cm rnlumn in a 180 ml linear gradient of 0"01 M vs 0~5 M ammonium bicarbonate-ammonium carbonate buffer, pH 8" 1, at l8 ml hr'' .
0.3
E 0
0 N O Ô
3000
oz
ô -020
a E
-alo E lution Volum~ (ml~)
E E t
1~YYo. 4. DEAE~,oi.o~ c»eol~wz+oaawrxx, secoxn ooLU~v . lurißcation of zone I (Fig. 3) on a 1 x20-cm oohunn in a 220 ml linear gradient of 005 M vs 0"25 M ammonium bicarbonate-ammonium carbonate buffer, pH 8"1, at 18 ml hr'1 . T~OXICON7973 Vol. II
486
J. SHILOAH, CHAYA KLIBANSKY and A. DE VRIES
Elution Volunw(mlI
FYo. 5. DFAE-ea?uLOSe c~o~~rooiurtn, amen wLU>m~. Purification of combined zone II (Fig . 3) and zoo III (Fig. 4) on a I x 20-cm column in a 220 ml linear gradient of O~I M vs 0'S M ammonium bicarbonate-ammonium carbonate buffer, pH 8'l, at i8 ml hr - ' .
Elution Volum~(mlI
Fx3. 6. CM-c~r v~.o~ cxiio~~roonerxY, sECOrw wLtn~v . Purification of zone II (Fig . 2) on a 1~3 x SO-cm column in 005 M ammonium acetate buffer, pH 5~8, at 40 ml hr - '. 71DYJCON 1973 Yol. 11
Naja naja
phospholipase isoenzymes
487
t I F41 . OISi-
3000 ~ ö
i i
e
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ô
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r
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1
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~~
200
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~~~. I 300 400
1 \
ô
I S00
Elution Volumelmll
Fto. 7. GEL FII.TRATION ON SErrunEx G-S0. Purification of zone 1 (Fig. 6) on a 1~Sx230-cm column in 0"1 M ammoniums bicarbonate buffer, pH 7" 1, at 10 ml hr' 1 .
1 - ~- T1 1 tF3) I
0.3
I
~~1~
I
3000
..
i In
0.2 H
Y 6 y
80
160 240 Elution Volume lmll
320
Flo. 8. GEt irn.:rn~noly olv SErttwnEx G-S0. Purification of zone II (Fig. ~ on a 1 "S x 250~In column in 0"l M ammonium bicarbonate buffer, pH 7"1, at 10 ml hr''. 710YICON 1973 Yd. II
488
J. SHILOAH, CHAYA KLIBANSKY and A. DE VRIES .6 0 0.16
-0 .14
E
0 o) N
0 0 i I-QIO ô E 'é E 0 -Q08 E Q
C Ô
F-0.06
Eluflon Volum~(ml)
FIG. 9. CM-CELLULOaB CEOtOMAi~OGRAPHY, TFDRD COLUMN .
Purification of zone III (Fig. 2) on a 1 ~6 x 30-cIn column in a 670 ml linear gradient of 005 M vs 0~2 M ammonium acetate buffer, pH 5'8, at 40 ml hr - '.
obtained from each column were routinely dialyud against distilled water and lyophiliud thereafter prior to further fractionation. Six phospholipase containing fractions (F1-6) were obtained. Criteria ofpurity
Each of the purified fractions (Fl-6) appeared to be fairly homogeneous as judged from the specific activity estimations at the last purification stages (Figs. 4, 5, 7, 8, 9). This evaluation was consistent with the results obtained by immunological studies. A single distinct arc was developed when either of the fractions was studied by immunoelectrophoresis with antiserum against the whole venom; when a mixture of all fractions was tested a single continuous arc was obtained (Fig. 10) indicating antigenic identity of the fractions. Furthermore, in double diffusion antigen-antibody reaction (Fig. 11) a single continuous precipitin line was formed when all fractions were tested simultaneously . On the other hand, on disc electrophoresis at basic pH, only 4 out of the 6 phospholipase fractions appeared to be fairly homogenous (Fig . 12). Molecular weight determination
On SDS acrylamide gel electrophoresis each of the 6 fractions appeared to comprise more than one component. Table 2 summaries the estimated molecular weights. A typical standardization curve for the molecular weight estimation of fraction F1 is given in Fig. 13. Thiol group estimation
No free thiol groups were found in any of the 6 phospholipase fractions.
71DXICON I973 Yol. II
F3
Fi -6
FIG . lO . ANALYTICAL IMMUNOELECTROPHORES1s . Upper wells : whole Naja naja venom; lower wells: phospholipase fractions Fl, F2, F3, F4, F5, F6 and the combined phospholipase fractions F1-6 . Trough in all slides : rabbit antiwhole Naja naja venom.
TOXlCON J973 Yol. 11
F.P . 488
f~s~~. . . F .,
l l . AGAR GEL DOUBLE DIFFUSION OF THE 6 PHOSPHOLIPASE FRACTIONS, The antibody against the whole venom was placed in the center well .
FIG.
FIG. ]Z. ANALYTICAL ACRYLAMIDE GEL ELECTROPHORESIS (pH S3) OF THE 6 PHOSPHOLIPASE FRACTIONS. 710XICON J973 Yol. JI
Naja naja
phospholipase iscenzymes
489
5.0 4.0
_ô, 2.5
Y
3 2 .0
L0 FIG.
13 .
0.2
0.4
0.6
0.8
L0
Mobility
SEMI-LOG PLOT OF MOLECULAR WEIGHTS AGAINST DL4TANCE OF MIGRATION ON SDS ACRYI.AMIDE GEL.
The arrows indicate the mobility of the 2 components of phospholipase fraction Fl . TAIIIE 2. MOLECULAR WEIGHT OF PHOSPHO1.iPASE ISOENZYME4 OF Naja ngja VENOM
Fraction Fl F2 F3 F4 FS F6
Molecular weights 14,400 ; 19,250 14,000 ; 19,000 11,150 ; 14,700 ; 17,900 13,400 ; 18,850 14,300 ; 20,100 16,000 ; 23,800
Heat resistance
Heating of each of the 6 enzyme preparations at 100° and pH 5~5 for 15 min did not significantly change their enzymatic activity . DISCUSSION
Although each of the 6 phospholipase fractions obtained in the present study from Naja naja venom showed heterogeneity on acrylamide gel electrophoresis, it may be concluded
that all fractions represented phospholipase isoenzymes only, since all fractions were pure and identical according to immunological criteria . Due to the similarity in physico-chemical properties ofthese various iscenzymes, their separation and isolation was extremely difficult, but on the basis of the acrylamide gel electrophoresic patterns one may estimate their number to be at least 14 . Since the molecular weights of the components in each fraction were not multiples of a common factor, it does not seem likely that the components represent monomers and dieters, however, such a possibility cannot be entirely excluded. The molecular weights of the 14 components ranged from 11000-24000 and are in good agreement with those reported by SALACH et al. (1971) and Gb1t1t1E et al. (1968) . TOXICON 1973 Vol. Il
490
J. SHILOAH, CHAYA KLIBANSKY and A. DE VR1ES
Acknowledgement-This study was supported by a grant from the U.S . Army through its European Research Office (contract No DAYA 37-70-C-0447). REFERENCES Bw~c nvce, B. M. and SMfex.+Y, Y. M. (1967) Multiple forms of cobra venom phospholipase A. Nature, Land. 216, 1210. .n, C. A. (1968) Crystalline phospholipase A associated with a Cunaue, B. T., O~x~r, D. E. and Bnoo cobra venom toxin. Nature, Load. 220, 371. Dwis, B. J. (1964) Disc electrophoresis 11 . Method and application to human serum proteins. Ann. N. Y. .lead. Sci. 121, 404. ELIa1AN, G. L. (1939) Tissue sulfhydryl groups. Archs Bioclum. Biaphys. 82, 70. LowxY, O. H., Rasi nraoROVax, N. J., Fexx, A. L. and R.axn~u t., R. J. (1951) Protein measurement with Folin-phenol reagent. J. biol. Clrem. 193, 265. OuCKretu .orrY, O. (1948) In vitro method for testing toxin-producing capacity of diphtheria bacteria . dera path . microbial. stand. 23, 186. S~cx, J. L, Tuxmt, P., St>JVO, R., HAVHF3e, J. and SINC3ER, P. (1971) Phospholipase A of snake venoms I. Isolation and molecular properties of isoenzymes from Na/a ngja and Yipera russelli venons. J blol. Chcm . 246, 331 . WEene, K. and Osaoxta, M. (1969) T'he reliability of molecular weight determinations by dodecyl sulfatepolyacrylamide gel electrophoresis. J. blot. Chem. 244, 4406 .
71DXICON 1973 Vol. 11
90. Per¢amon Prcss. Printed in Grcat Britain.
PHOSPHOLIPASE ISOENZYMES FROM NAJA NAJA VENOM -I. PURIFICATION AND PARTIAL CHARACTERIZATION J. SHILOAH, * CHAYA KLIBANSKY 8nd A. DE Vx1Es The RogofFWellcome Medical Research Institute, Tel-Aviv University, 13eilinson Medical Center, Petah Tilcva, Israel (Accepted jor pubi'icalton 14 May 1973)
AbstraM-Six phospholipase A~ontaining fractions were isolated from cobra (Nglo agja) venom by ion~atchange and gel filtration chromatography. Each fraction was homogeneous by immunological criteria and comprised more than 1 phospholipaae isoenzyme, their toW number amounting to 14 . The molecular weights of the isoenzymes ranged from 11000 to 24000. None of them contained free SH groups . The six fractions exh]bited similar specific activities. INTRODUCTION
years, cobra (Naja naja) venom has been shown to contain several phospholipase iscenzymes. BRAGANCA and SAMBRAY (1967) reported the presence of 7 phospholipase iscenzymes in Naja naja venom and obtained 1 phospholipase fraction which appeared homogeneous on starch gel electrophoresis . CURRIE et al. (1968) crystallized from this venom a phospholipase A fraction which was further separated into two phospholipase A components, but no additional information was given concerning their purity . SALACH et al. (1971), applying iscelectric focusing to a Naja naja phospholipase preparation obtained after heat-inactivation and gel filtration, concluded that there existed 9-11 phospholipase iscenzymes in the venom. Only one of these was purified to give a single band on disc electrophoresis . Jn the course of our studies on the specificity of snake venom phospholipases A, Naja naja phospholipase iscenzymes were obtained in 6 fractions. Their purification and molecular properties are described in the present communication. IN RECENT
EXPERIMENTAL PROCEDURE
Naja naja venom was purchased from L. Light and Co. Ltd., Colnbrook, England. Chromatographic techniques All procedures for purification of the enzyme were conducted at 4°. CM-cellulose (Whatman) was washed with alkali and acid, and equilibrated with ammonium acetate buffer, pH 5~8, at the appropriate ionic strength . DEAF-cellulose (Whatman) was washed with acid and alkali, and equilibrated with ammonium bicarbonate-ammonium carbonate buffer, pH 8~1, at the appropriate ionic strength . Sephadex G-50 (Pharmacia, Uppsala, Sweden) was allowed to swell in water for 24 hr, packed into a column under a pressure "In partial fulfilment of therequirements for a Ph .D. degree of the Feinberg Graduate School, the Weiunann Institute of Science, Rehovot, Israel. TOXICON 1973 Vol. Il
481
482
J. SHILOAH, CHAYA KLIBANSKY and A. DE VRIES
head of 50 cm water, and equilibrated overnight with O~l M ammonium bicarbonate pH 7~1 at a flow rate of 10 ml/hr. Protein concentrations were estimated by the method of LOWRY et al. (1951) using bovine serum albumin as a standard ; absorbance was read at 750 nm. Electrophoresis
Disc electrophoresis in polyacrylamide gel (Eastman Organic Chemicals, Rochester, N.Y.) was performed using the method of Dwls (1964) . The separation was carried out in a Shandon Disc Electrophoresis Apparatus (Shandon Scientific Co. Ltd., London, England) in 5 x 5 mm gels (15 per cent acrylamide in Tris-glycine buffer, pH 8~3). The amount of protein applied was 20-100 pg in 100 pl buffer solution containing one drop of glycerol. Proteins were stained with 1 per cent Amido Black in 7 per cent acetic acid for 30 min, and washed thereafter with the latter acid containing the anion exchange resin Dowex AG 1-x 8 (220-400 mesh Bio-Rad) . Immunization procedure
The antiserum against Naja raja venom was prepared using white New Zealand rabbits of about 3 kg weight . The animals were injected intradermally in several locations on the back and in each foot pad with 1 ml emulsion prepared from equal volumes of whole venom in saline (6 mg/ml) and complete Freund's adjuvant. Ten days later an antigen booster was given intramuscularly (1 mg of whole venom in complete Freund's adjuvant). The booster injection was repeated twice, at 4-week intervals. One week after each booster injection the animals were bled by cardiac puncture and sera were tested for precipitating antibodies. The antibody content of the immune sera was estimated by absorbance measurements at 280 nm after dissolving the washed specific precipitates in 0~1 N NaOH . Immunological techniques
Immunodi$'usion experiments were performed as described by Oucx~i.ox~t (1948) with 1 per tent Difco agar in 0025 M veronal buffer pH 8~2. The peripheral wells were filled with 001 ml of each phospholipase fraction (0~5 mg/ml) and the central well with 0025 ml rabbit antiserum. Diffusion was allowed to proceed for 2-3 days at 4° in a moist chamber. After extensive washing with saline for 3 days, the plates were covered with Whatman paper and dried at 37° for several hr. The paper was removed and the plates were placed for 5 min in a solution of 1 per cent Amido Black in 7 per cent acetic acid, and then rinsed in 3 changes of the latter solvent. For immunoelectrophoresis, glass slides (76 x 2~ were coated with 3~5 ml of 1 "5 per cent agar solution in 0"025 M veronal buffer pH 9"0. Whole venom and purified phospholipase fractions were placed in the holes and electrophoresis was performed for 120 min at 140 V and 4°. The concentration of the tray buffer was 0"OS M. After completion of the electrophoresis, anti-whole Naja naja serum was placed in the main trough and the slides were developed, stained and washed as described above for immunodiffusion. Molecular weight determination and Thiol-group estimation
The molecular weight determination of the phospholipases were tamed out by dodecyl sulfate polyacrylamide gel electrophoresis as described by WESEIt and OSHOxrt (1969), using RNase, lysozyme, performec acid-oxidized hemoglobin, trypsin, chymotrypsinogen A, pepsin and ovalbumin as molecular weight standards. TOXICON 1973 Yol. 11
483
Naja naja phospholipase isoenzymcs
Free thiol groups of purified phospholipases in 1 per cent SDS were determined with 5,5-dithio-bis (2-nitrobenzoic acid) according to the method of ELLMAN (1959) . Assay of phospholipase A activity For specific activity determinations at the various purification stages of the enzyme, the pH-stet method (TTA s Radiometer autotitrator type SBR2C/SBU 1) was employed, using dilute egg yolk (1 :20 with saline) as substrate. Liberation of acid was measured at pH 8"0 and 37° by titration with 0"005 N NaOH under a constant stream of argon. One unit of activity was defined as the amount of enzyme which liberates one prllole of free fatty acid per min under the above conditions . This was permissible since the activity of a given sample was found to be proportional to its concentration in the assay mixture, indicating that the substrate was present in large excess. , RESULTS
Pur~cation procedure Crude lyophilized Naja naja venom (2"16 g) was dissolved in 5 ml of 0005 M ammonium acetate buffer pH 5"8 and dialyzed at 4° overnight against 2 I . of the same buffer. The material was then subjected to serial ion-exchange and gel filtration chromatography, as summarized in Table 1 and illustrated by Figs. 1-9. The phospholipase containing fractions TARIE 1 . SUMMARY OF THE PURIFICATION OF PHOSPHOLIPAaBa FROM
Fraction
Protein (mg)
Lyophilized venom CM-cellulose Erst column (Fl-I-F2-}F3) " (zone I, Fig. 2) Sephadea G-SOt (Fl ~-F2~-F3) DF.AFrcelluloae first column (Fl+F2) (zone I, Fig. 3) F3 (zone II, Figl . 3) DEAE-cellulose second column Fl (zone I, Fig. 4) F2 (zone II, Fig. 4) DBAP-cellulose third column F3 (zone I, Fig. S) CM~ellulose first wlumn (F4+FS) (zone II, Fig. 2) CM-cellulose second column (F4) (zone I, Fig. 6) (FS) (zone II, Fig. 6) Sephadex G-50, F4 (zone I, Fig. 7) Sephadea G-S0, FS (zone I, Fig. 8)
CM-cellulose first column (F6) (zone III. Fig. 2) CM-oeiluloae third column (F6) (zone I, Fig. 9) Sephade~c G-50f, F6
2160
Specific activity (snits/mg protein) 831 600 385 Total activity (units)
Naja naja
Yield (%~ 100
VENOM
Ratio to specific activity of whole venom 1
43 "5
139 635
3210
16"8
8" 34
41~0
129 970
3170
15"6
8" 23
12 "9 23 "9
40 377 76122
3130 3185
4"9 9"2
8"13 8"27
4"2 5"0
l3 230 16000
3150 3200
1"59 l "92
8"18 8 "31
18 "5
58 460
3160
7"03
8"21
49ß
69 020
1400
8"3
3"64
12"6 19~6 3"5 9"0
14 918 35 672 l0 920 27 630
1184 1820 3120 3070
1 "8 4"3 1 "31 3"32
3"O8 4~73 8"10 7"97
85 "7
83 986
980
20 "1 16 "3
60 903 50 856
3030 3120
10~1 7"3 6~11
2"55 7"87 8"]0
"Fs in parenthesis represent eluates containing fractions separated in subsequent steps. tSephadal G-50 column which did not result in purification of materials applied on and one single peak was obtained. 71DXlCON 1973 Vol. !!
484
J. SHILOAH, CHAYA KLIHANSKY and A. DE VRIES clrc
za~E I
1*lFl .2)
zaE u
za+E m
clrc,x^~
I
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EFFI.IEHI'
ZOIE II
DEAEC,2~IlFp .41
SG-30 IFfq.0)
Zpi 11F4)
ZbIE I tF~
ZOIE I (F6) SG-d0 EFFtilE4lr (Fg)
\AEC.3°d
r-~---i~
ZOUE Ilfj) ZaEUtT~) m1E11
lFla.s)
ZOIE IlF3)
FIß. I. PtntISIC,~~noN sue. Abbreviations : S-Sephadex ; CMC-carboxymethyl xllulose ; DEAEC-diethylamincethyl cellulose; serial numbers indicate appropriate columns (see text). The elution buffers for SG-S0, DEAEC and CMC columns wen 0"1 M ammonium bicarbonate pH 7~1, ammonium bicarbonate-amawnium carbonate pH 8~1, ammonium acetate pH S"8, respectively. The elution gradients of the two latter buffers are given in the legends to Figs. 3-9.
3000
o.sa 26 22
IA
~â2
_I ae 0 N aa d
ale als 14 .1E 0
IOOOâ
Q4
W
L
a2
000
1600
2400 ~200 Elation Velonlelml)
4000
4000
.10 Q06 Q08
0
FIß. 2. CM~.LULOSe CIIJtOMA1nCiRAPHY, wear COLiJI~I . Purification of 2"16 g crude venom on a 2"2 x 90-cm column in a 4"2 L linear gradient of 0"OOS M vs 0"S M ammonium acetate buffer, pH S'8, at 112 ml hr -1 . 7~OXICON J973 Yo(. 11
Naja ngja phospholipase iscenzymes
485
~-0.30 i
-oao t0 . _ e¬0 ä E
-a10 ~~ É E
Elusion Volume (ml)
Fta. 3. DEAB~-cs.ctrcosE etneo~roan.~rt>r, ~nes"r coiv~nv. Puri&cation of zone I (Fig. 2), after gel filtration through Sephadex G-S0, on a 1 x 20-cm rnlumn in a 180 ml linear gradient of 0"01 M vs 0~5 M ammonium bicarbonate-ammonium carbonate buffer, pH 8" 1, at l8 ml hr'' .
0.3
E 0
0 N O Ô
3000
oz
ô -020
a E
-alo E lution Volum~ (ml~)
E E t
1~YYo. 4. DEAE~,oi.o~ c»eol~wz+oaawrxx, secoxn ooLU~v . lurißcation of zone I (Fig. 3) on a 1 x20-cm oohunn in a 220 ml linear gradient of 005 M vs 0"25 M ammonium bicarbonate-ammonium carbonate buffer, pH 8"1, at 18 ml hr'1 . T~OXICON7973 Vol. II
486
J. SHILOAH, CHAYA KLIBANSKY and A. DE VRIES
Elution Volunw(mlI
FYo. 5. DFAE-ea?uLOSe c~o~~rooiurtn, amen wLU>m~. Purification of combined zone II (Fig . 3) and zoo III (Fig. 4) on a I x 20-cm column in a 220 ml linear gradient of O~I M vs 0'S M ammonium bicarbonate-ammonium carbonate buffer, pH 8'l, at i8 ml hr - ' .
Elution Volum~(mlI
Fx3. 6. CM-c~r v~.o~ cxiio~~roonerxY, sECOrw wLtn~v . Purification of zone II (Fig . 2) on a 1~3 x SO-cm column in 005 M ammonium acetate buffer, pH 5~8, at 40 ml hr - '. 71DYJCON 1973 Yol. 11
Naja naja
phospholipase isoenzymes
487
t I F41 . OISi-
3000 ~ ö
i i
e
Î
ô
zooo ~;
oao~-
r
d
ä
1!I
o.oS
1
1 100
~~
200
1
loon
~~~. I 300 400
1 \
ô
I S00
Elution Volumelmll
Fto. 7. GEL FII.TRATION ON SErrunEx G-S0. Purification of zone 1 (Fig. 6) on a 1~Sx230-cm column in 0"1 M ammoniums bicarbonate buffer, pH 7" 1, at 10 ml hr' 1 .
1 - ~- T1 1 tF3) I
0.3
I
~~1~
I
3000
..
i In
0.2 H
Y 6 y
80
160 240 Elution Volume lmll
320
Flo. 8. GEt irn.:rn~noly olv SErttwnEx G-S0. Purification of zone II (Fig. ~ on a 1 "S x 250~In column in 0"l M ammonium bicarbonate buffer, pH 7"1, at 10 ml hr''. 710YICON 1973 Yd. II
488
J. SHILOAH, CHAYA KLIBANSKY and A. DE VRIES .6 0 0.16
-0 .14
E
0 o) N
0 0 i I-QIO ô E 'é E 0 -Q08 E Q
C Ô
F-0.06
Eluflon Volum~(ml)
FIG. 9. CM-CELLULOaB CEOtOMAi~OGRAPHY, TFDRD COLUMN .
Purification of zone III (Fig. 2) on a 1 ~6 x 30-cIn column in a 670 ml linear gradient of 005 M vs 0~2 M ammonium acetate buffer, pH 5'8, at 40 ml hr - '.
obtained from each column were routinely dialyud against distilled water and lyophiliud thereafter prior to further fractionation. Six phospholipase containing fractions (F1-6) were obtained. Criteria ofpurity
Each of the purified fractions (Fl-6) appeared to be fairly homogeneous as judged from the specific activity estimations at the last purification stages (Figs. 4, 5, 7, 8, 9). This evaluation was consistent with the results obtained by immunological studies. A single distinct arc was developed when either of the fractions was studied by immunoelectrophoresis with antiserum against the whole venom; when a mixture of all fractions was tested a single continuous arc was obtained (Fig. 10) indicating antigenic identity of the fractions. Furthermore, in double diffusion antigen-antibody reaction (Fig. 11) a single continuous precipitin line was formed when all fractions were tested simultaneously . On the other hand, on disc electrophoresis at basic pH, only 4 out of the 6 phospholipase fractions appeared to be fairly homogenous (Fig . 12). Molecular weight determination
On SDS acrylamide gel electrophoresis each of the 6 fractions appeared to comprise more than one component. Table 2 summaries the estimated molecular weights. A typical standardization curve for the molecular weight estimation of fraction F1 is given in Fig. 13. Thiol group estimation
No free thiol groups were found in any of the 6 phospholipase fractions.
71DXICON I973 Yol. II
F3
Fi -6
FIG . lO . ANALYTICAL IMMUNOELECTROPHORES1s . Upper wells : whole Naja naja venom; lower wells: phospholipase fractions Fl, F2, F3, F4, F5, F6 and the combined phospholipase fractions F1-6 . Trough in all slides : rabbit antiwhole Naja naja venom.
TOXlCON J973 Yol. 11
F.P . 488
f~s~~. . . F .,
l l . AGAR GEL DOUBLE DIFFUSION OF THE 6 PHOSPHOLIPASE FRACTIONS, The antibody against the whole venom was placed in the center well .
FIG.
FIG. ]Z. ANALYTICAL ACRYLAMIDE GEL ELECTROPHORESIS (pH S3) OF THE 6 PHOSPHOLIPASE FRACTIONS. 710XICON J973 Yol. JI
Naja naja
phospholipase iscenzymes
489
5.0 4.0
_ô, 2.5
Y
3 2 .0
L0 FIG.
13 .
0.2
0.4
0.6
0.8
L0
Mobility
SEMI-LOG PLOT OF MOLECULAR WEIGHTS AGAINST DL4TANCE OF MIGRATION ON SDS ACRYI.AMIDE GEL.
The arrows indicate the mobility of the 2 components of phospholipase fraction Fl . TAIIIE 2. MOLECULAR WEIGHT OF PHOSPHO1.iPASE ISOENZYME4 OF Naja ngja VENOM
Fraction Fl F2 F3 F4 FS F6
Molecular weights 14,400 ; 19,250 14,000 ; 19,000 11,150 ; 14,700 ; 17,900 13,400 ; 18,850 14,300 ; 20,100 16,000 ; 23,800
Heat resistance
Heating of each of the 6 enzyme preparations at 100° and pH 5~5 for 15 min did not significantly change their enzymatic activity . DISCUSSION
Although each of the 6 phospholipase fractions obtained in the present study from Naja naja venom showed heterogeneity on acrylamide gel electrophoresis, it may be concluded
that all fractions represented phospholipase isoenzymes only, since all fractions were pure and identical according to immunological criteria . Due to the similarity in physico-chemical properties ofthese various iscenzymes, their separation and isolation was extremely difficult, but on the basis of the acrylamide gel electrophoresic patterns one may estimate their number to be at least 14 . Since the molecular weights of the components in each fraction were not multiples of a common factor, it does not seem likely that the components represent monomers and dieters, however, such a possibility cannot be entirely excluded. The molecular weights of the 14 components ranged from 11000-24000 and are in good agreement with those reported by SALACH et al. (1971) and Gb1t1t1E et al. (1968) . TOXICON 1973 Vol. Il
490
J. SHILOAH, CHAYA KLIBANSKY and A. DE VR1ES
Acknowledgement-This study was supported by a grant from the U.S . Army through its European Research Office (contract No DAYA 37-70-C-0447). REFERENCES Bw~c nvce, B. M. and SMfex.+Y, Y. M. (1967) Multiple forms of cobra venom phospholipase A. Nature, Land. 216, 1210. .n, C. A. (1968) Crystalline phospholipase A associated with a Cunaue, B. T., O~x~r, D. E. and Bnoo cobra venom toxin. Nature, Load. 220, 371. Dwis, B. J. (1964) Disc electrophoresis 11 . Method and application to human serum proteins. Ann. N. Y. .lead. Sci. 121, 404. ELIa1AN, G. L. (1939) Tissue sulfhydryl groups. Archs Bioclum. Biaphys. 82, 70. LowxY, O. H., Rasi nraoROVax, N. J., Fexx, A. L. and R.axn~u t., R. J. (1951) Protein measurement with Folin-phenol reagent. J. biol. Clrem. 193, 265. OuCKretu .orrY, O. (1948) In vitro method for testing toxin-producing capacity of diphtheria bacteria . dera path . microbial. stand. 23, 186. S~cx, J. L, Tuxmt, P., St>JVO, R., HAVHF3e, J. and SINC3ER, P. (1971) Phospholipase A of snake venoms I. Isolation and molecular properties of isoenzymes from Na/a ngja and Yipera russelli venons. J blol. Chcm . 246, 331 . WEene, K. and Osaoxta, M. (1969) T'he reliability of molecular weight determinations by dodecyl sulfatepolyacrylamide gel electrophoresis. J. blot. Chem. 244, 4406 .
71DXICON 1973 Vol. 11