- Email: [email protected]
Isolation of a hemolytic, toxic phospholipase from the venom of the Australian red-bellied black snake (Pseudechis porphyriacus)
0041-0101/81/0101-0095 502.00/0
Todkai, Vol . 19, PP. 95-101 . p Pergaman Peen Ltd 19111 . Printed to Goat Britain .
ISOLATION OF A HEMOLYTIC, TOXIC PHOSPHOLIPASE FROM THE VENOM OF THE AUSTRALIAN RED-BELLIED BLACK SNAKE (PSEUDECHIS PORPHYRIACUS) GARY T. VAuGHAN, THOMAS B. SCULLEY and ROY TIRRELL School of Biochemistry, University of New South Wales, Kensington, N.S .W. 2033, Australia
(Accepted for publkwdon 27 June 1980) G.T. VAUGHAN, T . B. SCULLEY and R . TIA1tELL. Isolation ofa hemolytic, toxic phospholipase from the venom of the Australian red-bellied black snake (Pmudechis porphyriacns~ Taxiton 19, 95-101, 1981 .-A weak toxin was isolated from the venom of the Australian red-bellied black snake, Pswdechis porphyrlacm, by ion change chromatography on Bio-Rex 70, followed by gel filtration on Sephadex G-50. The toxin, pseudexin, accounts for 25% of the venom and has an LDsa (Lp.) of 480 pg/kg mouse. It is a polypeptide of 143 amino acid residues and has a formula weight of 16,659. Pseudexin has phospholipase A (phosphatidate aryl-hydrolase, EC 3.1 .1 .4) activity. Modification of the toxin with p-bromophenacyl bromide resulted in a 9949'/ low of phospholipase activity and a reduction of its toxicity. The toxin, like other phospholipases, câused indirect hemolysis of washed erythrocytes. In addition, pseudexin directly hemoyed erythrocytes under conditions in which phospholipases are normally non-hemolytic .
INTRODUCTION THE ENZYME phospholipase A (EC 3 .1 .1 .4) occurs widely in snake venoms (BROCKERHOFF and JENSEN, 1974) . The great majority of these phospholipases have been found to be of low
toxicity and to possess indirect hemolytic activity. However, a small group of venom phospholipases are highly toxic, due to acting presynaptically on neuromuscular junctions . Three of these have been isolated from the venoms of Australian snakes . They are : notexin (KARLSSON et al., 1972) and notechis 5 (HALPERT and EAKER,1976) from the Australian tiger snake (Notechis scutatus~ and taipoxin from the taipan (Oxyuranus scutellatus) (EAKER, 1975).
Although the venoms of the taipan and the Australian tiger snake have been investigated in some detail, few other Australian snake venoms have been studied in depth. The venom of the Australian red-bellied black snake, Pseudechis porphyriacus, was described by KELLAWAY (1938) as being feebly neurotoxic and powerfully hemolytic. However, the venom components were not separated and fewquantitative details weregiven by the author. In an effort to understand the properties of black snake venom, we have fractionated the venom and studied some of the properties of the most toxic fraction . MATERiA3g AND Nllrl'HUIJJ Dried Psardichis poMhyriatm venom was purchased from Eric Worrell's Australian Reptile Park, P.O . Box 192, Godard, New South Werks, 2230, Australia. Ion-exchange chromatography
Crude venom (200 mg) was dissolved in 5 ml of 0-06 M ammonium acetate, 95
pki 1-3.
tmtolnbie matmal was
96
GARY T. VAUGHAN, THOMAS B. SCULLEY and ROY TIRRELL
removed by centrifugation (12,500 g,15 min) . Theclarifiedvenom solution waschromatographed on the carboxylic acrylic type cation exchange resin Bio-Rex 70, 400 mesh (Bio-Rad Laboratories} The pretreatment and equilibration of the rain are described in detail by KAtu .ssoN and Emma (1972} The sample was eluted with an 800 ml concave gradient of0-09 vs 140 Mammonium acetate at pH 7-3. The gradient was achieved by the use of a two-cylinder mixing device. The diameter of the mixing cylinder was twice that of the cylinder containing terminal buffer. Gelfiltration
The most toxic fraction eluted from Bio-Rex 70 was further purified by filtration on a 2-5 x 60 cm column of Sephadex G-50 (Pharmacies) using 0'20 M ammonium acetate, pH 7-3, as an eluent .
Measurement of lethality
Lethalityofvenom fractions wasassayed by i.p . injection into whitemice weighing 20 t 1 & The mice were of an inbred strain from the University of New South Wales Animal Brooding Unit. Venom was dissolved in 0-9'/ saline and0-1 ml injected into each of five mice perdose level. Deaths occurring within 24 hr were recorded and i Dso values calculated by the method of REED and MUENcH (1938). Protein estimation
Absorbance of column elutee fractions was measured at 280 run, otherwise protein concentrations were determined according to the micro biuret method of ITzHAKj and Gnu. (1964 Phosphalipase A activity
The method of KocHALATY (1966) was used to measure phospholipaseA activity. to this method the fatty acids liberated from egg yolk emulsions by phospholipases are titrated with 0-02 M NaOH . Assays were performed in triplicate. Results are reported as means tS.D . Determination of molecular weight (a) Gelfiltration. Molecular weight was estimated by gel filtration
on Sephadea (3-100 (ANDREws, 1965 The . wt. The mol. wt of the toxin was calculated from a plot of column was calibrated using proteins of established mol Ve/Vo (elution volume/void volume) vs mol. wt. (b) Sodium dodecyl sulphate (SDS) polyacrylandde gel electrophoresis (PAGE) . Electrophoraû in 10'/ (w/v)_ polyacrylamide gels in the presence of 0-1% (w/v) SDS(WEBER and OsBoRN,1969) was used to estimate mol. wt andalso as a criterion of purity. Molecular weight was determined from a plot of mol. wt of reference proteins vs their mobility. The reference proteins used were : bovine serum albumin, chynrotrypsinogen A, myoglobin, lactate aehydrogenase and cytochrome C. The mol. wts of these proteins reported by WEBER and OsBoRN (1969) were used in calculations . Reduction and carboxymethylation
The method used was essentially that of CRHSrFmm et al. (1963) as modified by MooN and THompsoN (1969) except that 7 M guanidine-Ha was used instead of 8 M urea as a denaturinit ament. Reduction with 0ma+captoethanol was carried out for 18 hr before carboxymethylation with iodoacetate. Amino acid analyus
Reduced and S-caboxymethylated toxin was hydrolysed at 110°C in sealed evacuated tuba (CResrtmm et al, 1963) with redistilled 6 M HCl containing. 0-1 mg phenol/ml (SANoER and THomps^ 1963). Analyses were performed on aBeckman 121-M amino acid analyse and_peak areaswere integrated on a SpectraPhysics"System AA" integrator. Tryptophan was determined spectrophotometrically (BEAvAN and HouDAY, 1952) and eolorimetrically (SrrEs and CHAMBERS, 1949 Hemolytic activity
Themethod ofJo8HUA and ISHAY(1973) was used to estimate hemolyticactivity.Fordirect hemolytic activity, the assay mixture contained: 03 ml of 5'/ suspension of packed, washed mouse erythrocytes in phosphate-bul6ered saline (0-01 M phosphate buffer, pH 7-0, containing 0-15 M NaCl); 0-1 ml of phosphate-buffered saline containing venom or toxin ; 0-1 ml of phosphate-buffered saline. For indirect hemolytic activity, the phosphate-buffered saline was replaced by 0-1 ml of eggyolk emulsion (0-5 g egg yolk per 100 ml phosphate-saline containing 0-05 M Caa3~ Samola were incubated at 37°C for 2 hr at a constant shaking speed of 60 shakes/min. The absorbs= of the released hemoglobin was measured at 540nm and expressed as a percentage of that obtained by total hemolytis of the erythrocytes . Complete hemolytis was ensured by adding approximately 1 mg of saponin to the incubation mixture. HU so values (amount of toxin causing 50'/ hemolytis) were calculated by the method of DE titaaADo and LAYRME (1964), and were based on the assay conditions : 0-7 ml of 3-60% washed mouse erythrocyte suspension, pH 7-0,2 hr incubation at 37°C . Three determinations in duplicate were carriedout on amounts of venom ranging
Phospholipase from Pmudechis porphyriacus
97
EFFLUENT.mi FIG. 1. CHROMATOGRAPHY OF Pseudechis porphyriacw vENOM ON BIO-REx 70 . Venom (200 mg) was dissolved in 5 ml of 0-06 M ammonium acetate and the sample clarified by centrifugation (12,500g, 15 mini The 23 x 32 cm column of Bio-Rex 70 was equilibrated with 0-20 M ammonium acetate, pH 7 .3. Before application of the sample, the column was eluted with 50 ml of 0-06 M ammonium acetate to ensure binding of the sample to the resin.After application to thecolumn, the sample was eluted with 100 ml of0-09 M ammonium acetate, followed by 900 ml of a concave gradient (0-09 vs 1-40M ammonium acetate, pH 7-3~ A flow rate of 15 ml/hr was maintained.The effluent from the column was collected in 3 ml fractions and the absorbance of each was read at 280 nm.
from 0-5 pg/0-7 ml to 150 pgl0 -7 ml. Results are reported as means f S.D . Modfcation of phospWtowe A activüy p-Bromophenacyl bromide (Sigma)was used to block phospholipase A activity using the method of HALPERT et al. (1974 Modified toxin was separated from unreacted toxin by ion-exchange chromatography on a 2-5 x 30 cm column of Bio-Rex 70 with 0-39 M ammonium acetate, pH 7-3 as an eluent . RESULTS Fractionation of the venom and isolation of the main toxic component Dried Pseudechis porphyriacus venom was chromatographed on Bio-Rex
70 and eight hacctions (I-VIII) were collected (Fig. 1). Chromatography at this pH (7-3) gave a good separation of the basic toxins ; however, the more acidic compounds were poorly resolved. Wholevenom had an LD,o value of 1-30 mg/kg mouse and the most toxic fraction (fraction V) had an LDso value of0-48 mg/kgmouse. Mice injected with fraction V suffered paralysis ofthe hind legs and had difficulty in moving and breathing. Before death the mice lay immobilised. Mice which survived longer than 24 hr, and subsequently died, frequently showed signs of hemoglobinuria. Examination of fraction V by SDS-PAGE revealed the toxin to be almost pure . Small amounts of contaminants were removed by gel filtration on a 2-5 x 60 cm column of Sephadex G-50 using 0-25 M ammonium acetate as the eluent. The purified toxin, which comprised approximately 25% of the venom, was given the name Pseudexin (Pseudechis toxin Amino acid composition
The results of amino acid analyses of pseudexin are given in Table 1. Pseudexin was analysed in the reduced and S-carboxymethylated form (24, 48, 72 and 96 hr hydrolysates),
98
GARY T. VAUGHAN, THOMAS B. SCULLEY and ROY TIRRELL TABLE 1 . AMINO ACE) COMPOSITION OF PSEUDEXEd
Amino acid residue Asp Thr Ser Glu Pro Gly Ala 1 Cys (as CM-cysteine) Val Met lie Leu Tyr Phe Trp Lys His Arg NH 3 No . of residues Formula weight Mol. wt (SDS-PAGE) Mol. wt (gel filtration)
Residues/molecule of pseudexin 16 .6 t 0" 16" 7-9t 5 -8t 8-8 t 0.10 8-8 t 0-25 11 . 1 t 0 . 11 11-0 t 0. 10 13-8 f 0-26 39 t 009 1-3 t 0-11 4-9 t 0-05 6-0 t 0-05 9-2 t 0-15 6-0 t 0 .00 3$ 18-1 t 1-06 22 t 009 3-7 t 0-46 6ß t 0-77
Integers 17 8 6 9 9 11 11 14 4 1 5 6 9 6 3 18 2 4 6 142 16,659 16,900 16,200
Mean t SD. ; n = 5 . t Extrapolated from 25, 48, 72 and 96 hr values to zero hydrolysis time, t Determined spectrophotometrically (2-81) and colorialetrically (1 -98}
and in the native form. The values for threonine and serine were obtained by extrapolation to zero hydrolysis time. Tryptophan was determined spectrophotometrically to be 2-81 residues/molecule, and colorimetrically to be 1-98. A value of three residues for tryptophan is favoured because the colorimetric assay often gives low values. Molecular weight estimations
The moL wt of pseudexin, calculated by SDS-PAGE, was 16,900 ; by gel filtration, it was found to be 16,200. With both methods a single peak was the criterion for purity. SDS-PAGE also showed that the toxin existed as a single polypeptide chain. The formula weight (16,659) obtained from the amino acid analysis is in agreement with the mol. wt obtained by SDS-PAGE and by gel filtration . Phosphohpase activity
The specific phospholipase activity of pseudexin, 112 f 3-6 pmole free fatty acid/mg protein/min, is slightly higher than that for whole venom (97 t 2-5 pmole/mg/min), and is approximately halfthat ofthemost active phospholipase isolated from thevenom. The latter, isolated from peak 1, had a specific activity of 212 Eanole/mg protein/min (unpublished results). Hemolytic activity
Both wholevenom andpseudexin caused direct lysis ofmouse erythrocytes. Whole venom
Phospholipase from Pseudechis porphyriacus
EFFLUENT,rrd FIG. 2 . SEPARATION OF MODIFIED PSEUDEXIN FROM UNMODIFIED PSEUDEXIN ON BIO-REX 70. After incubation with p-bromophenacyl bromide, the products were dialysed and dissolved in 5 ml of 0. 39 M ammonium acetate, pH 7ß . The solution was applied to a 2-5 x 30 cm column ofBio-Rex 70 and eluted with Oß9 M ammonium acetate, pH 7 .3 . A flow rate of 12 ml/hr was maintained. The effluent was collected in 5 ml fractions and the absorbance measured at 280 nm.
wasmore strongly hemolytic than pseudexin. For direct hemolysis, the HU 50 values (amount ofhemolysin causing 50'/ hemolysis) were : whole venom, l l -8 f 0- 6,ug ; pseudexin, 21-4 t 2-3 pg. In the presence of egg yolk phospholipids, whole venom and pseudexin caused indirect lysis of erythrocytes : whole venom HU 50 , 1-7 t 0-2 ug ; pseudo HU50, 8-1 t 0-8 p& Modification of pseudexin
The relationship between the phospholipase activity of pseudexin and its hemolytic and toxic properties was examined by modification with p-bromophenacyl bromide. pBromophenacyl bromide blocks phospholipase activity by binding to a single histidine residue at the active site (VOLWERK et al., 1974 After modification, the modified toxin was separated from unreacted toxin by chromatography on Bio-Rex 70 (Fig. 2~ Comparison with the retention volume of unreacted pseudexin, using the same column, revealed the first peak to be the unmodified form . This conclusion was confirmed by phospholipase assays. Modification of pseudexin resulted in a loss of 99-9'/ of its phospholipase activity. When tested for toxicity, at a dose (6 mg/kg) which was 12 times the LD50 of pseudexin, modified pseudexin was not lethal. Modification also reduced the direct hemolytic activity of pseudexin. Pseudexin produced direct hemolysis of 50/ of the erythrocytes in the assay mixture at adose of21 -4,ug whereas the sameamount ofmodified pseudexin caused only 3% hemolysis. DISCUSSION
Pseudexin, isolated in a purified form, is a phospholipase consisting ofa single polypeptide chain . It has a mol. wt of approximately 16,500. The symptoms of paralysis ofthe hind legs and general flaccid paralysis shown by mice injected with pseudexin, are similar to those reported for notexin (KARLSSON et al., 1972). As with the presynaptically-acting toxins,
10 0
GARY T. VAUGHAN, THOMAS B. SCULLEY and ROY TIRRELL
notexin and ß-bungarotoxin, phospholipase activity is a requirement for toxicity of pseudexin . The evidence suggests that pseudexin may be a presynaptic neurotoxin and indeed preliminary neurophysiological studies on mouse diaphragm neuromuscular junctions have indicated that pseudexin acts presynaptically (unpublished results). Pseudexin has both direct and indirect hemolytic activity. Its indirect hemolytic activity in the presence of egg yolk phospholipids results from the production of lysophospholipids, which are known to be strongly hemolytic (CONDREA et al ., 1964). Crotoxin, and to a lesser extent ß-bungarotoxin, have indirect hemolytic activity (JENG et al ., 1978). Usually, phospholipases do not lyse erythrocytes in the absence of added phospholipid. However, in the presence of a direct lyric factor, or sublytic concentrations of detergents, or high Cat + concentrations, or albumin or in hypotonic media, direct hemolysis by phospholipases can occur (GUL et al., 1974). The conditions in which the direct hemolytic activity of pseudexin was tested were such that a typical phospholipase would not be hemolytic. Nevertheless, it would appear from the low direct hemolytic activity ofp-bromophenacyl bromide-modified pseudexin that the phospholipase activity plays a major role in the direct hemolysis by the toxin. Similarly, phospholipase action would appear to be involved in its toxicity . This is consistent with findings using presynaptically-acting toxins (HABERMANN and BRETTHAUPT, 1978). Acknowledgements-We express our thanks to Mr . A. Rvs and Mr. R. MANN for their assistance with amino acid analyses. This work was partly supported by an Australian Research Grants Committee grant (D2 67/16508). REFERENCES
ANDREWS, P. (1965) The gel filtration behaviour of proteins related to their molecular weights over a wide range. Biochem. J. 96, 595. BEAYAN, G. H. and HOLIDAY, E. R. (1952) Ultraviolet absorption spectraof proteins and amino acids. Ado. Protein Chem. 7, 319. BROCKERHOFF, H. and JENSEN, R. G. (1974) Lipolytic Enzymes, p. 197. New York : Academic Press. CoNDREA, E., DE VRIES, A. and MAGER, J. (1964) Hemolysis and splitting of human erythrocyte phospholipids by snake venoms. Biochim. biophys . Acts 84, 60. CRFSTFIELD, A. M., MOORE, S. and STEIN, W. H. (1963) The preparation and enzymic hydrolysis of reduced and Scarboxymethylated proteins. J . biol . Chem . 238, 622. DE HURTADO, I. and LAYRISSE, M. (1964) A quantitative method for the assay of snake venom hemolytic activity. Toxicon 2, 43. FAKER, D. (1975) Structural nature of pre-synaptic neurotoxins from Australian elapid venoms . Toxicon 13, 90. Gut, S, KHARA, J. S.and SMmI, A. D. (1974) Hemolysis of washed human redcells by various snakevenoms in the presence of albumin and Ca= +. Toxicon 12, 311. HABERMANN, E. and BREITHAUPT, H. (1978) The crotoxin complex-an exampleof biochemical and pharmacological complementation. Toxicon 16, 19 . HALPERT, J.and FAKER, D. (1976) Isolation and amino acid sequence of a neurotoxic phosolipase A from the venom of the Australian tiger snake, Notschis scutatus scutatus. J. biol. Chem. 251, 7343. HALPERT, J, FAKER, D. and KARLSSON, E. (1976) The role of phospholipase activity in the action of a presynaptic neurotoxin from the venom of Notschis scutatus scutatus (Australian tiger snake). FEBS Lett. 61, 72. ITZHAKI, R. F. and GILL, D. M. (1964) A micro-biuret method for estimating proteins . Analyt. Biochem . 9, 401. JENG, T. W., HENDON, R. A. and FRAENKEL-TONRAT, J. (1978) Search for relationships among the hemolytic, phospholipolytic, and neurotoxic activities of snake venoms. Proc . natn . Acad. Sci . U .S .A. 75, 600. JOSHUA, H. and ISHAY, J. (1973) The hemolytic properties of the oriental hornet venom. Acts Phasmac. Tox. 33,42. KARLssoN, E. and FAKER, D. (1972) Isolation of the principal neurotoxins of Naja naja subspecies from the Asian mainland. Toxicon 10, 217. KARLssoN, E, FAKER, D. and RYDEN, L. (1972) Purification of a presynaptic neurotoxin from the venom of the Australian tiger snake, Notschis scutatus scutatus. Toxicon 10, 405. KEId.AWAY, C. H. (1938) The symptomology and treatment of the bites of Australian snakes . Med . J . Aust. 2, 585. KOCHALATY, W. F. (1966) A rapid and simple phospholipase A assay. Toxicon 4, 1 . MOON,K. E. andTHOM PEON, E. O. P.(1969) Subunits from reduced and S-carboxymethylated ribulose diphosphate carboxylase (Fraction I protein) . Aust. J. Not. Sci. 22, 463. REED, L. D. and MUENCH, H. (1938) A simple method of estimating fifty per cent end points. Am . J . Hyg. 27, 493.
Phospholipase from Pmudechis porphyriacus
10 1
F. and THOMPSON, & O. P . (1963) Halogenation oftyrosine during acid hydrolysis. BiocMm . biophys. Acta 71,468. SPIES, J . R and CHAMBERS, D. C. (1949) Chemical determination of tryptophan in proteins. Anal. Chan . 21, 1249 . VOLwERI<, J. J, PIE[ERSON, W. A. and DE HMS, G. H . (1974) Histidine at the active site of phospholipase A= . Bioche nistry 13, 1446. WEBER, K. and OSBORN, M. (1969) The reliability of mol . wt determinations by dode yl sulfate-polyaaylamide gel electrophoresis. J. biol. Chan. 224, 4406. SANoER,
Todkai, Vol . 19, PP. 95-101 . p Pergaman Peen Ltd 19111 . Printed to Goat Britain .
ISOLATION OF A HEMOLYTIC, TOXIC PHOSPHOLIPASE FROM THE VENOM OF THE AUSTRALIAN RED-BELLIED BLACK SNAKE (PSEUDECHIS PORPHYRIACUS) GARY T. VAuGHAN, THOMAS B. SCULLEY and ROY TIRRELL School of Biochemistry, University of New South Wales, Kensington, N.S .W. 2033, Australia
(Accepted for publkwdon 27 June 1980) G.T. VAUGHAN, T . B. SCULLEY and R . TIA1tELL. Isolation ofa hemolytic, toxic phospholipase from the venom of the Australian red-bellied black snake (Pmudechis porphyriacns~ Taxiton 19, 95-101, 1981 .-A weak toxin was isolated from the venom of the Australian red-bellied black snake, Pswdechis porphyrlacm, by ion change chromatography on Bio-Rex 70, followed by gel filtration on Sephadex G-50. The toxin, pseudexin, accounts for 25% of the venom and has an LDsa (Lp.) of 480 pg/kg mouse. It is a polypeptide of 143 amino acid residues and has a formula weight of 16,659. Pseudexin has phospholipase A (phosphatidate aryl-hydrolase, EC 3.1 .1 .4) activity. Modification of the toxin with p-bromophenacyl bromide resulted in a 9949'/ low of phospholipase activity and a reduction of its toxicity. The toxin, like other phospholipases, câused indirect hemolysis of washed erythrocytes. In addition, pseudexin directly hemoyed erythrocytes under conditions in which phospholipases are normally non-hemolytic .
INTRODUCTION THE ENZYME phospholipase A (EC 3 .1 .1 .4) occurs widely in snake venoms (BROCKERHOFF and JENSEN, 1974) . The great majority of these phospholipases have been found to be of low
toxicity and to possess indirect hemolytic activity. However, a small group of venom phospholipases are highly toxic, due to acting presynaptically on neuromuscular junctions . Three of these have been isolated from the venoms of Australian snakes . They are : notexin (KARLSSON et al., 1972) and notechis 5 (HALPERT and EAKER,1976) from the Australian tiger snake (Notechis scutatus~ and taipoxin from the taipan (Oxyuranus scutellatus) (EAKER, 1975).
Although the venoms of the taipan and the Australian tiger snake have been investigated in some detail, few other Australian snake venoms have been studied in depth. The venom of the Australian red-bellied black snake, Pseudechis porphyriacus, was described by KELLAWAY (1938) as being feebly neurotoxic and powerfully hemolytic. However, the venom components were not separated and fewquantitative details weregiven by the author. In an effort to understand the properties of black snake venom, we have fractionated the venom and studied some of the properties of the most toxic fraction . MATERiA3g AND Nllrl'HUIJJ Dried Psardichis poMhyriatm venom was purchased from Eric Worrell's Australian Reptile Park, P.O . Box 192, Godard, New South Werks, 2230, Australia. Ion-exchange chromatography
Crude venom (200 mg) was dissolved in 5 ml of 0-06 M ammonium acetate, 95
pki 1-3.
tmtolnbie matmal was
96
GARY T. VAUGHAN, THOMAS B. SCULLEY and ROY TIRRELL
removed by centrifugation (12,500 g,15 min) . Theclarifiedvenom solution waschromatographed on the carboxylic acrylic type cation exchange resin Bio-Rex 70, 400 mesh (Bio-Rad Laboratories} The pretreatment and equilibration of the rain are described in detail by KAtu .ssoN and Emma (1972} The sample was eluted with an 800 ml concave gradient of0-09 vs 140 Mammonium acetate at pH 7-3. The gradient was achieved by the use of a two-cylinder mixing device. The diameter of the mixing cylinder was twice that of the cylinder containing terminal buffer. Gelfiltration
The most toxic fraction eluted from Bio-Rex 70 was further purified by filtration on a 2-5 x 60 cm column of Sephadex G-50 (Pharmacies) using 0'20 M ammonium acetate, pH 7-3, as an eluent .
Measurement of lethality
Lethalityofvenom fractions wasassayed by i.p . injection into whitemice weighing 20 t 1 & The mice were of an inbred strain from the University of New South Wales Animal Brooding Unit. Venom was dissolved in 0-9'/ saline and0-1 ml injected into each of five mice perdose level. Deaths occurring within 24 hr were recorded and i Dso values calculated by the method of REED and MUENcH (1938). Protein estimation
Absorbance of column elutee fractions was measured at 280 run, otherwise protein concentrations were determined according to the micro biuret method of ITzHAKj and Gnu. (1964 Phosphalipase A activity
The method of KocHALATY (1966) was used to measure phospholipaseA activity. to this method the fatty acids liberated from egg yolk emulsions by phospholipases are titrated with 0-02 M NaOH . Assays were performed in triplicate. Results are reported as means tS.D . Determination of molecular weight (a) Gelfiltration. Molecular weight was estimated by gel filtration
on Sephadea (3-100 (ANDREws, 1965 The . wt. The mol. wt of the toxin was calculated from a plot of column was calibrated using proteins of established mol Ve/Vo (elution volume/void volume) vs mol. wt. (b) Sodium dodecyl sulphate (SDS) polyacrylandde gel electrophoresis (PAGE) . Electrophoraû in 10'/ (w/v)_ polyacrylamide gels in the presence of 0-1% (w/v) SDS(WEBER and OsBoRN,1969) was used to estimate mol. wt andalso as a criterion of purity. Molecular weight was determined from a plot of mol. wt of reference proteins vs their mobility. The reference proteins used were : bovine serum albumin, chynrotrypsinogen A, myoglobin, lactate aehydrogenase and cytochrome C. The mol. wts of these proteins reported by WEBER and OsBoRN (1969) were used in calculations . Reduction and carboxymethylation
The method used was essentially that of CRHSrFmm et al. (1963) as modified by MooN and THompsoN (1969) except that 7 M guanidine-Ha was used instead of 8 M urea as a denaturinit ament. Reduction with 0ma+captoethanol was carried out for 18 hr before carboxymethylation with iodoacetate. Amino acid analyus
Reduced and S-caboxymethylated toxin was hydrolysed at 110°C in sealed evacuated tuba (CResrtmm et al, 1963) with redistilled 6 M HCl containing. 0-1 mg phenol/ml (SANoER and THomps^ 1963). Analyses were performed on aBeckman 121-M amino acid analyse and_peak areaswere integrated on a SpectraPhysics"System AA" integrator. Tryptophan was determined spectrophotometrically (BEAvAN and HouDAY, 1952) and eolorimetrically (SrrEs and CHAMBERS, 1949 Hemolytic activity
Themethod ofJo8HUA and ISHAY(1973) was used to estimate hemolyticactivity.Fordirect hemolytic activity, the assay mixture contained: 03 ml of 5'/ suspension of packed, washed mouse erythrocytes in phosphate-bul6ered saline (0-01 M phosphate buffer, pH 7-0, containing 0-15 M NaCl); 0-1 ml of phosphate-buffered saline containing venom or toxin ; 0-1 ml of phosphate-buffered saline. For indirect hemolytic activity, the phosphate-buffered saline was replaced by 0-1 ml of eggyolk emulsion (0-5 g egg yolk per 100 ml phosphate-saline containing 0-05 M Caa3~ Samola were incubated at 37°C for 2 hr at a constant shaking speed of 60 shakes/min. The absorbs= of the released hemoglobin was measured at 540nm and expressed as a percentage of that obtained by total hemolytis of the erythrocytes . Complete hemolytis was ensured by adding approximately 1 mg of saponin to the incubation mixture. HU so values (amount of toxin causing 50'/ hemolytis) were calculated by the method of DE titaaADo and LAYRME (1964), and were based on the assay conditions : 0-7 ml of 3-60% washed mouse erythrocyte suspension, pH 7-0,2 hr incubation at 37°C . Three determinations in duplicate were carriedout on amounts of venom ranging
Phospholipase from Pmudechis porphyriacus
97
EFFLUENT.mi FIG. 1. CHROMATOGRAPHY OF Pseudechis porphyriacw vENOM ON BIO-REx 70 . Venom (200 mg) was dissolved in 5 ml of 0-06 M ammonium acetate and the sample clarified by centrifugation (12,500g, 15 mini The 23 x 32 cm column of Bio-Rex 70 was equilibrated with 0-20 M ammonium acetate, pH 7 .3. Before application of the sample, the column was eluted with 50 ml of 0-06 M ammonium acetate to ensure binding of the sample to the resin.After application to thecolumn, the sample was eluted with 100 ml of0-09 M ammonium acetate, followed by 900 ml of a concave gradient (0-09 vs 1-40M ammonium acetate, pH 7-3~ A flow rate of 15 ml/hr was maintained.The effluent from the column was collected in 3 ml fractions and the absorbance of each was read at 280 nm.
from 0-5 pg/0-7 ml to 150 pgl0 -7 ml. Results are reported as means f S.D . Modfcation of phospWtowe A activüy p-Bromophenacyl bromide (Sigma)was used to block phospholipase A activity using the method of HALPERT et al. (1974 Modified toxin was separated from unreacted toxin by ion-exchange chromatography on a 2-5 x 30 cm column of Bio-Rex 70 with 0-39 M ammonium acetate, pH 7-3 as an eluent . RESULTS Fractionation of the venom and isolation of the main toxic component Dried Pseudechis porphyriacus venom was chromatographed on Bio-Rex
70 and eight hacctions (I-VIII) were collected (Fig. 1). Chromatography at this pH (7-3) gave a good separation of the basic toxins ; however, the more acidic compounds were poorly resolved. Wholevenom had an LD,o value of 1-30 mg/kg mouse and the most toxic fraction (fraction V) had an LDso value of0-48 mg/kgmouse. Mice injected with fraction V suffered paralysis ofthe hind legs and had difficulty in moving and breathing. Before death the mice lay immobilised. Mice which survived longer than 24 hr, and subsequently died, frequently showed signs of hemoglobinuria. Examination of fraction V by SDS-PAGE revealed the toxin to be almost pure . Small amounts of contaminants were removed by gel filtration on a 2-5 x 60 cm column of Sephadex G-50 using 0-25 M ammonium acetate as the eluent. The purified toxin, which comprised approximately 25% of the venom, was given the name Pseudexin (Pseudechis toxin Amino acid composition
The results of amino acid analyses of pseudexin are given in Table 1. Pseudexin was analysed in the reduced and S-carboxymethylated form (24, 48, 72 and 96 hr hydrolysates),
98
GARY T. VAUGHAN, THOMAS B. SCULLEY and ROY TIRRELL TABLE 1 . AMINO ACE) COMPOSITION OF PSEUDEXEd
Amino acid residue Asp Thr Ser Glu Pro Gly Ala 1 Cys (as CM-cysteine) Val Met lie Leu Tyr Phe Trp Lys His Arg NH 3 No . of residues Formula weight Mol. wt (SDS-PAGE) Mol. wt (gel filtration)
Residues/molecule of pseudexin 16 .6 t 0" 16" 7-9t 5 -8t 8-8 t 0.10 8-8 t 0-25 11 . 1 t 0 . 11 11-0 t 0. 10 13-8 f 0-26 39 t 009 1-3 t 0-11 4-9 t 0-05 6-0 t 0-05 9-2 t 0-15 6-0 t 0 .00 3$ 18-1 t 1-06 22 t 009 3-7 t 0-46 6ß t 0-77
Integers 17 8 6 9 9 11 11 14 4 1 5 6 9 6 3 18 2 4 6 142 16,659 16,900 16,200
Mean t SD. ; n = 5 . t Extrapolated from 25, 48, 72 and 96 hr values to zero hydrolysis time, t Determined spectrophotometrically (2-81) and colorialetrically (1 -98}
and in the native form. The values for threonine and serine were obtained by extrapolation to zero hydrolysis time. Tryptophan was determined spectrophotometrically to be 2-81 residues/molecule, and colorimetrically to be 1-98. A value of three residues for tryptophan is favoured because the colorimetric assay often gives low values. Molecular weight estimations
The moL wt of pseudexin, calculated by SDS-PAGE, was 16,900 ; by gel filtration, it was found to be 16,200. With both methods a single peak was the criterion for purity. SDS-PAGE also showed that the toxin existed as a single polypeptide chain. The formula weight (16,659) obtained from the amino acid analysis is in agreement with the mol. wt obtained by SDS-PAGE and by gel filtration . Phosphohpase activity
The specific phospholipase activity of pseudexin, 112 f 3-6 pmole free fatty acid/mg protein/min, is slightly higher than that for whole venom (97 t 2-5 pmole/mg/min), and is approximately halfthat ofthemost active phospholipase isolated from thevenom. The latter, isolated from peak 1, had a specific activity of 212 Eanole/mg protein/min (unpublished results). Hemolytic activity
Both wholevenom andpseudexin caused direct lysis ofmouse erythrocytes. Whole venom
Phospholipase from Pseudechis porphyriacus
EFFLUENT,rrd FIG. 2 . SEPARATION OF MODIFIED PSEUDEXIN FROM UNMODIFIED PSEUDEXIN ON BIO-REX 70. After incubation with p-bromophenacyl bromide, the products were dialysed and dissolved in 5 ml of 0. 39 M ammonium acetate, pH 7ß . The solution was applied to a 2-5 x 30 cm column ofBio-Rex 70 and eluted with Oß9 M ammonium acetate, pH 7 .3 . A flow rate of 12 ml/hr was maintained. The effluent was collected in 5 ml fractions and the absorbance measured at 280 nm.
wasmore strongly hemolytic than pseudexin. For direct hemolysis, the HU 50 values (amount ofhemolysin causing 50'/ hemolysis) were : whole venom, l l -8 f 0- 6,ug ; pseudexin, 21-4 t 2-3 pg. In the presence of egg yolk phospholipids, whole venom and pseudexin caused indirect lysis of erythrocytes : whole venom HU 50 , 1-7 t 0-2 ug ; pseudo HU50, 8-1 t 0-8 p& Modification of pseudexin
The relationship between the phospholipase activity of pseudexin and its hemolytic and toxic properties was examined by modification with p-bromophenacyl bromide. pBromophenacyl bromide blocks phospholipase activity by binding to a single histidine residue at the active site (VOLWERK et al., 1974 After modification, the modified toxin was separated from unreacted toxin by chromatography on Bio-Rex 70 (Fig. 2~ Comparison with the retention volume of unreacted pseudexin, using the same column, revealed the first peak to be the unmodified form . This conclusion was confirmed by phospholipase assays. Modification of pseudexin resulted in a loss of 99-9'/ of its phospholipase activity. When tested for toxicity, at a dose (6 mg/kg) which was 12 times the LD50 of pseudexin, modified pseudexin was not lethal. Modification also reduced the direct hemolytic activity of pseudexin. Pseudexin produced direct hemolysis of 50/ of the erythrocytes in the assay mixture at adose of21 -4,ug whereas the sameamount ofmodified pseudexin caused only 3% hemolysis. DISCUSSION
Pseudexin, isolated in a purified form, is a phospholipase consisting ofa single polypeptide chain . It has a mol. wt of approximately 16,500. The symptoms of paralysis ofthe hind legs and general flaccid paralysis shown by mice injected with pseudexin, are similar to those reported for notexin (KARLSSON et al., 1972). As with the presynaptically-acting toxins,
10 0
GARY T. VAUGHAN, THOMAS B. SCULLEY and ROY TIRRELL
notexin and ß-bungarotoxin, phospholipase activity is a requirement for toxicity of pseudexin . The evidence suggests that pseudexin may be a presynaptic neurotoxin and indeed preliminary neurophysiological studies on mouse diaphragm neuromuscular junctions have indicated that pseudexin acts presynaptically (unpublished results). Pseudexin has both direct and indirect hemolytic activity. Its indirect hemolytic activity in the presence of egg yolk phospholipids results from the production of lysophospholipids, which are known to be strongly hemolytic (CONDREA et al ., 1964). Crotoxin, and to a lesser extent ß-bungarotoxin, have indirect hemolytic activity (JENG et al ., 1978). Usually, phospholipases do not lyse erythrocytes in the absence of added phospholipid. However, in the presence of a direct lyric factor, or sublytic concentrations of detergents, or high Cat + concentrations, or albumin or in hypotonic media, direct hemolysis by phospholipases can occur (GUL et al., 1974). The conditions in which the direct hemolytic activity of pseudexin was tested were such that a typical phospholipase would not be hemolytic. Nevertheless, it would appear from the low direct hemolytic activity ofp-bromophenacyl bromide-modified pseudexin that the phospholipase activity plays a major role in the direct hemolysis by the toxin. Similarly, phospholipase action would appear to be involved in its toxicity . This is consistent with findings using presynaptically-acting toxins (HABERMANN and BRETTHAUPT, 1978). Acknowledgements-We express our thanks to Mr . A. Rvs and Mr. R. MANN for their assistance with amino acid analyses. This work was partly supported by an Australian Research Grants Committee grant (D2 67/16508). REFERENCES
ANDREWS, P. (1965) The gel filtration behaviour of proteins related to their molecular weights over a wide range. Biochem. J. 96, 595. BEAYAN, G. H. and HOLIDAY, E. R. (1952) Ultraviolet absorption spectraof proteins and amino acids. Ado. Protein Chem. 7, 319. BROCKERHOFF, H. and JENSEN, R. G. (1974) Lipolytic Enzymes, p. 197. New York : Academic Press. CoNDREA, E., DE VRIES, A. and MAGER, J. (1964) Hemolysis and splitting of human erythrocyte phospholipids by snake venoms. Biochim. biophys . Acts 84, 60. CRFSTFIELD, A. M., MOORE, S. and STEIN, W. H. (1963) The preparation and enzymic hydrolysis of reduced and Scarboxymethylated proteins. J . biol . Chem . 238, 622. DE HURTADO, I. and LAYRISSE, M. (1964) A quantitative method for the assay of snake venom hemolytic activity. Toxicon 2, 43. FAKER, D. (1975) Structural nature of pre-synaptic neurotoxins from Australian elapid venoms . Toxicon 13, 90. Gut, S, KHARA, J. S.and SMmI, A. D. (1974) Hemolysis of washed human redcells by various snakevenoms in the presence of albumin and Ca= +. Toxicon 12, 311. HABERMANN, E. and BREITHAUPT, H. (1978) The crotoxin complex-an exampleof biochemical and pharmacological complementation. Toxicon 16, 19 . HALPERT, J.and FAKER, D. (1976) Isolation and amino acid sequence of a neurotoxic phosolipase A from the venom of the Australian tiger snake, Notschis scutatus scutatus. J. biol. Chem. 251, 7343. HALPERT, J, FAKER, D. and KARLSSON, E. (1976) The role of phospholipase activity in the action of a presynaptic neurotoxin from the venom of Notschis scutatus scutatus (Australian tiger snake). FEBS Lett. 61, 72. ITZHAKI, R. F. and GILL, D. M. (1964) A micro-biuret method for estimating proteins . Analyt. Biochem . 9, 401. JENG, T. W., HENDON, R. A. and FRAENKEL-TONRAT, J. (1978) Search for relationships among the hemolytic, phospholipolytic, and neurotoxic activities of snake venoms. Proc . natn . Acad. Sci . U .S .A. 75, 600. JOSHUA, H. and ISHAY, J. (1973) The hemolytic properties of the oriental hornet venom. Acts Phasmac. Tox. 33,42. KARLssoN, E. and FAKER, D. (1972) Isolation of the principal neurotoxins of Naja naja subspecies from the Asian mainland. Toxicon 10, 217. KARLssoN, E, FAKER, D. and RYDEN, L. (1972) Purification of a presynaptic neurotoxin from the venom of the Australian tiger snake, Notschis scutatus scutatus. Toxicon 10, 405. KEId.AWAY, C. H. (1938) The symptomology and treatment of the bites of Australian snakes . Med . J . Aust. 2, 585. KOCHALATY, W. F. (1966) A rapid and simple phospholipase A assay. Toxicon 4, 1 . MOON,K. E. andTHOM PEON, E. O. P.(1969) Subunits from reduced and S-carboxymethylated ribulose diphosphate carboxylase (Fraction I protein) . Aust. J. Not. Sci. 22, 463. REED, L. D. and MUENCH, H. (1938) A simple method of estimating fifty per cent end points. Am . J . Hyg. 27, 493.
Phospholipase from Pmudechis porphyriacus
10 1
F. and THOMPSON, & O. P . (1963) Halogenation oftyrosine during acid hydrolysis. BiocMm . biophys. Acta 71,468. SPIES, J . R and CHAMBERS, D. C. (1949) Chemical determination of tryptophan in proteins. Anal. Chan . 21, 1249 . VOLwERI<, J. J, PIE[ERSON, W. A. and DE HMS, G. H . (1974) Histidine at the active site of phospholipase A= . Bioche nistry 13, 1446. WEBER, K. and OSBORN, M. (1969) The reliability of mol . wt determinations by dode yl sulfate-polyaaylamide gel electrophoresis. J. biol. Chan. 224, 4406. SANoER,