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Identification of the direct lytic factor from cobra venom as cardiotoxin
Toxlcon, 1969, Vol. 6. pp. 167-173 . PerQamo n Preas. Printed in Great Sritaia
IDENTIFICATION OF THE DIRECT LYTIC FACTOR FROM COBRA VENOM AS CARDIOTOXIN* KARL H. SLOTTA
and JAMES A .
VICK
Departments of Biochemistry and Medicine, University of Miami School of Medicine, Miami, Florida, U.S.A . and Neurology Branch Experimental Medicine Division, Medical Research, Edgwood Arsenal, Maryland, U.S .A . (Acceptedjorpublication 1 July 1968) Abstract-The most basic polypeptide in cobra (Ngja ngja) venom was isolated by chromatography on CM Sephadex columns as a chromatographically and electrophoretically uniform substance. This polypeptide comprises the total, rather low, direct lytic activity and also the total, very strong, cardiotoxic activity of the cobra venom. It should, therefore, be named `cardiotoxin' rather than 'direct lytic factor' (DLF). Cardiotoxin and phospholipase A display a synergistic action irr vitro. To determine whether this is also true In vivo, cardiotoxin was injected into dogs and monkeys by itself and together with phospholipase A. Furthermore, because of the interesting parallelisms of cobra and bee venom, analogous experiments with the polypeptides from bee venom were undertaken . The results showed that the lethality of cardiotoxin from cobra venom as well as that of melittin from bee venom increases strongly by additional injection of phospholipase A either before or after that of cardiotoxin or melittin . Thesecond injection is the decisive one in the mechanism of death ; if it consists of phospholipase A, the animals die due to respiratory failure, otherwise death is due to ventricular fibrillation.
Irt 1947 a substance from Naja raja venom was isolated by salt precipitation ; it stopped the perfused toad heart and arrested the heart beat of cats [1]. The molecular weight of this `cardiotoxin' was determined as 46,200 . When Naja naja venom was later dialysed, one part of the cardiotoxic factor was in the dialysate, while another one remained bound to the larger proteins and could be split off from them by paper chromatography [2] . These experiments made it obvious that `cardiotoxin' is one of the polypt;}ptides of Naja venom, not easily separable from the other components . The so-called `direct lytic factor' (DLF) of cobra venom is also one of the basic polypeptides so far separated only by paper electrophoresis [3J. The activity of DLF is rather low, particularly when compared with the corresponding substance, melittin, from bee (Apis mellifera) venom [4]. The aim of the present study was to prepare cardiotoxin and DLF in purest form and adequate supply, and then to test their activities in vitro and in vivo . Furthermore, phospholipase A from Naja raja venom was prepared and the activity of DLF by itself as well as together with phospholipase A was investigated . Finally, phospholipase A and melittin from bee venom were studied to show the similarities as well as the differences between these corresponding factors. "Supported by National Science Foundation grant GB-4445. 167
16R
KARL H . SLOTTA and JAMES A . VICK METHODS AND MATERIALS
Naja naja venom from the Miami Serpentarium, Miami, Florida and bee venom from Champlain Valley Apiaries, Middlebury, Vermont were used exclusively. Although column chromatography is the only practical method for the preparation of phospholipase A and DLF from Naja naja venom, electrophoresis on cellulose polyacetate strips is preferable for quantitative determinations of the two hemolytic factors. After staining the protein-polypeptide fractions the absorption curve can be drawn, the respective areas measured and the percentages ofall components calculated with sufficient accuracy. In this way we found in different samples of Naja venom 126, 145 and 146 per cent phospholipase A, and 234, 267 and 299 per cent DLF, respectively. Earlier experiments to isolate the two hemolytic factors on DEAF Sephadex columns were unsuccessful. Variations with CM Sephadex columns, such as changing buffers, pH and ionic strength did not provide satisfactory yields and degrees of purity either . This was finally achieved when batches of 500 mg raw venom in 2 ml water were applied on a column of 2~5 x SO cm CM Sephadex C 25 and the substances were eluted stepwise with the following buffers : A - Sodium acetate, pH 5~5, i.str. 001 ; B - Phosphate, pH 7~5, i .str. 0~ 1 +0~ 1 M NaCI ; C - Borate, pH 8~6, i.str. 0~ 1-}-025 M NaCI ; D - Borate, pH 8~6, i.str. 0~1-~1~0 M NaCI. The efljuent was collected in 2 ml fractions and the O.D. at 280 m~ recorded (Fig. 1). Thus, all proteins and polypeptides of the venom were obtained in a volume of 2S0-S00 ml.
Ç Ô
Tube No .
220 230
FIG . I . STEPWISE ELUTION OF NQjn Ifpja VENOM FROM CM SEPHADEX C TS COLUMN .
The solutions in the tubes corresponding to the first and last peak were tested for their phospholipase A and DLF activity, respectively . To assay phospholipase A, 10 ml from each tube were incubated at 37° with 0~2 ml ovolecithin suspension (1 mg per ml) in 1 ml of a 2 per cent suspension of washed, fresh, human erythrocytes in saline-phosphate buffer (4 :1), pH 7~5. Complete hemolytis after 30 min indicated high enzyme concentration in the respective tubes. For the assay of DLF, 10 ~I from each tube were incubated in an identical suspension of erythrocytes, but with 4 Wg phospholipase A (from Naja or bee venom) instead of the lecithin suspension. After 30 min of incubation the DLF-containing samples were completely hemolyzed. The contents of the tubes were combined into fractions according to the peaks of the
FIa. T. ELECTROPHORESIS OF (â~ NOjR pOja VENOM ; (b~ PHOSPHOLIPASE A FROM NnjO itpjpVENOM ; (C) DLF FROM Noja pOjü VENOM.
Tox. f.p. 168
DLF from Cobra Venom : Cardiotoxin
169
O.D . curve and the results of the testing for hemolysis. The solutions were acidified to pH 3 to 4, concentrated in vacuo to small volumes and the picrates precipitated with saturated, aqueous picric acid. They were separated by centrifugation, dried in vacuo, and transformed to hydrochlorides by stirring in acetone-hydrochloric acid (2 ml HCl in 1000 ml acetone). After pure DLF had been obtained by this technique, a standard curve could be computed and the polypeptide content of each of the DLF tubes could be determined quantitatively in further experiments by the Lowry method [5]. By using an adequate scale the curves of the polypeptide content and u .v.-absorption of the DLF became pracrically identical (Fig. 1). Under the conditions described above, phospholipase A and DLF represent the first and last of about twelve fractions and are uniform and free of by-products and salts. The yields of both were low in relation to the theoretical content of the venom. Generally not more than 55 mg phospholipase and 84 mg DLF (about 80 and 60 per cent of the theory, respectively) could be isolated in pure form . In earlier experiments with higher pH and ionic strength the electrophoresis of DLF showed one or two very small, hardly distinguishable lines beside that of the main fraction, and the DLF activity was significantly lower. To remove traces of these strongly adhering by-products, the DLF from three or four experiments was re-chromatographed, and the different fractions were re-precipitated separately. However, the losses by re-chromatography were very high . The purity of phospholipase A and DLF was tested by electrophoresis on cellulose polyacetate strips (Sepraphore III) in 005 M Veronal-Tris buffer, pH 8~8, 400 V, 45 min [4], (Fig. 2a). Phospholipase A from the column always appeared as a rather broad band in electrophoresis (Fig. 2b). This was the case even when it was re-chromatographed twice, or when only the first few tubes ofthe effluent from the column were used and tested separately. Different phospholipase A preparations, however, with similar electrophoresis patterns showed the same phospholipase activity. By determining the phosphoesterase activity of phospholipase from bee and Naja venom with the same method [6] we found that 2 wg of the pure enzymes from both sources equally hydrolyzed 80 per cent of the ovolecithin in suspension in 20 min. Whereas there are many methods for quantitative evaluation of phospholipase A activity, the determination of the relatively very weak activity of DLF from Naja venom presented a problem. Based on the discovery of the synergistic effect of phospholipase A on DLF [3], the activity of DLF was assayed against erythrocytes in the presence of a minute quantity of phospholipase A: to test-tubes (10 x 75 mm) with 1 ml of a 2 per cent suspension of washed, fresh, human erythrocytes in saline-phosphate buffer (4:1), pH 7~5, 4 wg of phospholipase A and different amounts of DLF preparations (from 5 to 20 fig) were added. The tubes were incubated at 37° for exactly 30 min and checked for complete hemolysis. We determined the lowest amount of chromatographically and electrophoretically uniform DLF (Fig. 2c) necessary for complete hemolysis to be 8 wg. Preparation of phospholipase A and melittin from bee venom was performed by established methods [7, 8]. The purity of melittin so obtained was tested by amino acid analyses ; the results were identical to the values published by others [7, 8]. DLF and melittin as well as phospholipase A from cobra venom were studied on 12 adult mongrel dogs and 14 rhesus monkeys. Simultaneous and continuous recordings of cerebral cortical electrical activity (EEG), blood pressure, electrocardiogram (EKG), heart rate and respiratory rate were made from time of injection until death. All injections were made directly into a femoral vein of dogs and monkeys [9].
170
KARL H. SLOTTA and JAMES A. vICK
The effect ofcardiotoxin was studied using the isolated canine heart preparation perfused with autologous blood [10]. Adult mongrel dogs, weighing between 10 and 15 kg, were anesthetized with 30 mg per kg Na pentobarbital (Nembutal) . A large bore polyethylene catheter was placed in the right femoral artery to allow for the rapid removal of blood. Heparin sodium, 1000 U.S.P . units per kg of body weight, was injected into the right femoral vein to prevent clotting. Artificial respiration was maintained using a Starling pump equipped with appropriate endotracheal fittings . The chest was opened by a midGne incision, and all major arteries in the aortic arch ligated . The heart was then quickly removed from the chest cavity and perfused with oxygenated blood maintained at 32-36° . Perfusion (pump) pressure was continuously monitored using an E & M pressure transducer. Needle-tipped electrodes were placed into either ventricle to monitor EKG and heart rate. Force of contraction was measured by means of a Walton-Brodie strain gauge arch sutured to the left ventricle of the heart. All injections of drugs were made directly into the venous side of the Sigma motor pump perfusing the heart. RESULTS
Direct lyticfactor (DLF)from Naja venom 3 mg per kg DLF administered i .v . to dogs produced little or no effect on any of the parameters monitored, and all animals survived . The same doses (3 mg per kg) given to monkeys produced a slight drop in arterial pressure with some narrowing of the pulse pressure . This effect, however, was followed by severe cardiac arrhythmia and ventricular fibrillation at approximately 45 sec. During this extremelybriefperiod oftime there appeared to be an associated loss of cortical electrical activity, arrest of respiration and decrease in heart rate . All monkeys receiving this dose of DLF died within 15 min following injection. When 1 mg DLF was injected into two monkeys, severe cardiac arrhythmia and some fall in arterial blood pressure occurred . However, these changes returned to normal 2 to 4 hr after injection, and both monkeys survived . Melittin The direct lytic factor from bee venom, when injected into dogs, produced only a slight and transient decrease in arterial blood pressure immediately after the injection of 3 mg per kg. Recovery to normal followed within 3 to 5 min, and all dogs survived . In contrast to DLF from cobra venom, 3 mg per kg of bee melittin produced neither severe arrhythmia nor death in monkeys. All animals survived . Phospholipase A Intravenously administered phospholipase A in doses of 2 mg per kg from cobra venom as well as that from bee venom showed very similar effects in both dogs and monkeys. Arterial blood pressure decreased from 160/100 to a low of 40/10 ; heart rate decreased from 130 to 60 beats per min ; and respiration rate fell from 12 per min to 2 per min, 30 sec after injection. The most remarkable effect of phospholipase A, however, was the dramatic loss of all cortical electrical activity within 30-60 sec afterinjection . From 15 to 30 min after injection all parameters began to return to normal, reaching control or near control levels at approximately 40-90 min post injection. None of the animals expired. It is important to note that phospholipase A from Naja venom had much less effect in monkeys than in dogs.
bLF
from Cobra Venom : Cardiotoxin
171
Combination ofsubletlral doses oflyticfactors The injection of 2 mg per kg phospholipase A (Naja) into either dogs or monkeys, 60 min after 1 mg per kg DLF, resulted in a sharp decrease in blood pressure, heart rate and cortical activity within 8 to 12 min . The injection of the two lytic factors in reverse order into the dog or the monkey produced very different results . Both species of animals became sensitized to the `cardiotoxic action' ofDLF by prior administration ofphospholipase A. When 2 mg per kg cobra venom phospholipase A were followed in 60 min by I mg per kg DLF, immediate death due to ventricular fibrillation resulted . Cardiotoxic activity The injection of 30 mg DLF (Naja) into the perfused dog heart was followed by a progressive decrease in force of contraction, terminating in its complete loss at approximately 37500 sec. During this period of time the heart rate increased from 78 per min to 180 per min, falling to 0 per min at 400 sec. Marked changes in EKG occurred at 240 sec and were characterized by extrasystoles, multifocal discharges and premature beats which lead to complete cardiac arrest at 400 sec (Fig. 3). FORCE OF CONTRACTION
40 l1
20
~1,1 . _J 78/M in
HEART RATE EKG
wvi~
PERFUSION PRESSURE (mm Hg)
~2
~ ,.
',~~~,
. .
. .
.
FA~-e
200100~~ 0 ~-rrrr-r~rrr-r~-rr~-rrr-,-r-~-r10 30 50 70 90 240 SEC DLF (Naja) (30 mg)
FIG. 3.
. ,
180/Min 12/Min 0/Min. .
CARDIOTOXIC
ACTIVrrY
375
400 Sec.
mg
AFTER INJECriON OF 3O CARDIOTOXIN=DLF FROM NOjA RafO VENOM INTO PERFU3ED DOCi HEART .
The cardiotoxic effect of melittin is very similar to that of DLF (Naja) but much less potent. Injection of 100 mg melittin produced an increase in perfusion pressure and heart rate at 30 sec, followed by arrhythmias and a decrease in force of contraction . Irreversible ventricular fibrillation followed multifocal discharges and premature ventricular beats noted on the EKG tracing. An obvious loss ofcontractile force accompanied the ventricular fibrillation (Fig. 4). Similar experiments with raw cobra and bee venom showed activity one-quarter of that produced by the isolated cardiotoxins. This is in accordance with the fact mentioned earlier, that the cardiotoxin fraction comprises about one-quarter of the weight of the raw venom. Other fractions, including phospholipase A from both bee and cobra venom, had no erect on the isolated perfused heart .
17 2
KARL H. SLOTTA and JAMES A. VICK
20FORCE OF CONTRACTION 0 : HEART RATE EKG
PERFUSION PRESSURE (mm Hg)
ylii ivv
w
200 : ]00' 0-
-rr
MELITTIN (100mg)
30
60
90
120 Sec
FIG. 4. CARDIOTOXIC ACTIVITY AFTER INJECTION OF 100 mg MELTTTIN FROM BEE VENOM INTO PERFUSED DOG HEART. DISCUSSION
The most basic polypeptide in Naja venom has a specific and highly toxic effect on the heart and a very weak direct hemolytic activity on erythrocytes . Thus, we suggest calling this substance `cardiotoxin' rather than DLF. It is completely different, not only in its activities but also in its chemical structure from neurotoxin, the most toxic polypeptide of cobra venom recently isolated in pure form by different investigators [1 l, 12]. Whereas the lethality of neurotoxin depends on the integrity of the disulfide bonds [13], cardiotoxin is free of sulfur-containing amino acids . It seems significant from the analytical point of view that cardiotoxin and melittin contain lysine and arginine but no histidine. This was found by amino acid analyses of hydrolysates from cardiotoxin and melittin by column chromatography and staining with ninhydrin [14]. The most fascinating fact brought to light by these studies is the interaction of cardiotoxin with phospholipase A. This synergism had previously been detected in vitro by others [3] and one of us applied it to the detection of minimal amounts of cardiotoxin as a basis for the quantitative determination of the direct lytic activity [4]. We had, however, not expected that the injection of phospholipase A and cardiotoxin into animals in sublethal doses would produce synergistic effects similar to those observed in vitro. While phospholipase A is one of the enzymes normally present in brain [15], the injection of additional phospholipase A produces a severe disruption in the cortical electric activity. The injection of phospholipase A also sensitizes the organism to a subsequent injection of cardiotoxin. The reverse is also true. The second injection always determines the ultimate mechanism of death ; if it is cardiotoxin, the animals die due to ventricular fibrillation ; if it is phospholipase A, they die of respiratory failure. Nothing is known about the mechanism by which cardiotoxin exerts its special effect on the heart as well as on the membrane of erythrocytes . It seems that the direct lytic activity is linked to the fact that the red cell membranes have a great capacity for binding cardia toxin ; this can be concluded from experiments with IIaI labelled DLF [l6] . It is doubtful
DLF from Cobra Venom: Cardiotoxin
173
that the lytic action of such basic polypeptides can be simply interpreted as being due to their `tensid' structure or to their effect as that of an `invert soap' [17] . REFERENCES
[1] SARKAR, N. K Existence of a cardiotoxic principle in cobra venom. Ann. Bi'ochinr. exp. Med. 8, 11, 1947 . [2] RAUDONAT, H. W. and HOLLER, B., Heart-active component of cobra venom (cardiotoxin). Arch. exp. Path . Pharmak. 233, 431, 1958 . (3] CONDREA, E., DE VRIFS, A. and MAGER, J., Hemolysis and splitting of human erythrocyte phospholipids by snake venoms . Blochim. biophys. Acta 84, 60, 1964. [4] SLOTTA, K. H., GONZALEZ, J. A. and RoTH, S. C., Thedirect and indirect hemolytic factors from animal venoms . In : Animal Toxtns. (RUSSELL, F. E. and SAUNDERS, P. R., Eds.) . Pergamon Press : Oxford, PP~ 369-377,1967. [S] LOWRY, O. H., R06ENBROUGH, N. O., FARR, A. L. and RANDALL, R. J., Protein measurement With the folin phenol reagent. J. biol. Chern.193, 265,1951 . [6] MAGEE, W. L. and THOMPSON, R. H. S., The estimation of phospholipase A activity in aqueous systems. Biochenr . .i. 77, 526, 1960 . [7] HAHERMANN, E. and Re~z, K. G., A new method for the separation of the components of bee venom, especially of the centrally active peptide, apamine. Bioche»r. Z. 341, 451, 1965 . Biochemistry of the bee poison peptides melittin and apamine. Biochern . Z. 343, 192, 1965 . [8] KREIL, G., Preparation and characterization of melittin, the main toxin of bee poison . Morratsh . Cheer. 96, 2061, 1965 . [9] Vrcx, J. A., CivcrrrA, N. P. and POLLEY, E. N., Effect of snake venom and endotoxin on cortical electrical activity . Nature,Lond. 203,1387,1964. [10] VrcK, J. A, and PERRY, J. F., JR ., Vascular effects of exotoxins (snake venoms) in heart and lung . Fedn Proc. Fedn Am. Soes exp.Biol . 22,431,1963 . [11] YANG, C. C., Crystallization and properties of cobra toxin from Formosan cobra venom. J. biol. Chem. 240, 1616, 1965 . [12] KARLSSON, E., FAKER, D. L. and PORATH,J., Purification of a neurotoxin from the venomof Ngja nigrfcollis . Bioclrirn . biophys. Acta 127, SOS, 1966 . [l3] YANG, C. C., The disulfide bonds of cobratoxin and their relationship to lethality. Biochlm. biophys. Acta 133, 346, 1967. [14] MOORS, S., SPACKMAN, A. H. and STEIN, W. H., Chromatography of amino acids on sulfurated polysterene resins . Analyt . Cheer. 30, 1185, 1958 . [15] GALLAI-HATCHARD, J., MAGEE, W. L., THOMPSON, R. H. $. and WEHSTER, $. R., The formation of lysophosphatides from di-acyl phosphatides by brain preparations . J. Neurochenr. 9, 454, 1962. [16] COIYDREA, E., KENDZERSKY, l, and DE VRIFS, A., Binding of Ringhals venom direct hemolytic factor to erythrocytes and osmotic ghosts of various animal species. F .xperientia 21, 461, 1965. (17] HABERMANN, E. Snd JENTSCH, J., Sequential analysis of melittin from tryptic and peptic fragments. Biochenr . Z. 345, 37, 1967.
IDENTIFICATION OF THE DIRECT LYTIC FACTOR FROM COBRA VENOM AS CARDIOTOXIN* KARL H. SLOTTA
and JAMES A .
VICK
Departments of Biochemistry and Medicine, University of Miami School of Medicine, Miami, Florida, U.S.A . and Neurology Branch Experimental Medicine Division, Medical Research, Edgwood Arsenal, Maryland, U.S .A . (Acceptedjorpublication 1 July 1968) Abstract-The most basic polypeptide in cobra (Ngja ngja) venom was isolated by chromatography on CM Sephadex columns as a chromatographically and electrophoretically uniform substance. This polypeptide comprises the total, rather low, direct lytic activity and also the total, very strong, cardiotoxic activity of the cobra venom. It should, therefore, be named `cardiotoxin' rather than 'direct lytic factor' (DLF). Cardiotoxin and phospholipase A display a synergistic action irr vitro. To determine whether this is also true In vivo, cardiotoxin was injected into dogs and monkeys by itself and together with phospholipase A. Furthermore, because of the interesting parallelisms of cobra and bee venom, analogous experiments with the polypeptides from bee venom were undertaken . The results showed that the lethality of cardiotoxin from cobra venom as well as that of melittin from bee venom increases strongly by additional injection of phospholipase A either before or after that of cardiotoxin or melittin . Thesecond injection is the decisive one in the mechanism of death ; if it consists of phospholipase A, the animals die due to respiratory failure, otherwise death is due to ventricular fibrillation.
Irt 1947 a substance from Naja raja venom was isolated by salt precipitation ; it stopped the perfused toad heart and arrested the heart beat of cats [1]. The molecular weight of this `cardiotoxin' was determined as 46,200 . When Naja naja venom was later dialysed, one part of the cardiotoxic factor was in the dialysate, while another one remained bound to the larger proteins and could be split off from them by paper chromatography [2] . These experiments made it obvious that `cardiotoxin' is one of the polypt;}ptides of Naja venom, not easily separable from the other components . The so-called `direct lytic factor' (DLF) of cobra venom is also one of the basic polypeptides so far separated only by paper electrophoresis [3J. The activity of DLF is rather low, particularly when compared with the corresponding substance, melittin, from bee (Apis mellifera) venom [4]. The aim of the present study was to prepare cardiotoxin and DLF in purest form and adequate supply, and then to test their activities in vitro and in vivo . Furthermore, phospholipase A from Naja raja venom was prepared and the activity of DLF by itself as well as together with phospholipase A was investigated . Finally, phospholipase A and melittin from bee venom were studied to show the similarities as well as the differences between these corresponding factors. "Supported by National Science Foundation grant GB-4445. 167
16R
KARL H . SLOTTA and JAMES A . VICK METHODS AND MATERIALS
Naja naja venom from the Miami Serpentarium, Miami, Florida and bee venom from Champlain Valley Apiaries, Middlebury, Vermont were used exclusively. Although column chromatography is the only practical method for the preparation of phospholipase A and DLF from Naja naja venom, electrophoresis on cellulose polyacetate strips is preferable for quantitative determinations of the two hemolytic factors. After staining the protein-polypeptide fractions the absorption curve can be drawn, the respective areas measured and the percentages ofall components calculated with sufficient accuracy. In this way we found in different samples of Naja venom 126, 145 and 146 per cent phospholipase A, and 234, 267 and 299 per cent DLF, respectively. Earlier experiments to isolate the two hemolytic factors on DEAF Sephadex columns were unsuccessful. Variations with CM Sephadex columns, such as changing buffers, pH and ionic strength did not provide satisfactory yields and degrees of purity either . This was finally achieved when batches of 500 mg raw venom in 2 ml water were applied on a column of 2~5 x SO cm CM Sephadex C 25 and the substances were eluted stepwise with the following buffers : A - Sodium acetate, pH 5~5, i.str. 001 ; B - Phosphate, pH 7~5, i .str. 0~ 1 +0~ 1 M NaCI ; C - Borate, pH 8~6, i.str. 0~ 1-}-025 M NaCI ; D - Borate, pH 8~6, i.str. 0~1-~1~0 M NaCI. The efljuent was collected in 2 ml fractions and the O.D. at 280 m~ recorded (Fig. 1). Thus, all proteins and polypeptides of the venom were obtained in a volume of 2S0-S00 ml.
Ç Ô
Tube No .
220 230
FIG . I . STEPWISE ELUTION OF NQjn Ifpja VENOM FROM CM SEPHADEX C TS COLUMN .
The solutions in the tubes corresponding to the first and last peak were tested for their phospholipase A and DLF activity, respectively . To assay phospholipase A, 10 ml from each tube were incubated at 37° with 0~2 ml ovolecithin suspension (1 mg per ml) in 1 ml of a 2 per cent suspension of washed, fresh, human erythrocytes in saline-phosphate buffer (4 :1), pH 7~5. Complete hemolytis after 30 min indicated high enzyme concentration in the respective tubes. For the assay of DLF, 10 ~I from each tube were incubated in an identical suspension of erythrocytes, but with 4 Wg phospholipase A (from Naja or bee venom) instead of the lecithin suspension. After 30 min of incubation the DLF-containing samples were completely hemolyzed. The contents of the tubes were combined into fractions according to the peaks of the
FIa. T. ELECTROPHORESIS OF (â~ NOjR pOja VENOM ; (b~ PHOSPHOLIPASE A FROM NnjO itpjpVENOM ; (C) DLF FROM Noja pOjü VENOM.
Tox. f.p. 168
DLF from Cobra Venom : Cardiotoxin
169
O.D . curve and the results of the testing for hemolysis. The solutions were acidified to pH 3 to 4, concentrated in vacuo to small volumes and the picrates precipitated with saturated, aqueous picric acid. They were separated by centrifugation, dried in vacuo, and transformed to hydrochlorides by stirring in acetone-hydrochloric acid (2 ml HCl in 1000 ml acetone). After pure DLF had been obtained by this technique, a standard curve could be computed and the polypeptide content of each of the DLF tubes could be determined quantitatively in further experiments by the Lowry method [5]. By using an adequate scale the curves of the polypeptide content and u .v.-absorption of the DLF became pracrically identical (Fig. 1). Under the conditions described above, phospholipase A and DLF represent the first and last of about twelve fractions and are uniform and free of by-products and salts. The yields of both were low in relation to the theoretical content of the venom. Generally not more than 55 mg phospholipase and 84 mg DLF (about 80 and 60 per cent of the theory, respectively) could be isolated in pure form . In earlier experiments with higher pH and ionic strength the electrophoresis of DLF showed one or two very small, hardly distinguishable lines beside that of the main fraction, and the DLF activity was significantly lower. To remove traces of these strongly adhering by-products, the DLF from three or four experiments was re-chromatographed, and the different fractions were re-precipitated separately. However, the losses by re-chromatography were very high . The purity of phospholipase A and DLF was tested by electrophoresis on cellulose polyacetate strips (Sepraphore III) in 005 M Veronal-Tris buffer, pH 8~8, 400 V, 45 min [4], (Fig. 2a). Phospholipase A from the column always appeared as a rather broad band in electrophoresis (Fig. 2b). This was the case even when it was re-chromatographed twice, or when only the first few tubes ofthe effluent from the column were used and tested separately. Different phospholipase A preparations, however, with similar electrophoresis patterns showed the same phospholipase activity. By determining the phosphoesterase activity of phospholipase from bee and Naja venom with the same method [6] we found that 2 wg of the pure enzymes from both sources equally hydrolyzed 80 per cent of the ovolecithin in suspension in 20 min. Whereas there are many methods for quantitative evaluation of phospholipase A activity, the determination of the relatively very weak activity of DLF from Naja venom presented a problem. Based on the discovery of the synergistic effect of phospholipase A on DLF [3], the activity of DLF was assayed against erythrocytes in the presence of a minute quantity of phospholipase A: to test-tubes (10 x 75 mm) with 1 ml of a 2 per cent suspension of washed, fresh, human erythrocytes in saline-phosphate buffer (4:1), pH 7~5, 4 wg of phospholipase A and different amounts of DLF preparations (from 5 to 20 fig) were added. The tubes were incubated at 37° for exactly 30 min and checked for complete hemolysis. We determined the lowest amount of chromatographically and electrophoretically uniform DLF (Fig. 2c) necessary for complete hemolysis to be 8 wg. Preparation of phospholipase A and melittin from bee venom was performed by established methods [7, 8]. The purity of melittin so obtained was tested by amino acid analyses ; the results were identical to the values published by others [7, 8]. DLF and melittin as well as phospholipase A from cobra venom were studied on 12 adult mongrel dogs and 14 rhesus monkeys. Simultaneous and continuous recordings of cerebral cortical electrical activity (EEG), blood pressure, electrocardiogram (EKG), heart rate and respiratory rate were made from time of injection until death. All injections were made directly into a femoral vein of dogs and monkeys [9].
170
KARL H. SLOTTA and JAMES A. vICK
The effect ofcardiotoxin was studied using the isolated canine heart preparation perfused with autologous blood [10]. Adult mongrel dogs, weighing between 10 and 15 kg, were anesthetized with 30 mg per kg Na pentobarbital (Nembutal) . A large bore polyethylene catheter was placed in the right femoral artery to allow for the rapid removal of blood. Heparin sodium, 1000 U.S.P . units per kg of body weight, was injected into the right femoral vein to prevent clotting. Artificial respiration was maintained using a Starling pump equipped with appropriate endotracheal fittings . The chest was opened by a midGne incision, and all major arteries in the aortic arch ligated . The heart was then quickly removed from the chest cavity and perfused with oxygenated blood maintained at 32-36° . Perfusion (pump) pressure was continuously monitored using an E & M pressure transducer. Needle-tipped electrodes were placed into either ventricle to monitor EKG and heart rate. Force of contraction was measured by means of a Walton-Brodie strain gauge arch sutured to the left ventricle of the heart. All injections of drugs were made directly into the venous side of the Sigma motor pump perfusing the heart. RESULTS
Direct lyticfactor (DLF)from Naja venom 3 mg per kg DLF administered i .v . to dogs produced little or no effect on any of the parameters monitored, and all animals survived . The same doses (3 mg per kg) given to monkeys produced a slight drop in arterial pressure with some narrowing of the pulse pressure . This effect, however, was followed by severe cardiac arrhythmia and ventricular fibrillation at approximately 45 sec. During this extremelybriefperiod oftime there appeared to be an associated loss of cortical electrical activity, arrest of respiration and decrease in heart rate . All monkeys receiving this dose of DLF died within 15 min following injection. When 1 mg DLF was injected into two monkeys, severe cardiac arrhythmia and some fall in arterial blood pressure occurred . However, these changes returned to normal 2 to 4 hr after injection, and both monkeys survived . Melittin The direct lytic factor from bee venom, when injected into dogs, produced only a slight and transient decrease in arterial blood pressure immediately after the injection of 3 mg per kg. Recovery to normal followed within 3 to 5 min, and all dogs survived . In contrast to DLF from cobra venom, 3 mg per kg of bee melittin produced neither severe arrhythmia nor death in monkeys. All animals survived . Phospholipase A Intravenously administered phospholipase A in doses of 2 mg per kg from cobra venom as well as that from bee venom showed very similar effects in both dogs and monkeys. Arterial blood pressure decreased from 160/100 to a low of 40/10 ; heart rate decreased from 130 to 60 beats per min ; and respiration rate fell from 12 per min to 2 per min, 30 sec after injection. The most remarkable effect of phospholipase A, however, was the dramatic loss of all cortical electrical activity within 30-60 sec afterinjection . From 15 to 30 min after injection all parameters began to return to normal, reaching control or near control levels at approximately 40-90 min post injection. None of the animals expired. It is important to note that phospholipase A from Naja venom had much less effect in monkeys than in dogs.
bLF
from Cobra Venom : Cardiotoxin
171
Combination ofsubletlral doses oflyticfactors The injection of 2 mg per kg phospholipase A (Naja) into either dogs or monkeys, 60 min after 1 mg per kg DLF, resulted in a sharp decrease in blood pressure, heart rate and cortical activity within 8 to 12 min . The injection of the two lytic factors in reverse order into the dog or the monkey produced very different results . Both species of animals became sensitized to the `cardiotoxic action' ofDLF by prior administration ofphospholipase A. When 2 mg per kg cobra venom phospholipase A were followed in 60 min by I mg per kg DLF, immediate death due to ventricular fibrillation resulted . Cardiotoxic activity The injection of 30 mg DLF (Naja) into the perfused dog heart was followed by a progressive decrease in force of contraction, terminating in its complete loss at approximately 37500 sec. During this period of time the heart rate increased from 78 per min to 180 per min, falling to 0 per min at 400 sec. Marked changes in EKG occurred at 240 sec and were characterized by extrasystoles, multifocal discharges and premature beats which lead to complete cardiac arrest at 400 sec (Fig. 3). FORCE OF CONTRACTION
40 l1
20
~1,1 . _J 78/M in
HEART RATE EKG
wvi~
PERFUSION PRESSURE (mm Hg)
~2
~ ,.
',~~~,
. .
. .
.
FA~-e
200100~~ 0 ~-rrrr-r~rrr-r~-rr~-rrr-,-r-~-r10 30 50 70 90 240 SEC DLF (Naja) (30 mg)
FIG. 3.
. ,
180/Min 12/Min 0/Min. .
CARDIOTOXIC
ACTIVrrY
375
400 Sec.
mg
AFTER INJECriON OF 3O CARDIOTOXIN=DLF FROM NOjA RafO VENOM INTO PERFU3ED DOCi HEART .
The cardiotoxic effect of melittin is very similar to that of DLF (Naja) but much less potent. Injection of 100 mg melittin produced an increase in perfusion pressure and heart rate at 30 sec, followed by arrhythmias and a decrease in force of contraction . Irreversible ventricular fibrillation followed multifocal discharges and premature ventricular beats noted on the EKG tracing. An obvious loss ofcontractile force accompanied the ventricular fibrillation (Fig. 4). Similar experiments with raw cobra and bee venom showed activity one-quarter of that produced by the isolated cardiotoxins. This is in accordance with the fact mentioned earlier, that the cardiotoxin fraction comprises about one-quarter of the weight of the raw venom. Other fractions, including phospholipase A from both bee and cobra venom, had no erect on the isolated perfused heart .
17 2
KARL H. SLOTTA and JAMES A. VICK
20FORCE OF CONTRACTION 0 : HEART RATE EKG
PERFUSION PRESSURE (mm Hg)
ylii ivv
w
200 : ]00' 0-
-rr
MELITTIN (100mg)
30
60
90
120 Sec
FIG. 4. CARDIOTOXIC ACTIVITY AFTER INJECTION OF 100 mg MELTTTIN FROM BEE VENOM INTO PERFUSED DOG HEART. DISCUSSION
The most basic polypeptide in Naja venom has a specific and highly toxic effect on the heart and a very weak direct hemolytic activity on erythrocytes . Thus, we suggest calling this substance `cardiotoxin' rather than DLF. It is completely different, not only in its activities but also in its chemical structure from neurotoxin, the most toxic polypeptide of cobra venom recently isolated in pure form by different investigators [1 l, 12]. Whereas the lethality of neurotoxin depends on the integrity of the disulfide bonds [13], cardiotoxin is free of sulfur-containing amino acids . It seems significant from the analytical point of view that cardiotoxin and melittin contain lysine and arginine but no histidine. This was found by amino acid analyses of hydrolysates from cardiotoxin and melittin by column chromatography and staining with ninhydrin [14]. The most fascinating fact brought to light by these studies is the interaction of cardiotoxin with phospholipase A. This synergism had previously been detected in vitro by others [3] and one of us applied it to the detection of minimal amounts of cardiotoxin as a basis for the quantitative determination of the direct lytic activity [4]. We had, however, not expected that the injection of phospholipase A and cardiotoxin into animals in sublethal doses would produce synergistic effects similar to those observed in vitro. While phospholipase A is one of the enzymes normally present in brain [15], the injection of additional phospholipase A produces a severe disruption in the cortical electric activity. The injection of phospholipase A also sensitizes the organism to a subsequent injection of cardiotoxin. The reverse is also true. The second injection always determines the ultimate mechanism of death ; if it is cardiotoxin, the animals die due to ventricular fibrillation ; if it is phospholipase A, they die of respiratory failure. Nothing is known about the mechanism by which cardiotoxin exerts its special effect on the heart as well as on the membrane of erythrocytes . It seems that the direct lytic activity is linked to the fact that the red cell membranes have a great capacity for binding cardia toxin ; this can be concluded from experiments with IIaI labelled DLF [l6] . It is doubtful
DLF from Cobra Venom: Cardiotoxin
173
that the lytic action of such basic polypeptides can be simply interpreted as being due to their `tensid' structure or to their effect as that of an `invert soap' [17] . REFERENCES
[1] SARKAR, N. K Existence of a cardiotoxic principle in cobra venom. Ann. Bi'ochinr. exp. Med. 8, 11, 1947 . [2] RAUDONAT, H. W. and HOLLER, B., Heart-active component of cobra venom (cardiotoxin). Arch. exp. Path . Pharmak. 233, 431, 1958 . (3] CONDREA, E., DE VRIFS, A. and MAGER, J., Hemolysis and splitting of human erythrocyte phospholipids by snake venoms . Blochim. biophys. Acta 84, 60, 1964. [4] SLOTTA, K. H., GONZALEZ, J. A. and RoTH, S. C., Thedirect and indirect hemolytic factors from animal venoms . In : Animal Toxtns. (RUSSELL, F. E. and SAUNDERS, P. R., Eds.) . Pergamon Press : Oxford, PP~ 369-377,1967. [S] LOWRY, O. H., R06ENBROUGH, N. O., FARR, A. L. and RANDALL, R. J., Protein measurement With the folin phenol reagent. J. biol. Chern.193, 265,1951 . [6] MAGEE, W. L. and THOMPSON, R. H. S., The estimation of phospholipase A activity in aqueous systems. Biochenr . .i. 77, 526, 1960 . [7] HAHERMANN, E. and Re~z, K. G., A new method for the separation of the components of bee venom, especially of the centrally active peptide, apamine. Bioche»r. Z. 341, 451, 1965 . Biochemistry of the bee poison peptides melittin and apamine. Biochern . Z. 343, 192, 1965 . [8] KREIL, G., Preparation and characterization of melittin, the main toxin of bee poison . Morratsh . Cheer. 96, 2061, 1965 . [9] Vrcx, J. A., CivcrrrA, N. P. and POLLEY, E. N., Effect of snake venom and endotoxin on cortical electrical activity . Nature,Lond. 203,1387,1964. [10] VrcK, J. A, and PERRY, J. F., JR ., Vascular effects of exotoxins (snake venoms) in heart and lung . Fedn Proc. Fedn Am. Soes exp.Biol . 22,431,1963 . [11] YANG, C. C., Crystallization and properties of cobra toxin from Formosan cobra venom. J. biol. Chem. 240, 1616, 1965 . [12] KARLSSON, E., FAKER, D. L. and PORATH,J., Purification of a neurotoxin from the venomof Ngja nigrfcollis . Bioclrirn . biophys. Acta 127, SOS, 1966 . [l3] YANG, C. C., The disulfide bonds of cobratoxin and their relationship to lethality. Biochlm. biophys. Acta 133, 346, 1967. [14] MOORS, S., SPACKMAN, A. H. and STEIN, W. H., Chromatography of amino acids on sulfurated polysterene resins . Analyt . Cheer. 30, 1185, 1958 . [15] GALLAI-HATCHARD, J., MAGEE, W. L., THOMPSON, R. H. $. and WEHSTER, $. R., The formation of lysophosphatides from di-acyl phosphatides by brain preparations . J. Neurochenr. 9, 454, 1962. [16] COIYDREA, E., KENDZERSKY, l, and DE VRIFS, A., Binding of Ringhals venom direct hemolytic factor to erythrocytes and osmotic ghosts of various animal species. F .xperientia 21, 461, 1965. (17] HABERMANN, E. Snd JENTSCH, J., Sequential analysis of melittin from tryptic and peptic fragments. Biochenr . Z. 345, 37, 1967.