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Titration of antiserum to South American rattlesnake (Crotalus durissus terrificus) venom by inhibition of phospholipase A2 activity
sos.aoro
coal-0lol~zo~ow3-m p 1982 Perpmm leer
Toslron, VoL 20, No. 3, pp. 563-569, 19ß2
Prmoed in Gmat Brihm.
Ltd.
TITRATION OF ANTISERUM TO SOUTH AMERICAN RATTLESNAKE (CROTALUS DURISSUS TERRIFICUS) VENOM BY MEASURING INHIBITION OF PHOSPHOLIPASE A2 ACTIVITY MARIA HELENA DA SILVA* and OTTO GUILHERME BIER WHO/PAHO Immunobgy Research and Training Center, Insdtuto Botenfan, Sâo Paulo, SP, Brazil
(Acceptedfor publication 22 October 1981) M. H . DA SavA and O. G. BmL Titration of antiaerum to South American rattle®ake (Crotalesdivisors terrificus) venom by inhibition of phospholipese A Z activity. Toxicon 20, 563-569, 1982.-Horse antiserum to the venom of Crotales derissus terrificus, a South American rattlesnake, inhibits the phosphofipase activity of the crude venom. There is a close relationship between this inhibitory property and the neutralizing potency of the antiserum in viva. This may provide the basis for a rigorous standardization of anticrotalic venom in vitro.
INTRODUCTION TI->E vENOM of Crotalus durissus temficus, the South American rattlesnake, like other snake venoms is a complex mixture of polypeptides and proteins with toxic and enzymatic
properties (MEBS,1970 ; ZLOTxIN,1973). Its main toxic component, crotoxin, was obtained in 1938 by $LOTTA and FRAENKEL-CONRAT, as a crystalline protein having phospholipase activity. In fact crotoxin is a complex between a highly basic phospholipase A Z (phosphatidate 2-acylhydrolase EC 3.1 .1 .4) and an acidic polypeptide, crotapotin, which potentiates the toxicity but inhibits the enzymatic activity of the phopholipase (HAHIItMANN and Rt)ssA1~N, 1970 ; RÜBSAMEN et al., 1971 ; HENDON and FRAENxEL-CONRAT, 1971).
Crotoxin represents 65-68% (w/w) of the crude venom and shows an LD SO of about 0.070mg/kg after i .p. inoculation in mice . It accounts for the high toxicity of the crude venom : i .p . LD SO, 0.090mg/kg, in mice (HANASHIROet al., 1978). In the same species the i .v. ~so of crotamine, another neurotoxic component present in venoms from specimens collected in Southern Brazil and Argentina (GoxçALVES and VIEIRA, 1950 ; SCHENBERG, 1959), is 4 mg/kg (HABERMANN and CIIElvc-RAUDE, 1975). It has been previously shown that rabbit anti-Crotalus phospholipase A z neutralizes the enzymatic and neurotoxic activities of the purified enzyme as well as of crotoxin and crude
venom (HANASHIRO et al., 1978). This led us to investigate whether the inhibition of phospholipase activity in vitro would parallel the neutralization of toxicity in viva .
+Fellow of CNPq, the Brazilian Council for Research Development and Technology. 563
564
MARIA HELENA DA SILVA and OTTO GUILHERME BIER MATERIALS AND METHODS
Venom One batch of crotatnine positive C. durissus terrificus venom was used throughout the experiments . It was prepared at Butantan Institute (Säo Paulo, Brazil) by pooling venom from animals captured in the Southern region of Brazil and drying it over CaCh under normal pressure, at 37°C. The dried venom was removed from the desiecator and kept in tightly closed vials of 4°C. Two samples of this batch (lO.Omg each), after exposure to the room atmosphere for 24 hr, were transferred to a second deaiocator and maintained over CaCh under normal pressure, at room temperature, until constant weight. The samples were dissohrod in 0.15 M NaCI and used to establish a standard curve by plotting absorbency at 280 nm vs venom ooneentration in mg per mL The concentration of venom solutions used throughout the experiments was obtained by reference to this curve. All venom solutions were kept at - 20°C until use. Antiphospholipase A s serum Antiphospholipase A~ serum was prepared by injecting rabbits with purified Crotalus phospholipase A, isolated from crotoxin, as dearibed previously (HANASHIRO et aL, 1978). The whole serum was lyophilized and dissolved in distilled water immediately before use. Anticrotalic t+erwm sera Six diüerent batches of anticrotalic serum, as prepared at Butantan Institute for serumtherapy, were used . These sera were prepared in horses according to a routine immunization procedure which involves s.c. injections of aotamine positive venom emulsified in Freund's complete or incomplete adjuvant on two consecutive weeks, followed by booster injections of antigen without adjuvant. Sera showed potencies corresponding to the neutralization of 2 .0-4 .5 mg of crude venom per ml of andvenom, when tested into pigeons . Sera were dialyzed against saline (0.15 M NaCI) to remove phenol and kept frozen at - 20°C. Corrections for dilution doting the process were made by precipitating proteins with ammonium sulfate at 50/ saturation, dissolving the precipitate in 0.25 M acetic acid and determining the absorbency of the solution at 280 nm. Phospholipase A Z assay Phaspholipase A 2 activity in venom solutions or mixtures of venom plus antivenom, was determined by the egg yolk-hemolysis test, as described by GRASSMAN and H~rnvtG (1954 with slight modifications : O.SOmI of a 6 .4% (w/w) egg yolk emulsion in saline containing 0.02 sodium suds, was added to SOOpI of the test solution and incubated at 37°C. After a defined hydrolysis tithe (30 thin-16 hr) 30 pl samples of the hydrolyaates were transferred to 0.30 ail of a standardized sheep red blood cell suspension (4.5-5.0 x l0s cells/ml) plus 26 tail of saline. After 1 hr at 37°C, suspensions were centrifuged for 15 mirr at 400 g and the absorbanciea of the supernatants read at 540 nm. Lysis (/) was estimated by reference to a completely hemolyzod mixture obtained by adding 0.30 m1 of the standardized cells to 26 ml of distilled water. The test solutions containing venom plus antivenom were prepared by mixing constant volumes of venom solution with variable vohtmes of antiaenrm and saline to the final volurrte of 500 W. In most experiments normal horse serum (30 pl) was added to the teat solutions. All test solutions (venom solutions or venom plus antivenom mixtures) were pre-incubated for 45 min at 37°C before addition of the egg yolk emulsion, in the same way as were the trrixturea prepared for the neutralization test in mice . Neutralization test in mice and pigeons .20 ml of venom solution or venom plus antivenom rrtixtures per Male Swiss mice of 20-25 g were injected with 0 10 g of body wei~t . The mixtures were pro-incubated for 45 thin at 37°C before being injected. Mortality was recorded up to 24 hr. r u se and its confidence limits (for 19/20 probability) were estimated by the method of Lrfct~p and wucoxoN (1949) on the basis of seven points on the regression line. Injections were given i.p. and six animals per done wero leafed. The neutralizing potency of horse antisera was determined by Vital Brazil's method by injecting pigeons of 250-350g with mixtures of 1 .Om1 of undiluted serum plus LOmI of variable dilutions of crude venom (MLD Cor pigeons is 0.015 mg). After 30 min of incubation at 37°C, mixtures were injected i.v . Deaths were recorded 24 hr aater .
RESULTS
Phospholipase AZ activity ojthe whole venom. Effect ojnormal serum on phospholipase activity Phospholipase activity could not be detected up to 120 min with 27 pg of crude venom in saline . Addition of 30Et1 of normal horse or rabbit serum to the test solution led to 100'/ hemolysis within 90 min. This activation effect was not observed if the serum was previously dialyzed (0% hemolysis after 120 min of hydrolysis). In either case, l6 hr hydrolysates caused 100'/ hemolysis.
56 5
Standardization of AnticrotaGc Venom Serum
Inhibition of phospholipase A Z activity by horse anticrotalic serum Horse hyperimmune serum raised against the whole venom inhibited its phospholipase activity. In a typical experiment, 27 ug of venom were mixed with variable volumes of antiserum diluted 1 :10. The phospholipase activity ofthe mixtures decreased as the volume of antiserum increased . With SOpI of serum "E" (batch C-23-77-118, neutralizing potency in pigeons 4.5 mg ofvenom per ml) only 10~ hymolysis was detected after 16 hr ofhydrolysis. At a higher level of antiserum (70p1) no lysis was obtained (0'~ hemolysis~ Residual phospholipase activity of partially inhibited mixtures was enhanced by addition of normal serum For instance, the mixture containing 50~1 of serum "E~ caused 100'/ hymolysis when 30p1 of normal serum were also added to the test solution. No enhancement effect was detected in mixtures containing 701 of antiserum "E" and 30p1 of normal serum (0'~ hemolysis). Similar results were obtained with five other batches of antiserum. The volumes of antiserum necessary to inhibit the phospholipase activity of an equal amount of venom varied from serum to serum depending on its neutralizing potency . In Fig . 1, the minimum volumes of antiserum necessary to inhibit the phospholipase activity of different amounts of venom (up to 16 hr of hydrolysis) were plotted against the amount of venom assayed . Straight lines were obtained whose slopes corresponded approximately to the neutralizing potencies of the sera, as determined by the pigeon test. Inhibition of phosphoüpase AZ activity 6y rabbit anti-Crotalus phospholipase serum Variable volumes of rabbit antiphospholipase serum were added to constant amounts of crude venom and the phospholipase activity of the mixture determined by the egg yolk hemolysis test, in 16 hr hydrolysates. The minimum volumes of antiserum necessary to inhibit
io
so
30
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so
VENOAA
(Ny)
FIG. I . INHIBr17ON ~ I~iOBPHOLIPASE A= ACI'IVrrY OF C. "WIJ3Y3 ClfrifICU.f VENOM BY HOR.4E ANrICROTALIC VENOM SERA WITH DffFFFFREENt' l'O'IENC[F3 ~ NEUTRALIZATION.
Titres given in mg of neutralized venom per ml of serum, tents carried out in pigeats : A(C-17-77-93) and C(C-28-76A1~ 2.Omg/ml ; D(G28-77-128) and F(G22-77-117 3.5 mg/ml ; E(C-23-77-118) and G(C-21-77-113 4 .5 mg/mL Points represent the minimum volume of serum (1 :10 dilution) which inhibits the enzyme activity of the ceding assayed venom sample. Hydrolysis time 16 hr . Data points represent average vahles of duplicate experiments except for sera A and E. The points related to these two sera represent the mean values of fî-10 experiments. Vertical bars indicate the standard deviations about the mesas.
566
MARIA HELENA DA SILVA and OTTO GUILHERME BIER
~o FiG.
so '
ao
so
ao
V E N OIN
2 INHIBITION OF PHOSPHOLIPASE A2 ACTIVITY OF C. dwissus ANTIPHOSPHOLIPASE SERUM
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VENOM HY RABBIT
Each point represents the minimum volume of antiserum which inhibits the enzyme activity of the assayed venom sample . Hydrolysis time 16hr. Average of two determinations .
TABLE 1 . NEUTRALIZATION OF CRUDE VENOM BY RABBIT ANTISERUM TO PURIFIED C. durissus terrificus PHOSPHOLIPASE A=
Antiserum plus venom mixtures per g mouse Antiserum" Venomt pl LDSO pg 0.475 0.475 0 .475 0 .950 0 .950 0 .950 1 .425 1 .425 1 .425 1 .900 1 .900 1 .900
(1 .0) (2 .0) (3 .0) (4 .0)
0 .078 0.160 0.241 0 .157 0 .241 0 .321 0 .257 0 .343 0 .401 0 .321 0 .401 0 .471
1 .0 2 .1 3.1 20
3.1 4 .2 3.3 4.5 5.2 4.2 5.2 6.2
Mortality (24 hr) Dead/Total 0/6 3/6 5/6 0/6 3/6 6/6 0/6 4/6 5/6 0/6 3/6 4/6
Figures in parentheses are numbers of tnso neutralized at that level of antiserum as estimated by inhibition of the phospholipase activity of corresponding amounts of venom 1The LDSO for this batch of C. dratiwus ttrrifrcus venom was 0.077 (0.048-0.123) mg,/kg. Teats carried out i .p . in mice.
Standardization of Anticrotalic Venom Serum
567
the phospholipase activity were plotted against the venom concentration (Fig. 2~ A straight line was obtained. The "antiphosphofipase potency" was determined by extrapolation to 1.0 ml of serum. Neutralization of venom lethality by rabbit anti-Crotalus phosphotipase A Z serum
Lethality of mixtures containing venom plus antiphospholipase serum was tested in mice. Mixtures were prepared taking into account the volume of serum necessary to inhibit the phospholipase activity ofacertain amount ofcrude venom as determined previously (Fig. 2} Tests were performed at several levels of antiserum and venom At a first level of antiserum, a volume of serum sufficient to inhibit the phospholipase activity of an amount of venom corresponding to 1 Las so was mixed with venom equivalent to 1, 2 and 3 Lasso . At a second level, a volume ofserum sufficient to inhibitthe phospholipase activity ofvenom equivalent to 2 ~sn was mixed with amounts of venom corresponding to 2, 3 and 4 Lasso . Similar procedure was adopted for twohigher levels ofantiserum. Results are summarized inTable 1 . At the first level of antiserum, the mixture containing 0.475 Etl of serum to 0.078 ~g ofvenom caused 0~ mortality indicating that 1 Lasso was neutralized. The mixture containing the same volume of serum to 0.160 hg of venom (about 2 tn so ) resulted in 50'/ mortality indicating, in agreement with the preceding, that l ~so was neutralized while 1 ip so remained free. With 3 i.D SO , venom equivalent to 2 Las so remained free and caused 100' mortality. Similar results were obtained at the three levels of antiserum. TABLE
2.
NEUTRALIZATION OF C. 4l1lß3W3 tQITiflgl8 VENOM BY HORSE AN'rICROTALIC VENOM SERUM A'
Antiserum plus venom mixtures P~ 8 mouse Antiserum (1 :10)t Venom ; ul Las so Wg 0 .242 0.242 0.242 0.484 0.484 0.484 0.727 0.727 0.727 0.969 0.969 0.969 2.358 4 .716 4 .804
(1 .0) (2.0) (3.1) (4.2) (10.1) (202) (20 .6)
0.078 0 .157 0.235 0.157 0 .235 0 .315 0 .230 0.307 0 .384 0 .307 0.384 0 .461 0 .772 1 .543 1 .543
1 .0 2.0 3 .0 2 .0 3.0 4.0 3 .0 4 .0 5 .0 4.0 5 .0 6.0 10 .0 20.0 20 .0
Mortality (24 hr) Dead/Total 0/6 3/6 6/6 0/6 3/6 6/6 0/6 0/6 5/6 0/6 1/6 6/6 0/6 2/6 0/6
'Batch C-17-77-93 with a neutralizing potency of 2.0 mg of venom per ml of serum Teats carried out i.v . in pigeon . tFigures in par~theais are numbers of Lasso neutralized at that level of antiserum as estimated by inhibition of phosphohpase activity of corresponding amounts of venom. $the Loso for this batch of crude venom was 0.077 (0.048-0 .123) mg/kg. Tests carried out i.p. in mix .
568
MARIA HELENA DA SILVA and OTTO GUILHERME BIER TAHLE
3.
NEUTRALIZATION OF C. d>Qissu.* ICIIifiCY3 VENOM BY HORSE ANrICROTALIC VENOM SERUM E "
Antiserum plus venom mixtures per g mouse Andserumt Venom$ (1 :10) ltl Rg ~so 0.133 0.133 0.133 0.266 0.266 0.266 0.398 0.398 0.398 0.512 0.512 0.512 0.683 0.683 0.683 1 .313 2.626 3.938
(1 .0) (2.0) (3.0) (3.9) (5.2) (10.0) (20.0) (30.0)
0 .080 0 .160 0.239 0 .160 0.239 0 .295 0 .221 0 .295 0.369 0.301 0.401 0 .502 0 .401 0.502 0.602 0 .772 1 .544 2 .316
1 .0 2.1 3.1 2.1 3.1 3.8 2.9 3.8 4.8 3.9 5.2 6.5 5.2 6.5 7.8 10.0 2R0 30.0
Mortality (24 hr) Deadfl'otal 0/6 5/6 6/6 0/6 2/6 4/6 0/6 3/6 5/6 0/6 3/6 4/6 0/6 4/6 4/6 0/6 0/6 2/6
" Batch C-23-77-118 with a neutralizing potency of 4S nlg of venom per ml of serum. Tests carried out i .v. in pigeon . tFigures in parantheses are numbers of I,o so neutralized at that level of antiserum as estimated by inhibition of phophoGpase activity of corresponding amounts of venom. $ The tnso for this batch of crude venom was 0.077 (0.048 - 0.123) mgng. Teats carried out i .p. is mice.
Neutralization of crude venom lethality by horse anticrotalic sera
Neutralization tests with anticrotalic sera were performed according to the same procedure adopted for antiphospholipase serum Results obtained with sera of low and high neutralization potencies are summarized in Tables 2 and 3, respectively. Antiphospholipase activity and neutralizing capacity of antisera are closely related. DISCUSSION
Both crotoxin and crude venom exhibit phospholipase AZ activity, indicating that some molecules of the enzyme are not inhibited by crotapotin. The phophofipase activity is enhanced by normal horse or rabbit serum and is inhibited by specific antiserum against purified Crotalus phospholipase A2 or against the whole venom The enhancement of the phospholipase activity by dialyzable constituents of normal serum is attributed to the activation ofphosphofipase molecules not inhibited by crotapotin. This could also be due to an increased number of free phospholipase molecules resulting from the dissociation of phospholipase~:rotapotin complexes in the presence of normal serum. Inhibition experiments with antisera did not confirm this hypothesis. The volumes of antiserum which completely inhibited the enzyme activity were the same, either in the presence or in the
Standardization of Antiaotalic Venom Serum
569
absence of normal serum The dialyzable activating factor may be related to divalent rations which are known to be required for the catalytic activity of the enzyme (BRErrHAVPT,1976) . Antiphospholipase serum also neutralizes the toxic activity ofcrotoxin (HANASHIRO et a!, 1978). Since crotoxin does not dissociate in serum, it is inferred thatantigenicdeterminants of the immunogen - the phospholipase molecule - are not significantly affected by its bonding with crotapotin. Phospholipase complexed with crotapotin can compete with unbound phospholipase for antibody molecules. The neutralization of crotoxin should parallel the inhibition of phospholipase. Antiphospholipase serum also neutralizes venom lethality. This was expected since crotoxin is qualitatively and quantitatively the main component of the venom. Experiments with anticrotalic sera have also demonstrated the close relationship betwcen antiphospholipase activity and neutralizing potency. These results raise the possibility of titratiog anticrotalic serum in vitro by measuring its capacity to inhibit the phospholipase activity of the venom Although the method estimates antibodies to only onevenom component, it is applicable to titration ofcommercial antisera since it evaluates the neutralization of crotoxin, the component responsible for C. durissus terrificus venom lethality. In addition, hyperimmune sera contain antibodies to all venom components which are neutralized concomitantly with crotoxin . This is inferred since, at least in the range of venom concentration tested, C. durissus terrificus venom interacts with anticrotalic serum as a system ofonly one component. Further work is required to verify the reliability of the method REFERENCES Breranr~urr, H. (1976) Enzymatic characteri~tics of Crotahia phospholipase Az and the crotoxin complex. 'Toxicon 14, 221. GoN~ni.ves,J. M. and Vmaw, L. G. (1950) Estudoa sobre venmos de serpentea brasileiraa I. Anélise electroforética. Anu Aced . fires. Cifnc. 22, 141. Ga~ssetnxN, W. and H~tvw~c, K. (1954) Elektrophoretische Unterwchungen an Schlangen- and Insektentoxinen . Hoppe-Stykr's Z. physiol. Chan. 296, 30. H~aeat~xr~, E. and Ctu:xGR~unt; D. (1975) Central neurotoxicity of apamin, aotamin, phoephoGpase A aad aamanitin. Toxicon 13, 465. H~eest~xN, E. and Rüasat~N, K. (1970) Biochemical and pharmacological analysis of the socalled crotoxin. In Toxins ojAnimaf andPlant Origin, Vol. l, p. 333. Pros of the 2nd International Symposium on Animal and Plant Toxins, Tel Aviv . London : Gordon and Breach. HANASHIRO~ M. A, DA Saves, M. H. and B~ex, O. G. (1978) Neutralization of crotoxin and crude venom by rabbit antiserum to Crotalus phospholipase A. Invnunochemistry 13, 745. HEnmora, R A. and Fw~Nt~et.-Corrnwr, H. (1971) Biological roles of the two oomponenta of ~TOtoxin. Proc. natn . Aced. Sci. U.S.A . 68, 1560. Lrrctn'~, J. T. and wu.coxoni, F. (1949) A simplified method of evaluating dose eBcet experiments. J. Pharmac. txp. Thtr. %, 99. Meal, D. (1970) A comparative study of enzyme activities in snake venoms . Int. J. Biochtm. 1, 335. Rües~s~t~, K., Hnerrtuurr, H. and H~seautaNx, E. (1971) Biochemistry and pharmacology of the crotoxin complex. I. Subfiactionation and recombination of the crotoxin complex. Naunyn-Sehmiede6ergs Arch. exp. Path. Pharmak. 270, 274. Scr~xaeaq S. (1959) Geographical pattern of crotamine distribution in the same rattlesnake subspecies . Science 129, 1361 . Sw~ru, K.H. andFx~tvxn.,-Coxs:~T, H. (1938) Schlangen-gifte. III. Mitteilung : Reinigung undKryatallisation des Klapperschlangen~rittes . Ba. dt. them. Ges. 71, 1076 .
coal-0lol~zo~ow3-m p 1982 Perpmm leer
Toslron, VoL 20, No. 3, pp. 563-569, 19ß2
Prmoed in Gmat Brihm.
Ltd.
TITRATION OF ANTISERUM TO SOUTH AMERICAN RATTLESNAKE (CROTALUS DURISSUS TERRIFICUS) VENOM BY MEASURING INHIBITION OF PHOSPHOLIPASE A2 ACTIVITY MARIA HELENA DA SILVA* and OTTO GUILHERME BIER WHO/PAHO Immunobgy Research and Training Center, Insdtuto Botenfan, Sâo Paulo, SP, Brazil
(Acceptedfor publication 22 October 1981) M. H . DA SavA and O. G. BmL Titration of antiaerum to South American rattle®ake (Crotalesdivisors terrificus) venom by inhibition of phospholipese A Z activity. Toxicon 20, 563-569, 1982.-Horse antiserum to the venom of Crotales derissus terrificus, a South American rattlesnake, inhibits the phosphofipase activity of the crude venom. There is a close relationship between this inhibitory property and the neutralizing potency of the antiserum in viva. This may provide the basis for a rigorous standardization of anticrotalic venom in vitro.
INTRODUCTION TI->E vENOM of Crotalus durissus temficus, the South American rattlesnake, like other snake venoms is a complex mixture of polypeptides and proteins with toxic and enzymatic
properties (MEBS,1970 ; ZLOTxIN,1973). Its main toxic component, crotoxin, was obtained in 1938 by $LOTTA and FRAENKEL-CONRAT, as a crystalline protein having phospholipase activity. In fact crotoxin is a complex between a highly basic phospholipase A Z (phosphatidate 2-acylhydrolase EC 3.1 .1 .4) and an acidic polypeptide, crotapotin, which potentiates the toxicity but inhibits the enzymatic activity of the phopholipase (HAHIItMANN and Rt)ssA1~N, 1970 ; RÜBSAMEN et al., 1971 ; HENDON and FRAENxEL-CONRAT, 1971).
Crotoxin represents 65-68% (w/w) of the crude venom and shows an LD SO of about 0.070mg/kg after i .p. inoculation in mice . It accounts for the high toxicity of the crude venom : i .p . LD SO, 0.090mg/kg, in mice (HANASHIROet al., 1978). In the same species the i .v. ~so of crotamine, another neurotoxic component present in venoms from specimens collected in Southern Brazil and Argentina (GoxçALVES and VIEIRA, 1950 ; SCHENBERG, 1959), is 4 mg/kg (HABERMANN and CIIElvc-RAUDE, 1975). It has been previously shown that rabbit anti-Crotalus phospholipase A z neutralizes the enzymatic and neurotoxic activities of the purified enzyme as well as of crotoxin and crude
venom (HANASHIRO et al., 1978). This led us to investigate whether the inhibition of phospholipase activity in vitro would parallel the neutralization of toxicity in viva .
+Fellow of CNPq, the Brazilian Council for Research Development and Technology. 563
564
MARIA HELENA DA SILVA and OTTO GUILHERME BIER MATERIALS AND METHODS
Venom One batch of crotatnine positive C. durissus terrificus venom was used throughout the experiments . It was prepared at Butantan Institute (Säo Paulo, Brazil) by pooling venom from animals captured in the Southern region of Brazil and drying it over CaCh under normal pressure, at 37°C. The dried venom was removed from the desiecator and kept in tightly closed vials of 4°C. Two samples of this batch (lO.Omg each), after exposure to the room atmosphere for 24 hr, were transferred to a second deaiocator and maintained over CaCh under normal pressure, at room temperature, until constant weight. The samples were dissohrod in 0.15 M NaCI and used to establish a standard curve by plotting absorbency at 280 nm vs venom ooneentration in mg per mL The concentration of venom solutions used throughout the experiments was obtained by reference to this curve. All venom solutions were kept at - 20°C until use. Antiphospholipase A s serum Antiphospholipase A~ serum was prepared by injecting rabbits with purified Crotalus phospholipase A, isolated from crotoxin, as dearibed previously (HANASHIRO et aL, 1978). The whole serum was lyophilized and dissolved in distilled water immediately before use. Anticrotalic t+erwm sera Six diüerent batches of anticrotalic serum, as prepared at Butantan Institute for serumtherapy, were used . These sera were prepared in horses according to a routine immunization procedure which involves s.c. injections of aotamine positive venom emulsified in Freund's complete or incomplete adjuvant on two consecutive weeks, followed by booster injections of antigen without adjuvant. Sera showed potencies corresponding to the neutralization of 2 .0-4 .5 mg of crude venom per ml of andvenom, when tested into pigeons . Sera were dialyzed against saline (0.15 M NaCI) to remove phenol and kept frozen at - 20°C. Corrections for dilution doting the process were made by precipitating proteins with ammonium sulfate at 50/ saturation, dissolving the precipitate in 0.25 M acetic acid and determining the absorbency of the solution at 280 nm. Phospholipase A Z assay Phaspholipase A 2 activity in venom solutions or mixtures of venom plus antivenom, was determined by the egg yolk-hemolysis test, as described by GRASSMAN and H~rnvtG (1954 with slight modifications : O.SOmI of a 6 .4% (w/w) egg yolk emulsion in saline containing 0.02 sodium suds, was added to SOOpI of the test solution and incubated at 37°C. After a defined hydrolysis tithe (30 thin-16 hr) 30 pl samples of the hydrolyaates were transferred to 0.30 ail of a standardized sheep red blood cell suspension (4.5-5.0 x l0s cells/ml) plus 26 tail of saline. After 1 hr at 37°C, suspensions were centrifuged for 15 mirr at 400 g and the absorbanciea of the supernatants read at 540 nm. Lysis (/) was estimated by reference to a completely hemolyzod mixture obtained by adding 0.30 m1 of the standardized cells to 26 ml of distilled water. The test solutions containing venom plus antivenom were prepared by mixing constant volumes of venom solution with variable vohtmes of antiaenrm and saline to the final volurrte of 500 W. In most experiments normal horse serum (30 pl) was added to the teat solutions. All test solutions (venom solutions or venom plus antivenom mixtures) were pre-incubated for 45 min at 37°C before addition of the egg yolk emulsion, in the same way as were the trrixturea prepared for the neutralization test in mice . Neutralization test in mice and pigeons .20 ml of venom solution or venom plus antivenom rrtixtures per Male Swiss mice of 20-25 g were injected with 0 10 g of body wei~t . The mixtures were pro-incubated for 45 thin at 37°C before being injected. Mortality was recorded up to 24 hr. r u se and its confidence limits (for 19/20 probability) were estimated by the method of Lrfct~p and wucoxoN (1949) on the basis of seven points on the regression line. Injections were given i.p. and six animals per done wero leafed. The neutralizing potency of horse antisera was determined by Vital Brazil's method by injecting pigeons of 250-350g with mixtures of 1 .Om1 of undiluted serum plus LOmI of variable dilutions of crude venom (MLD Cor pigeons is 0.015 mg). After 30 min of incubation at 37°C, mixtures were injected i.v . Deaths were recorded 24 hr aater .
RESULTS
Phospholipase AZ activity ojthe whole venom. Effect ojnormal serum on phospholipase activity Phospholipase activity could not be detected up to 120 min with 27 pg of crude venom in saline . Addition of 30Et1 of normal horse or rabbit serum to the test solution led to 100'/ hemolysis within 90 min. This activation effect was not observed if the serum was previously dialyzed (0% hemolysis after 120 min of hydrolysis). In either case, l6 hr hydrolysates caused 100'/ hemolysis.
56 5
Standardization of AnticrotaGc Venom Serum
Inhibition of phospholipase A Z activity by horse anticrotalic serum Horse hyperimmune serum raised against the whole venom inhibited its phospholipase activity. In a typical experiment, 27 ug of venom were mixed with variable volumes of antiserum diluted 1 :10. The phospholipase activity ofthe mixtures decreased as the volume of antiserum increased . With SOpI of serum "E" (batch C-23-77-118, neutralizing potency in pigeons 4.5 mg ofvenom per ml) only 10~ hymolysis was detected after 16 hr ofhydrolysis. At a higher level of antiserum (70p1) no lysis was obtained (0'~ hemolysis~ Residual phospholipase activity of partially inhibited mixtures was enhanced by addition of normal serum For instance, the mixture containing 50~1 of serum "E~ caused 100'/ hymolysis when 30p1 of normal serum were also added to the test solution. No enhancement effect was detected in mixtures containing 701 of antiserum "E" and 30p1 of normal serum (0'~ hemolysis). Similar results were obtained with five other batches of antiserum. The volumes of antiserum necessary to inhibit the phospholipase activity of an equal amount of venom varied from serum to serum depending on its neutralizing potency . In Fig . 1, the minimum volumes of antiserum necessary to inhibit the phospholipase activity of different amounts of venom (up to 16 hr of hydrolysis) were plotted against the amount of venom assayed . Straight lines were obtained whose slopes corresponded approximately to the neutralizing potencies of the sera, as determined by the pigeon test. Inhibition of phosphoüpase AZ activity 6y rabbit anti-Crotalus phospholipase serum Variable volumes of rabbit antiphospholipase serum were added to constant amounts of crude venom and the phospholipase activity of the mixture determined by the egg yolk hemolysis test, in 16 hr hydrolysates. The minimum volumes of antiserum necessary to inhibit
io
so
30
+o
so
VENOAA
(Ny)
FIG. I . INHIBr17ON ~ I~iOBPHOLIPASE A= ACI'IVrrY OF C. "WIJ3Y3 ClfrifICU.f VENOM BY HOR.4E ANrICROTALIC VENOM SERA WITH DffFFFFREENt' l'O'IENC[F3 ~ NEUTRALIZATION.
Titres given in mg of neutralized venom per ml of serum, tents carried out in pigeats : A(C-17-77-93) and C(C-28-76A1~ 2.Omg/ml ; D(G28-77-128) and F(G22-77-117 3.5 mg/ml ; E(C-23-77-118) and G(C-21-77-113 4 .5 mg/mL Points represent the minimum volume of serum (1 :10 dilution) which inhibits the enzyme activity of the ceding assayed venom sample. Hydrolysis time 16 hr . Data points represent average vahles of duplicate experiments except for sera A and E. The points related to these two sera represent the mean values of fî-10 experiments. Vertical bars indicate the standard deviations about the mesas.
566
MARIA HELENA DA SILVA and OTTO GUILHERME BIER
~o FiG.
so '
ao
so
ao
V E N OIN
2 INHIBITION OF PHOSPHOLIPASE A2 ACTIVITY OF C. dwissus ANTIPHOSPHOLIPASE SERUM
U~yi
rCffiflCYS
VENOM HY RABBIT
Each point represents the minimum volume of antiserum which inhibits the enzyme activity of the assayed venom sample . Hydrolysis time 16hr. Average of two determinations .
TABLE 1 . NEUTRALIZATION OF CRUDE VENOM BY RABBIT ANTISERUM TO PURIFIED C. durissus terrificus PHOSPHOLIPASE A=
Antiserum plus venom mixtures per g mouse Antiserum" Venomt pl LDSO pg 0.475 0.475 0 .475 0 .950 0 .950 0 .950 1 .425 1 .425 1 .425 1 .900 1 .900 1 .900
(1 .0) (2 .0) (3 .0) (4 .0)
0 .078 0.160 0.241 0 .157 0 .241 0 .321 0 .257 0 .343 0 .401 0 .321 0 .401 0 .471
1 .0 2 .1 3.1 20
3.1 4 .2 3.3 4.5 5.2 4.2 5.2 6.2
Mortality (24 hr) Dead/Total 0/6 3/6 5/6 0/6 3/6 6/6 0/6 4/6 5/6 0/6 3/6 4/6
Figures in parentheses are numbers of tnso neutralized at that level of antiserum as estimated by inhibition of the phospholipase activity of corresponding amounts of venom 1The LDSO for this batch of C. dratiwus ttrrifrcus venom was 0.077 (0.048-0.123) mg,/kg. Teats carried out i .p . in mice.
Standardization of Anticrotalic Venom Serum
567
the phospholipase activity were plotted against the venom concentration (Fig. 2~ A straight line was obtained. The "antiphosphofipase potency" was determined by extrapolation to 1.0 ml of serum. Neutralization of venom lethality by rabbit anti-Crotalus phosphotipase A Z serum
Lethality of mixtures containing venom plus antiphospholipase serum was tested in mice. Mixtures were prepared taking into account the volume of serum necessary to inhibit the phospholipase activity ofacertain amount ofcrude venom as determined previously (Fig. 2} Tests were performed at several levels of antiserum and venom At a first level of antiserum, a volume of serum sufficient to inhibit the phospholipase activity of an amount of venom corresponding to 1 Las so was mixed with venom equivalent to 1, 2 and 3 Lasso . At a second level, a volume ofserum sufficient to inhibitthe phospholipase activity ofvenom equivalent to 2 ~sn was mixed with amounts of venom corresponding to 2, 3 and 4 Lasso . Similar procedure was adopted for twohigher levels ofantiserum. Results are summarized inTable 1 . At the first level of antiserum, the mixture containing 0.475 Etl of serum to 0.078 ~g ofvenom caused 0~ mortality indicating that 1 Lasso was neutralized. The mixture containing the same volume of serum to 0.160 hg of venom (about 2 tn so ) resulted in 50'/ mortality indicating, in agreement with the preceding, that l ~so was neutralized while 1 ip so remained free. With 3 i.D SO , venom equivalent to 2 Las so remained free and caused 100' mortality. Similar results were obtained at the three levels of antiserum. TABLE
2.
NEUTRALIZATION OF C. 4l1lß3W3 tQITiflgl8 VENOM BY HORSE AN'rICROTALIC VENOM SERUM A'
Antiserum plus venom mixtures P~ 8 mouse Antiserum (1 :10)t Venom ; ul Las so Wg 0 .242 0.242 0.242 0.484 0.484 0.484 0.727 0.727 0.727 0.969 0.969 0.969 2.358 4 .716 4 .804
(1 .0) (2.0) (3.1) (4.2) (10.1) (202) (20 .6)
0.078 0 .157 0.235 0.157 0 .235 0 .315 0 .230 0.307 0 .384 0 .307 0.384 0 .461 0 .772 1 .543 1 .543
1 .0 2.0 3 .0 2 .0 3.0 4.0 3 .0 4 .0 5 .0 4.0 5 .0 6.0 10 .0 20.0 20 .0
Mortality (24 hr) Dead/Total 0/6 3/6 6/6 0/6 3/6 6/6 0/6 0/6 5/6 0/6 1/6 6/6 0/6 2/6 0/6
'Batch C-17-77-93 with a neutralizing potency of 2.0 mg of venom per ml of serum Teats carried out i.v . in pigeon . tFigures in par~theais are numbers of Lasso neutralized at that level of antiserum as estimated by inhibition of phosphohpase activity of corresponding amounts of venom. $the Loso for this batch of crude venom was 0.077 (0.048-0 .123) mg/kg. Tests carried out i.p. in mix .
568
MARIA HELENA DA SILVA and OTTO GUILHERME BIER TAHLE
3.
NEUTRALIZATION OF C. d>Qissu.* ICIIifiCY3 VENOM BY HORSE ANrICROTALIC VENOM SERUM E "
Antiserum plus venom mixtures per g mouse Andserumt Venom$ (1 :10) ltl Rg ~so 0.133 0.133 0.133 0.266 0.266 0.266 0.398 0.398 0.398 0.512 0.512 0.512 0.683 0.683 0.683 1 .313 2.626 3.938
(1 .0) (2.0) (3.0) (3.9) (5.2) (10.0) (20.0) (30.0)
0 .080 0 .160 0.239 0 .160 0.239 0 .295 0 .221 0 .295 0.369 0.301 0.401 0 .502 0 .401 0.502 0.602 0 .772 1 .544 2 .316
1 .0 2.1 3.1 2.1 3.1 3.8 2.9 3.8 4.8 3.9 5.2 6.5 5.2 6.5 7.8 10.0 2R0 30.0
Mortality (24 hr) Deadfl'otal 0/6 5/6 6/6 0/6 2/6 4/6 0/6 3/6 5/6 0/6 3/6 4/6 0/6 4/6 4/6 0/6 0/6 2/6
" Batch C-23-77-118 with a neutralizing potency of 4S nlg of venom per ml of serum. Tests carried out i .v. in pigeon . tFigures in parantheses are numbers of I,o so neutralized at that level of antiserum as estimated by inhibition of phophoGpase activity of corresponding amounts of venom. $ The tnso for this batch of crude venom was 0.077 (0.048 - 0.123) mgng. Teats carried out i .p. is mice.
Neutralization of crude venom lethality by horse anticrotalic sera
Neutralization tests with anticrotalic sera were performed according to the same procedure adopted for antiphospholipase serum Results obtained with sera of low and high neutralization potencies are summarized in Tables 2 and 3, respectively. Antiphospholipase activity and neutralizing capacity of antisera are closely related. DISCUSSION
Both crotoxin and crude venom exhibit phospholipase AZ activity, indicating that some molecules of the enzyme are not inhibited by crotapotin. The phophofipase activity is enhanced by normal horse or rabbit serum and is inhibited by specific antiserum against purified Crotalus phospholipase A2 or against the whole venom The enhancement of the phospholipase activity by dialyzable constituents of normal serum is attributed to the activation ofphosphofipase molecules not inhibited by crotapotin. This could also be due to an increased number of free phospholipase molecules resulting from the dissociation of phospholipase~:rotapotin complexes in the presence of normal serum. Inhibition experiments with antisera did not confirm this hypothesis. The volumes of antiserum which completely inhibited the enzyme activity were the same, either in the presence or in the
Standardization of Antiaotalic Venom Serum
569
absence of normal serum The dialyzable activating factor may be related to divalent rations which are known to be required for the catalytic activity of the enzyme (BRErrHAVPT,1976) . Antiphospholipase serum also neutralizes the toxic activity ofcrotoxin (HANASHIRO et a!, 1978). Since crotoxin does not dissociate in serum, it is inferred thatantigenicdeterminants of the immunogen - the phospholipase molecule - are not significantly affected by its bonding with crotapotin. Phospholipase complexed with crotapotin can compete with unbound phospholipase for antibody molecules. The neutralization of crotoxin should parallel the inhibition of phospholipase. Antiphospholipase serum also neutralizes venom lethality. This was expected since crotoxin is qualitatively and quantitatively the main component of the venom. Experiments with anticrotalic sera have also demonstrated the close relationship betwcen antiphospholipase activity and neutralizing potency. These results raise the possibility of titratiog anticrotalic serum in vitro by measuring its capacity to inhibit the phospholipase activity of the venom Although the method estimates antibodies to only onevenom component, it is applicable to titration ofcommercial antisera since it evaluates the neutralization of crotoxin, the component responsible for C. durissus terrificus venom lethality. In addition, hyperimmune sera contain antibodies to all venom components which are neutralized concomitantly with crotoxin . This is inferred since, at least in the range of venom concentration tested, C. durissus terrificus venom interacts with anticrotalic serum as a system ofonly one component. Further work is required to verify the reliability of the method REFERENCES Breranr~urr, H. (1976) Enzymatic characteri~tics of Crotahia phospholipase Az and the crotoxin complex. 'Toxicon 14, 221. GoN~ni.ves,J. M. and Vmaw, L. G. (1950) Estudoa sobre venmos de serpentea brasileiraa I. Anélise electroforética. Anu Aced . fires. Cifnc. 22, 141. Ga~ssetnxN, W. and H~tvw~c, K. (1954) Elektrophoretische Unterwchungen an Schlangen- and Insektentoxinen . Hoppe-Stykr's Z. physiol. Chan. 296, 30. H~aeat~xr~, E. and Ctu:xGR~unt; D. (1975) Central neurotoxicity of apamin, aotamin, phoephoGpase A aad aamanitin. Toxicon 13, 465. H~eest~xN, E. and Rüasat~N, K. (1970) Biochemical and pharmacological analysis of the socalled crotoxin. In Toxins ojAnimaf andPlant Origin, Vol. l, p. 333. Pros of the 2nd International Symposium on Animal and Plant Toxins, Tel Aviv . London : Gordon and Breach. HANASHIRO~ M. A, DA Saves, M. H. and B~ex, O. G. (1978) Neutralization of crotoxin and crude venom by rabbit antiserum to Crotalus phospholipase A. Invnunochemistry 13, 745. HEnmora, R A. and Fw~Nt~et.-Corrnwr, H. (1971) Biological roles of the two oomponenta of ~TOtoxin. Proc. natn . Aced. Sci. U.S.A . 68, 1560. Lrrctn'~, J. T. and wu.coxoni, F. (1949) A simplified method of evaluating dose eBcet experiments. J. Pharmac. txp. Thtr. %, 99. Meal, D. (1970) A comparative study of enzyme activities in snake venoms . Int. J. Biochtm. 1, 335. Rües~s~t~, K., Hnerrtuurr, H. and H~seautaNx, E. (1971) Biochemistry and pharmacology of the crotoxin complex. I. Subfiactionation and recombination of the crotoxin complex. Naunyn-Sehmiede6ergs Arch. exp. Path. Pharmak. 270, 274. Scr~xaeaq S. (1959) Geographical pattern of crotamine distribution in the same rattlesnake subspecies . Science 129, 1361 . Sw~ru, K.H. andFx~tvxn.,-Coxs:~T, H. (1938) Schlangen-gifte. III. Mitteilung : Reinigung undKryatallisation des Klapperschlangen~rittes . Ba. dt. them. Ges. 71, 1076 .