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Detoxification of Russell's viper (Vipera russellii) and water moccasin (Agkistrodon piscivorus) venoms by photooxidation
Toxfcon, 1966, Vol . 3, pp. 197-194.
Perpamon Press Ltd., Printed in Groat Britain
DETOXIFICATION OF RUSSELL'S VIPER (VIPERA RUSSELLII) AND WATER MOCCASIN (AGKISTRODON PISCIVORUS) VENOMS BY PHOTOOXIDATION WALTER KOCHOLATY 8IId BILLY D. ASHLEY Biochemistry Division, U.S . Army Medical Research Laboratory, Fort Knox, Kentucky, U.S.A . (Acceptedjor publication 16 September 1965) Abstract-Photooxidation of six venoms of domestic and foreign snakes resulted in the detoxification of all venoms . Immunization of rabbits with two of the detoxified venoms, Agkistrodon piscivorus and Vlpera russellü, resulted in the production of antibodies which protected mice against the action of unaltered venom. INTRODUCTION
it was reported that Crotalus atrox venom ca.n be detoxified by photooxidation with visible light in the presence of methylene blue, and that the detoxified material will give rise to antibodies in rabbits which in turn will protect mice from the toxic effects of this venom. Extending these observations to the venoms of other snakes selected for their diverse manifestations of toxic effects has shown that photooxidation will also detoxify these venoms in the crude stage. The present investigation is concerned with these observations and with the immunogenic properties of two of the detoxified venoms. PREVIOUSLY [1]
MATERIALS AND METHODS
Starting material The bulk of Russell's viper (Vipers russellü) venom was purchased from the Miami Serpentarium Laboratories, Miami, Florida. One fourth of the venom was collected at the U.S . Army Medical Research Laboratory snake colony. The dried venoms were combined and sieved similarly as described in the above report . The dried venom did not dissolve completely in distilled water or in 0 ~85 per cent NaC 1 on prolonged stimng . The undissolved material, which was removed by centrifugation, consisted of mucid material and cell debris . The protein content of the centrifuged and filtered solution (Millipore filter of 0 ~45 ~m pore size) determined by biuret assay as in the previous paper was found to be 57 per cent of the dry weight of the venom. The venom of the cottonmouth water moccasin (Agkistrodon piscivorus) which was also collected locally was dissolved in distilled water with the formation of a small precipitate. The protein content of the filtrate was 74 per cent. Apparatus and generalprocedure The venoms were detoxified using WEII,'S [2, 3] photooxidation procedure catalyzed by methylene blue . The procedure was carried out in a Gilson differential respirometer Model GRP 20, the light source of which provided 1,000-1,400 foot candles according to the manufacturer. 187
l 88
WALTER KOCHOLATY and BILLY D. ASHLEY
Photooxidation Photooxidation was carried out on the centrifuged and filtered venoms in conical Warburg flasks at 37° . The final reaction mixture contained 5 to 10 mg of venom protein, 0 ~ 1 mg methylene blue, in a total volume of 2 ml in 0 ~ 1 M TRIS (Tris-(Hydroxymethyl) aminomethane) buffer ofpH 8 ~5 for the A. piscivorus and pH 7 ~4 for the V. russellü venom. Assays Proteolytic, esterase, and phospholipase A activities were determined with casein, TAME (p-toluene sulfonyl-L-arginine methyl ester) and egg yolk as substrate as previously described . Toxicity tests White mice of about 20 g weight were used in the assays for the unaltered and photooxidized venom using 0 ~2-0~25 ml for the intravenous (LV .) and 0~5 ml for the intraperitoneal (LP.) route. The calculations for the LDbo dose were done according to RED and MuExcx [4] . RESULTS
Effect ojphotooxidation on the proteolytic, esterase, and phospholipase A activity of A . piscivorus venom The venom ofthe water moccasin had a strong proteolytic activity withcasein as substrate and hydrolyzed TAME with great rapidity . The phospholipase A activity was of about the same magnitude as that of C. atrox venom. During photooxidation of A. piscivorus a rapid loss of these enzyme activities occurred. After only 15 min of photooxidation during which Std OQ were taken up per mg venom protein (Table 1), the proteolytic and TABLE 1 .
LOSS OF PROTEOLYTIC, ESTERASE AND PHOSPHOLIPASE A Agkistrodon p%SClYOrUS VENOM DURING PHOTOOXIDATION
ACrMT'Y
OF
P.O . pl O, takenup Per cent loss of activity LD time permg venom tolerated (min) protein proteolytic esterase phospholipase A 20 g mouse 15 30
8 13
73 99
88 99
99 -
6 22
esterase activities were reduced to low levels and the phospholipase A activity was abolished . Concomitant with the Os uptake, a reduction in toxicity took place and after an uptake of 13 .1 O$ per mg venom protein a 22-fold LD6o was tolerated by a 20 g mouse . Crude A. piscivorus venom did take up a small amount of 08 in the absence of methylene blue and light . This amounted to only 141 O$ per 10 mg venom protein in 90 min and was not considered when using shorter photooxidation times, which were found to be adequate to detoxify the venom . Effect of photooxidation on the phospholipase A and esterase activity of V. russellü venom No proteolytic activity could be demonstrated in this venom when casein was used as a substrate although a weak esterase activity was shown . The photooxidation of this venom was carried out at pH 7 ~4 (see next page) . As seen in Table 2, no esterase activity remained
Venoms DetoxiSed by Photooxidation TABr.E
2.
189
PHOTOOXIDATION OF Vipera rassellti vexoM. LO33 of ENZYM ACIIYITIS4 AND TOXIQTY AS RELATED TO OXYGEN UPTAKE
P.O . time (min)
~.1 O, taken up per S mg venom protein pH T4
esterase
15 30 45 60 90
31 44 47 48 56
99 -
Per cent loss of activity
LD, o
tolerated phospholipase A 20 g mouse 88 96 96 98 99
8 25 -
after 15 min of photooxidation and the phospholipase A activity was almost abolished within 30 min . With increasing O a uptake a detoxification of the venom occurred, and when 9~1 O $ were taken up per mg venom protein mice survived a 25-fold LD bo. When the photooxidation of this venom was carried out at pH 8 ~4, all other conditions being equal, about double the amount of O $ was taken up at identical time intervals as those shown in Table 2. A precipitate formed in the early stages ofphotooxidation which increased with additional O s uptake. Immunization of mice and rabbits with photooxidized venom of A . piscivores and V. russellü The venom of V. russellü was photooxidized to an uptake of 9 ~1 O, per mg protein (20 mg venom in 0 ~ 1 M TRIS buffer, pH 7 ~4, at 37°) and the venom of A. piscivores to an O, uptake of 13 ~l O $ per mg protein (30 mg in 0 ~ 1 M TRIS buffer, pH 8 ~5, at 37°). The final volume of the reaction mixtures was 2~0 ml and included 0~ 1 mg methylene blue. The photooxidized venoms, diluted in physiological saline to twice the desired concentrations, were mixed before the injection with an equal amount ofAlgivant (Colab Laboratories, Inc.), an adjuvant consisting of 4 per cent Na-alginate, 067 per cent Ca-gluconate. The methylene blue of these photooxidized venoms was not removed . All photooxidized venoms were stored in ice and in the dark prior to the injections. The weekly immunization schedule for mice and rabbits was identical. Each animal received 3 injections (Mon., Wed ., Fri.) Mice received this schedule for 3 consecutive weeks and after a rest period of one week the mice were challenged with unaltered venom (see below) . Rabbits received this schedule for 4 consecutive weeks and after a rest of one week were bled to death by heart puncture. Prior to immunization, all rabbits were bled by heart puncture and the normal serum examined by qualitative precipitin test for antibody to A. piscivores and V. russellü venom. These tests were negative. Mice. Fifteen mature Swiss mice of 30 to 40 g were immunized with either venom. All injections were made subcutaneously. For the mice receiving the photooxidized venom of A. piscivores the dose was 0~5 mg protein for each of the first 2 injections and 1 ~0 mg for the following 7 administrations . To the mice receiving the photooxidized venom of V. russellü 62 ~5 y ofthis protein was given at each ofthe 9 injections. The volume ofthe injection was 0 ~ 1 ml in all cases . Thus the mice received a total of either 0-56 mg of photooxidized V. russellü or 8 mg ofphotooxidized A. piscivores venom in 3 weeks. Five ofthe mice injected with photooxidized V. russellü venom died during the injection schedule, and of these, 4 mice died within 16 hr after the first injection . The same number ofdeaths occurred among the mice receiving photooxidized A. piscivores venom ; one at the third injection (higher
l90
WALTER KOCHOLATY and BILLY D. ASHLEY
dose), the others during the course of the injection period. All mice receiving the photooxidized venom developed lesions, ranging from chafing of the skin (V. russellli) to open sores (A.piscivorus) . Seven days after the last injection the mice were challenged with 2~5 to ]0 t.nbo's of A . piscivorus and V. russellli venom, respectively, given intravenously with appropriate controls of unaltered venom . Survivors were determined after 24 hr (A . piscivorus) and 48 hr ( V. russellli). While protection is evident in the A . piscivorus experiments, the results with the V. russellli are ambiguous (Table 3). TABLE
3.
NEUTRALIZATION OF Agkis7rodon piscivorus AND HY MICE IMMUNIZED WITH PHOTOOXIDIZED VENOMS
Ylpera russellil VENOM LD 6o 1 2 2'S 5 T5 0
Venom controls A. piscivorus V. russellli A. piscivorus V. russellli 0/2 2/0 1/2 2/4
2/4 1/4 2/4
0/2 2/2
1/2 1/2
Numbers are deaths over the total number of mice used . Readings were done 24 hr (A . piscivorus) and 48 hr (V. russellli) after injection.
Rabbits . New Zealand whites weighing 2 to 3 kg were injected intramuscularly with photooxidized venom . The same weekly schedule used on mice was employed and the site of injections were the inner thighs ofthe hind legs. Ten animals were used with each venom. The injection volume was 1 ~0 ml for the photooxidized V. russellil venom and 1 ~ 1 to 2 ~2 ml for the photooxidized A. piscivorus venom . The amounts of V. russellil venom administered were 2 mg for the first 3 injections. In the fourth injection halfofthe animals received 2 mg, the other half 4 mg. The rest of the injections were 4 mg for the whole group for a total of 40 and 42 mg venom protein, respectively. With the photooxidized A . piscivorus venom the first 2 doses were 16 ~5 mg and the next, l0, 33 mg eachfor a total of 363 mgvenom protein in 4 weeks . During the injection schedule light to pronounced swelling and edema was noticed in some animals, particularly with A. piscivorus venom. The swelling subsided after 24 to48 hr. One rabbit died within 5 min after the fourth injection with2 mg ofphotooxidized V. russellil venom, another within 2 min after the tenth injection with A. piscivorus venom, and another rabbit had to be destroyed. Qualitative precipitin tests with the rabbit sera were carried out starting with a 1 :250 dilution of venom in the first tube and continuing with 5-fold dilutions. With A. piscivorus venom, precipitates were obtained in the fifth (2 sera) and in the sixth (6 sera) dilution. With V, russellil venom, precipitates were obtained. i n the fifth (6 sera) and sixth (3 sera) dilution For both groups of rabbits immunized with the photooxidized venoms y-globulin fractions were prepared from the pooled sera by repeated salting out with ammonium sulfate according to CAMPBELL et al. [5] and the dialyzed material concentrated with cellugel* in the cold. The protection studies with the y-globulins were carried out by mining 4-16 " Cellugel Super (3000), Svenska Cellulose. Aktiebolaget, Kemiska Produkter, Eesoik, Sweden.
191
Venoms Detoxified by Photooxidation
intravenous doses* with 100 mg of each of the globulins in a total volume of 1 ml. Controls were carried out in a similar manner with normal rabbit y-globulint . After incubation at 37° for 60 min 0 ~25 ml of each mixture equivalent to 2S mg y-globulin was injected intravenously into 3 mice. Physiological saline was the solvent throughout including the controls of unaltered venom. The results of this experiment are shown in Table 4. The capacity of the immune globulins was also investigated for its ability to passively protect against a fatal dose of venom. In this experiment 3-6 mice were injected intravenously with 25 mg of the appropriate y-globulin and challenged 30 min later with 1~ Ln su of unaltered A. piscivorus or V. russellü venom and compared with corresponding controls . Table 5 shows the results of these experiments which indicate that both immune globulins impart definite protection against these venoms . In order to explore the potentialities of photooxidation in the detoxification of crude snake venom 7 venoms showing a variety of toxic manifestations have been selected in this study. A comparison of their proteolytic (casein), esterase (TAME), and phospholipase Ln ao
TAHLE 4 .
NEUTRALIZATION OF Agkistrodon piscivores exn Vipera ressellü v>~"toM nv MtCE Bv IAQKIINa OLOBULIN FROM RAHHITS D~AlIJNIZED WITfi PHOTbOXIDAZ® VENOMS
A piscivores LD
Immune* globulin
1 2 3 4
0/3 0/3 0/3 1/3
V. russellü
Normal globulin control
Venom control
Immune* globulin
3/3 4/4
5/7 7/7
0/3 0/3 1/3 1/3
Normal globulin control
Venom control
4/4 4/4
4/7 6/6
*From rabbits immunized with A, piscivores and V. russellti venoms respectively. Numbers are deaths over tho total number of mice used . Readings were done 24 hr (A . piscivores) and 48 hr (V. ressellü) after injection . Throe or more mice received either 25 mg immune rabbit globulin or normal rabbit globulin and tho Lv,o as stated above.
TABLe S . PROT6CTiON oF Mice eaeu~rsr AgkLstrodon piscivores AND Vipera russellü vFavoMS wITII RABBrr IMAIVNB aLOSVLU"t Venom controls
Lo bo
A . piscivores
Y. ressellü
A . piscivores
V. ressellü
1 2 3 4 5 6
0/3 0/3 1/3 3/3 -
0/6 0/6 0/6 0/6 0/3 2/3
3/3 3/3
4/6 6/6
Numbers are deaths over the total number of mico used . Readings wore done 24 hr (A . piscivores) and 48 hr (Y. russellü) after injection. 25 mg ofrabbit y-globulin was injected(LV.) in a volume of 0 ~25 ml, followed in 30 min by(LP.) injection of 1-6 Ln 6o of the venoms . Controls received venom only (LP.) .
" 1 Lnbo A . piscivores I . V .=45 y, I. P.=90 y . I LDbo V. russellü I . V.=S y, I. P.=9 y . t Fraction II from the Pentex Laboratories, Inc., I{ankakee, Illinois .
192
WALTER KOCHOLATY and BILLY D . ASHLEY
A activity is shown in Table 6. C. atrox venom appeared the most proteolytic o.these venoms followed by A . piscivorus and Bothrops atrox. These 3 venoms were also the only ones of this group displaying activity for all the 3 enzymes tested. All 3 venoms also displayed considerable esterase (TAME) activity . Phospholipase A activity, an enzyme common to all 7 venoms in varying degree, was most pronounced with Micrurus fulvius which lacked proteolytic and esterase activity . Naja raja somewhat resembled this venom showing a similar phospholipase activity ; V. russellü did not have proteolytic activity but displayed a weak esterase activity . Crotalus terrificus venom was unique in having the lowest phospholipase A activity of all venoms tested but possessed a very high esterase and no proteolytic activity. The Os uptake ofthese crude venoms during photooxidation was recorded against time using identical conditions of protein concentration, pH, and light intensity (Table 7). In general the rapid initial rate of the 0E consumption is followed by a gradual decrease . An exception formed the rate of OE uptake of C. terrifrcus venom which was practically linear for the first 60 min . Several of the venoms formed precipitates at pH 8 ~5 as indicated in the table. The nature of the precipitates was not investigated . Photooxidation with subsequent detoxification may be carried out at other pH values, however, as shown with TABLE
6.
PROTEOLYTIC (CASEIN, ESTERASE (TAME), AND PHOSPHOLIPASE A ACTIVITY OF SOME SNAKE VENOMS
Venom Agkistrodonpiscirorrrr Bothrops atrox Crotalus atrox Crotalus terrificus Micrurus frrlvirrs Najaxaja Vipera russellü
Casein
Phospholipase A' m Eq acid
I " 17 0 " 87 2 " 00 0 "00 0 " 00 0 " 00 0 "00
0 " 32 0 " 33 0 " 30 0 "25 3 " 36 2 "51 2 " 51
TCA-OD~ o"
Units'
TAME 650 518 433 637 0 0 86
" Per 1 mg venom protein . B. atrox and M. fulvius venoms were of commercial origin . The venom of C . terrifrcus was a gift from Dr . Karl H . Slotta, University of Miami, School of Medicine . The rest of the venoms were collected at the USAMRL snake colony . TABLE
7.
RATE OF OXYGEN UPTAKE OF VARIOUS SNAKE VENOMS DURING PHOTOOXIDATION IN THE PRESENCE OF METHYLENE BLUE
Venom Agkistrodon piscivores Bothrops atrox Crotalus adarnanteus' Crotalus atrox Crotalus terrifrcus" Micrurus fulvius' Naja ngja Yipera russellü "
Photooxidation time (min) 30 45 60 90 ~1 O, uptake/10 mg venom protein
IS
80 71 92 74 26 80 59 61
129 126 130 I 15 58 130 87 84
163 161 155 142 77 147 108 99
190 188 177 165 100 173 123 113
245 243 211 200 135 213 150 136
Reaction mixture : 10 mg venom protein, 0 " l mg methylene blue in 2 ml 0'1 M TRIS buffer, pH 8 " 5 . Numbers are ~1 O, taken up by 10 mg venom protein . 'These venoms formed precipitates during photooxidation at pH 8 " 5.
Venoms Detoxified by Photooxidation
19 3
the venom of V. russellü. While the O~ uptakes varied by not mush more than double when calculated in a mg protein basis, vast differences in the detoxification were obtained at identical 0 9 consumption . Table 8 shows that all 7 venoms could be detoxified by photooxidation . While the venoms of C. terr~cus and M. julvius were detoxified to an extent that 150 Lo bo were tolerated at an OE uptake of only 8 and 13 ~l per mg venom protein, the detoxification of N. raja venom proceeded at a considerably slower and almost linear rate with comparatively large O Q consumption. TABLE S.
EFFECT OF OXYGEN UPTAKE DURING PHOTOOXIDATION ON LOSS OF TOXICITY iN SEVERAL VENOMS
Agkistrodon Bothrops Crotales Crolalus piscivores afrox afrox terrlfrcus F
LU,o
20 g mouse, LV . (y)
8 13 -
6 22 45
3~5 7~0 29
1 35 -
10~0 I1 ~5 12~5 14~0 - - -
2~5 2~5 6~0 20~0 8~0150~0 - -
70
2
Micrurrrs fulvius Fal O, LD,o 8 13 -
40 I50 8
Fal
Naja naja Oa LDso 2 4 8 16
5 l0 20 27 8
Vlpera rrrssellil F<1 O, LD,o 6 9 -
8 25 5
0,=taken up by 1 mg of crude venom protein during photooxidation . Number of LD,o tolerated ; 3 mice out of 3 surviving 24 hr (A . piscivores, C. atrox)~8 hr (the other five). Wl
DISCUSSION
The purpose of these investigations was to establish whether or not crude venoms could be detoxified by photooxidation and whether the detoxified venoms were capable of giving rise to immune bodies. Although the results were disappointing in the mice experiments, the protection experiments with the isolated y-globulins from rabbits immunized with photooxidized venoms of A . piscivorus and V. russellü produced results similar to those reported for C. afrox venom [I] . Again, as with C. afrox, optimal conditions may not have been employed. A. piscivorus venom at a lower pH and temperature of photooxidation and somewhat higher O$ uptake might yield a product producing less of the deleterious effects in mice. The pH of 7 ~4 used for the photooxidation of V, russellü venom was selected arbitrarily because of precipitate formation at pH 8 ~5 . The immunization schedule of 3 injections a week for 3~ weeks may well be a "heavy" one and unnecessary and the particular adjuvant used may not be the most desirable . The measure of detoxification used in these studies was the amount of O$ uptake as related to toxicity. The abolishment of the proteolytic and phospholipase A activity during photooxidation together with a considerable reduction of toxicity may be coincidental. Also, the number of LD6p tolerated in mice following photooxidation (Table 8) does not represent the absolute minimum but is rather on the conservative side, since the limited amounts of some venoms prevented the establishment of the uppermost levels oftolerance . The detoxification obtained in all these venoms using such a gentle method as photooxidation gives hope of similar responses in the production of immune bodies . SU MMARY
1 . Photooxidation of A. piscivorus and V. russellü venom by visible light in the presence of methylene blue resulted in the detoxification ofthese venoms .
194
WALTER KOCHOLATY and BILLY D. ASHLEY
2. Immunization of rabbits with these detoxified venoms resulted in the production of antibodies which protected mice against the action of the unaltered venoms . 3. Four additional snake venoms were shown to be detoxified by photooxidation and a comparison of al] seven venoms was made with respect to their proteolytic, esterase, and phospholipase A activities and behavior during photooxidation . Pathologic findings
ADDENDUM
Grossly, the effects of repeated intramuscular injections of the photooxidized venoms of V. russellü and A . piscivorus was similar in that there was inflammation of the musculature varying from small focal lesions to abscesses 3 cm in dia. Microscopically, the findings for the Y. russellü, and A. piscivorus venoms were also similar. The reaction of the musculature surrounding the areas of inflammation or abscessation were of particular interest. Surrounding the areas of complete necrosis of muscle there were zones of inflammatory cell infiltrations consisting mostly of plasma cells, eosinophiles and small round cells. Many multinucleate giant cells were also present . The giant cells appeared to be formed by the syncytium of the nuclei of degenerate perimysial tissue as well as nuclei of muscle cells. Finely granular mineralization was seen throughout the necrotic areas. In the lungs there was a medial hyperplasia of the muscular arteries and ballooning of endothelium was noted. A few sections of myocardium showed areas of degeneration and focal collections of inflammatory cells that may have been associated with intracardiac punctures. No other significant lesions were noted in the duodenum, pancreas, adrenals, liver, or kidney . This information was furnished by Col. M. A. Ross, Head, Department of Pathology. Acknowledgements-The authors acknowledge the technical assistance of C. R. Sxn:r us and T. A. Bn t naas. REFERENCES [1 ] Kocxoc.~rv, W., Detoxification of Crotalus atrox venomby photooxidation in thepresence of methylene blue, Toxicon, 3, 175,1966 . [2] Wsa,, L. and Mate, J., Photodynamic action of methylene blue on nicotine and its derivatives. Archs Biochem. Biophys., 29, 241, 1950. [3] WEn., L., Goxnox, W. G. and Bucter, A. R., Photooxidation of amino acids in the presence of methylene blue. Archs Blochem. Biophys., 33, 90, 1951 . [4] Ran, L. J. and Muavcx, H., A simple method of estimating fifty per cent end points. Am. J. Hyg., 21, 493,1938 . [S] Ca~sEta., D. H., GARVEY, J. S., CRS, N. E. and Sussnoxr, D. H., Methodsin Immunology, p. 118, W. A. Benjamin, New York, 1963 .
Perpamon Press Ltd., Printed in Groat Britain
DETOXIFICATION OF RUSSELL'S VIPER (VIPERA RUSSELLII) AND WATER MOCCASIN (AGKISTRODON PISCIVORUS) VENOMS BY PHOTOOXIDATION WALTER KOCHOLATY 8IId BILLY D. ASHLEY Biochemistry Division, U.S . Army Medical Research Laboratory, Fort Knox, Kentucky, U.S.A . (Acceptedjor publication 16 September 1965) Abstract-Photooxidation of six venoms of domestic and foreign snakes resulted in the detoxification of all venoms . Immunization of rabbits with two of the detoxified venoms, Agkistrodon piscivorus and Vlpera russellü, resulted in the production of antibodies which protected mice against the action of unaltered venom. INTRODUCTION
it was reported that Crotalus atrox venom ca.n be detoxified by photooxidation with visible light in the presence of methylene blue, and that the detoxified material will give rise to antibodies in rabbits which in turn will protect mice from the toxic effects of this venom. Extending these observations to the venoms of other snakes selected for their diverse manifestations of toxic effects has shown that photooxidation will also detoxify these venoms in the crude stage. The present investigation is concerned with these observations and with the immunogenic properties of two of the detoxified venoms. PREVIOUSLY [1]
MATERIALS AND METHODS
Starting material The bulk of Russell's viper (Vipers russellü) venom was purchased from the Miami Serpentarium Laboratories, Miami, Florida. One fourth of the venom was collected at the U.S . Army Medical Research Laboratory snake colony. The dried venoms were combined and sieved similarly as described in the above report . The dried venom did not dissolve completely in distilled water or in 0 ~85 per cent NaC 1 on prolonged stimng . The undissolved material, which was removed by centrifugation, consisted of mucid material and cell debris . The protein content of the centrifuged and filtered solution (Millipore filter of 0 ~45 ~m pore size) determined by biuret assay as in the previous paper was found to be 57 per cent of the dry weight of the venom. The venom of the cottonmouth water moccasin (Agkistrodon piscivorus) which was also collected locally was dissolved in distilled water with the formation of a small precipitate. The protein content of the filtrate was 74 per cent. Apparatus and generalprocedure The venoms were detoxified using WEII,'S [2, 3] photooxidation procedure catalyzed by methylene blue . The procedure was carried out in a Gilson differential respirometer Model GRP 20, the light source of which provided 1,000-1,400 foot candles according to the manufacturer. 187
l 88
WALTER KOCHOLATY and BILLY D. ASHLEY
Photooxidation Photooxidation was carried out on the centrifuged and filtered venoms in conical Warburg flasks at 37° . The final reaction mixture contained 5 to 10 mg of venom protein, 0 ~ 1 mg methylene blue, in a total volume of 2 ml in 0 ~ 1 M TRIS (Tris-(Hydroxymethyl) aminomethane) buffer ofpH 8 ~5 for the A. piscivorus and pH 7 ~4 for the V. russellü venom. Assays Proteolytic, esterase, and phospholipase A activities were determined with casein, TAME (p-toluene sulfonyl-L-arginine methyl ester) and egg yolk as substrate as previously described . Toxicity tests White mice of about 20 g weight were used in the assays for the unaltered and photooxidized venom using 0 ~2-0~25 ml for the intravenous (LV .) and 0~5 ml for the intraperitoneal (LP.) route. The calculations for the LDbo dose were done according to RED and MuExcx [4] . RESULTS
Effect ojphotooxidation on the proteolytic, esterase, and phospholipase A activity of A . piscivorus venom The venom ofthe water moccasin had a strong proteolytic activity withcasein as substrate and hydrolyzed TAME with great rapidity . The phospholipase A activity was of about the same magnitude as that of C. atrox venom. During photooxidation of A. piscivorus a rapid loss of these enzyme activities occurred. After only 15 min of photooxidation during which Std OQ were taken up per mg venom protein (Table 1), the proteolytic and TABLE 1 .
LOSS OF PROTEOLYTIC, ESTERASE AND PHOSPHOLIPASE A Agkistrodon p%SClYOrUS VENOM DURING PHOTOOXIDATION
ACrMT'Y
OF
P.O . pl O, takenup Per cent loss of activity LD time permg venom tolerated (min) protein proteolytic esterase phospholipase A 20 g mouse 15 30
8 13
73 99
88 99
99 -
6 22
esterase activities were reduced to low levels and the phospholipase A activity was abolished . Concomitant with the Os uptake, a reduction in toxicity took place and after an uptake of 13 .1 O$ per mg venom protein a 22-fold LD6o was tolerated by a 20 g mouse . Crude A. piscivorus venom did take up a small amount of 08 in the absence of methylene blue and light . This amounted to only 141 O$ per 10 mg venom protein in 90 min and was not considered when using shorter photooxidation times, which were found to be adequate to detoxify the venom . Effect of photooxidation on the phospholipase A and esterase activity of V. russellü venom No proteolytic activity could be demonstrated in this venom when casein was used as a substrate although a weak esterase activity was shown . The photooxidation of this venom was carried out at pH 7 ~4 (see next page) . As seen in Table 2, no esterase activity remained
Venoms DetoxiSed by Photooxidation TABr.E
2.
189
PHOTOOXIDATION OF Vipera rassellti vexoM. LO33 of ENZYM ACIIYITIS4 AND TOXIQTY AS RELATED TO OXYGEN UPTAKE
P.O . time (min)
~.1 O, taken up per S mg venom protein pH T4
esterase
15 30 45 60 90
31 44 47 48 56
99 -
Per cent loss of activity
LD, o
tolerated phospholipase A 20 g mouse 88 96 96 98 99
8 25 -
after 15 min of photooxidation and the phospholipase A activity was almost abolished within 30 min . With increasing O a uptake a detoxification of the venom occurred, and when 9~1 O $ were taken up per mg venom protein mice survived a 25-fold LD bo. When the photooxidation of this venom was carried out at pH 8 ~4, all other conditions being equal, about double the amount of O $ was taken up at identical time intervals as those shown in Table 2. A precipitate formed in the early stages ofphotooxidation which increased with additional O s uptake. Immunization of mice and rabbits with photooxidized venom of A . piscivores and V. russellü The venom of V. russellü was photooxidized to an uptake of 9 ~1 O, per mg protein (20 mg venom in 0 ~ 1 M TRIS buffer, pH 7 ~4, at 37°) and the venom of A. piscivores to an O, uptake of 13 ~l O $ per mg protein (30 mg in 0 ~ 1 M TRIS buffer, pH 8 ~5, at 37°). The final volume of the reaction mixtures was 2~0 ml and included 0~ 1 mg methylene blue. The photooxidized venoms, diluted in physiological saline to twice the desired concentrations, were mixed before the injection with an equal amount ofAlgivant (Colab Laboratories, Inc.), an adjuvant consisting of 4 per cent Na-alginate, 067 per cent Ca-gluconate. The methylene blue of these photooxidized venoms was not removed . All photooxidized venoms were stored in ice and in the dark prior to the injections. The weekly immunization schedule for mice and rabbits was identical. Each animal received 3 injections (Mon., Wed ., Fri.) Mice received this schedule for 3 consecutive weeks and after a rest period of one week the mice were challenged with unaltered venom (see below) . Rabbits received this schedule for 4 consecutive weeks and after a rest of one week were bled to death by heart puncture. Prior to immunization, all rabbits were bled by heart puncture and the normal serum examined by qualitative precipitin test for antibody to A. piscivores and V. russellü venom. These tests were negative. Mice. Fifteen mature Swiss mice of 30 to 40 g were immunized with either venom. All injections were made subcutaneously. For the mice receiving the photooxidized venom of A. piscivores the dose was 0~5 mg protein for each of the first 2 injections and 1 ~0 mg for the following 7 administrations . To the mice receiving the photooxidized venom of V. russellü 62 ~5 y ofthis protein was given at each ofthe 9 injections. The volume ofthe injection was 0 ~ 1 ml in all cases . Thus the mice received a total of either 0-56 mg of photooxidized V. russellü or 8 mg ofphotooxidized A. piscivores venom in 3 weeks. Five ofthe mice injected with photooxidized V. russellü venom died during the injection schedule, and of these, 4 mice died within 16 hr after the first injection . The same number ofdeaths occurred among the mice receiving photooxidized A. piscivores venom ; one at the third injection (higher
l90
WALTER KOCHOLATY and BILLY D. ASHLEY
dose), the others during the course of the injection period. All mice receiving the photooxidized venom developed lesions, ranging from chafing of the skin (V. russellli) to open sores (A.piscivorus) . Seven days after the last injection the mice were challenged with 2~5 to ]0 t.nbo's of A . piscivorus and V. russellli venom, respectively, given intravenously with appropriate controls of unaltered venom . Survivors were determined after 24 hr (A . piscivorus) and 48 hr ( V. russellli). While protection is evident in the A . piscivorus experiments, the results with the V. russellli are ambiguous (Table 3). TABLE
3.
NEUTRALIZATION OF Agkis7rodon piscivorus AND HY MICE IMMUNIZED WITH PHOTOOXIDIZED VENOMS
Ylpera russellil VENOM LD 6o 1 2 2'S 5 T5 0
Venom controls A. piscivorus V. russellli A. piscivorus V. russellli 0/2 2/0 1/2 2/4
2/4 1/4 2/4
0/2 2/2
1/2 1/2
Numbers are deaths over the total number of mice used . Readings were done 24 hr (A . piscivorus) and 48 hr (V. russellli) after injection.
Rabbits . New Zealand whites weighing 2 to 3 kg were injected intramuscularly with photooxidized venom . The same weekly schedule used on mice was employed and the site of injections were the inner thighs ofthe hind legs. Ten animals were used with each venom. The injection volume was 1 ~0 ml for the photooxidized V. russellil venom and 1 ~ 1 to 2 ~2 ml for the photooxidized A. piscivorus venom . The amounts of V. russellil venom administered were 2 mg for the first 3 injections. In the fourth injection halfofthe animals received 2 mg, the other half 4 mg. The rest of the injections were 4 mg for the whole group for a total of 40 and 42 mg venom protein, respectively. With the photooxidized A . piscivorus venom the first 2 doses were 16 ~5 mg and the next, l0, 33 mg eachfor a total of 363 mgvenom protein in 4 weeks . During the injection schedule light to pronounced swelling and edema was noticed in some animals, particularly with A. piscivorus venom. The swelling subsided after 24 to48 hr. One rabbit died within 5 min after the fourth injection with2 mg ofphotooxidized V. russellil venom, another within 2 min after the tenth injection with A. piscivorus venom, and another rabbit had to be destroyed. Qualitative precipitin tests with the rabbit sera were carried out starting with a 1 :250 dilution of venom in the first tube and continuing with 5-fold dilutions. With A. piscivorus venom, precipitates were obtained in the fifth (2 sera) and in the sixth (6 sera) dilution. With V, russellil venom, precipitates were obtained. i n the fifth (6 sera) and sixth (3 sera) dilution For both groups of rabbits immunized with the photooxidized venoms y-globulin fractions were prepared from the pooled sera by repeated salting out with ammonium sulfate according to CAMPBELL et al. [5] and the dialyzed material concentrated with cellugel* in the cold. The protection studies with the y-globulins were carried out by mining 4-16 " Cellugel Super (3000), Svenska Cellulose. Aktiebolaget, Kemiska Produkter, Eesoik, Sweden.
191
Venoms Detoxified by Photooxidation
intravenous doses* with 100 mg of each of the globulins in a total volume of 1 ml. Controls were carried out in a similar manner with normal rabbit y-globulint . After incubation at 37° for 60 min 0 ~25 ml of each mixture equivalent to 2S mg y-globulin was injected intravenously into 3 mice. Physiological saline was the solvent throughout including the controls of unaltered venom. The results of this experiment are shown in Table 4. The capacity of the immune globulins was also investigated for its ability to passively protect against a fatal dose of venom. In this experiment 3-6 mice were injected intravenously with 25 mg of the appropriate y-globulin and challenged 30 min later with 1~ Ln su of unaltered A. piscivorus or V. russellü venom and compared with corresponding controls . Table 5 shows the results of these experiments which indicate that both immune globulins impart definite protection against these venoms . In order to explore the potentialities of photooxidation in the detoxification of crude snake venom 7 venoms showing a variety of toxic manifestations have been selected in this study. A comparison of their proteolytic (casein), esterase (TAME), and phospholipase Ln ao
TAHLE 4 .
NEUTRALIZATION OF Agkistrodon piscivores exn Vipera ressellü v>~"toM nv MtCE Bv IAQKIINa OLOBULIN FROM RAHHITS D~AlIJNIZED WITfi PHOTbOXIDAZ® VENOMS
A piscivores LD
Immune* globulin
1 2 3 4
0/3 0/3 0/3 1/3
V. russellü
Normal globulin control
Venom control
Immune* globulin
3/3 4/4
5/7 7/7
0/3 0/3 1/3 1/3
Normal globulin control
Venom control
4/4 4/4
4/7 6/6
*From rabbits immunized with A, piscivores and V. russellti venoms respectively. Numbers are deaths over tho total number of mice used . Readings were done 24 hr (A . piscivores) and 48 hr (V. ressellü) after injection . Throe or more mice received either 25 mg immune rabbit globulin or normal rabbit globulin and tho Lv,o as stated above.
TABLe S . PROT6CTiON oF Mice eaeu~rsr AgkLstrodon piscivores AND Vipera russellü vFavoMS wITII RABBrr IMAIVNB aLOSVLU"t Venom controls
Lo bo
A . piscivores
Y. ressellü
A . piscivores
V. ressellü
1 2 3 4 5 6
0/3 0/3 1/3 3/3 -
0/6 0/6 0/6 0/6 0/3 2/3
3/3 3/3
4/6 6/6
Numbers are deaths over the total number of mico used . Readings wore done 24 hr (A . piscivores) and 48 hr (Y. russellü) after injection. 25 mg ofrabbit y-globulin was injected(LV.) in a volume of 0 ~25 ml, followed in 30 min by(LP.) injection of 1-6 Ln 6o of the venoms . Controls received venom only (LP.) .
" 1 Lnbo A . piscivores I . V .=45 y, I. P.=90 y . I LDbo V. russellü I . V.=S y, I. P.=9 y . t Fraction II from the Pentex Laboratories, Inc., I{ankakee, Illinois .
192
WALTER KOCHOLATY and BILLY D . ASHLEY
A activity is shown in Table 6. C. atrox venom appeared the most proteolytic o.these venoms followed by A . piscivorus and Bothrops atrox. These 3 venoms were also the only ones of this group displaying activity for all the 3 enzymes tested. All 3 venoms also displayed considerable esterase (TAME) activity . Phospholipase A activity, an enzyme common to all 7 venoms in varying degree, was most pronounced with Micrurus fulvius which lacked proteolytic and esterase activity . Naja raja somewhat resembled this venom showing a similar phospholipase activity ; V. russellü did not have proteolytic activity but displayed a weak esterase activity . Crotalus terrificus venom was unique in having the lowest phospholipase A activity of all venoms tested but possessed a very high esterase and no proteolytic activity. The Os uptake ofthese crude venoms during photooxidation was recorded against time using identical conditions of protein concentration, pH, and light intensity (Table 7). In general the rapid initial rate of the 0E consumption is followed by a gradual decrease . An exception formed the rate of OE uptake of C. terrifrcus venom which was practically linear for the first 60 min . Several of the venoms formed precipitates at pH 8 ~5 as indicated in the table. The nature of the precipitates was not investigated . Photooxidation with subsequent detoxification may be carried out at other pH values, however, as shown with TABLE
6.
PROTEOLYTIC (CASEIN, ESTERASE (TAME), AND PHOSPHOLIPASE A ACTIVITY OF SOME SNAKE VENOMS
Venom Agkistrodonpiscirorrrr Bothrops atrox Crotalus atrox Crotalus terrificus Micrurus frrlvirrs Najaxaja Vipera russellü
Casein
Phospholipase A' m Eq acid
I " 17 0 " 87 2 " 00 0 "00 0 " 00 0 " 00 0 "00
0 " 32 0 " 33 0 " 30 0 "25 3 " 36 2 "51 2 " 51
TCA-OD~ o"
Units'
TAME 650 518 433 637 0 0 86
" Per 1 mg venom protein . B. atrox and M. fulvius venoms were of commercial origin . The venom of C . terrifrcus was a gift from Dr . Karl H . Slotta, University of Miami, School of Medicine . The rest of the venoms were collected at the USAMRL snake colony . TABLE
7.
RATE OF OXYGEN UPTAKE OF VARIOUS SNAKE VENOMS DURING PHOTOOXIDATION IN THE PRESENCE OF METHYLENE BLUE
Venom Agkistrodon piscivores Bothrops atrox Crotalus adarnanteus' Crotalus atrox Crotalus terrifrcus" Micrurus fulvius' Naja ngja Yipera russellü "
Photooxidation time (min) 30 45 60 90 ~1 O, uptake/10 mg venom protein
IS
80 71 92 74 26 80 59 61
129 126 130 I 15 58 130 87 84
163 161 155 142 77 147 108 99
190 188 177 165 100 173 123 113
245 243 211 200 135 213 150 136
Reaction mixture : 10 mg venom protein, 0 " l mg methylene blue in 2 ml 0'1 M TRIS buffer, pH 8 " 5 . Numbers are ~1 O, taken up by 10 mg venom protein . 'These venoms formed precipitates during photooxidation at pH 8 " 5.
Venoms Detoxified by Photooxidation
19 3
the venom of V. russellü. While the O~ uptakes varied by not mush more than double when calculated in a mg protein basis, vast differences in the detoxification were obtained at identical 0 9 consumption . Table 8 shows that all 7 venoms could be detoxified by photooxidation . While the venoms of C. terr~cus and M. julvius were detoxified to an extent that 150 Lo bo were tolerated at an OE uptake of only 8 and 13 ~l per mg venom protein, the detoxification of N. raja venom proceeded at a considerably slower and almost linear rate with comparatively large O Q consumption. TABLE S.
EFFECT OF OXYGEN UPTAKE DURING PHOTOOXIDATION ON LOSS OF TOXICITY iN SEVERAL VENOMS
Agkistrodon Bothrops Crotales Crolalus piscivores afrox afrox terrlfrcus F
LU,o
20 g mouse, LV . (y)
8 13 -
6 22 45
3~5 7~0 29
1 35 -
10~0 I1 ~5 12~5 14~0 - - -
2~5 2~5 6~0 20~0 8~0150~0 - -
70
2
Micrurrrs fulvius Fal O, LD,o 8 13 -
40 I50 8
Fal
Naja naja Oa LDso 2 4 8 16
5 l0 20 27 8
Vlpera rrrssellil F<1 O, LD,o 6 9 -
8 25 5
0,=taken up by 1 mg of crude venom protein during photooxidation . Number of LD,o tolerated ; 3 mice out of 3 surviving 24 hr (A . piscivores, C. atrox)~8 hr (the other five). Wl
DISCUSSION
The purpose of these investigations was to establish whether or not crude venoms could be detoxified by photooxidation and whether the detoxified venoms were capable of giving rise to immune bodies. Although the results were disappointing in the mice experiments, the protection experiments with the isolated y-globulins from rabbits immunized with photooxidized venoms of A . piscivorus and V. russellü produced results similar to those reported for C. afrox venom [I] . Again, as with C. afrox, optimal conditions may not have been employed. A. piscivorus venom at a lower pH and temperature of photooxidation and somewhat higher O$ uptake might yield a product producing less of the deleterious effects in mice. The pH of 7 ~4 used for the photooxidation of V, russellü venom was selected arbitrarily because of precipitate formation at pH 8 ~5 . The immunization schedule of 3 injections a week for 3~ weeks may well be a "heavy" one and unnecessary and the particular adjuvant used may not be the most desirable . The measure of detoxification used in these studies was the amount of O$ uptake as related to toxicity. The abolishment of the proteolytic and phospholipase A activity during photooxidation together with a considerable reduction of toxicity may be coincidental. Also, the number of LD6p tolerated in mice following photooxidation (Table 8) does not represent the absolute minimum but is rather on the conservative side, since the limited amounts of some venoms prevented the establishment of the uppermost levels oftolerance . The detoxification obtained in all these venoms using such a gentle method as photooxidation gives hope of similar responses in the production of immune bodies . SU MMARY
1 . Photooxidation of A. piscivorus and V. russellü venom by visible light in the presence of methylene blue resulted in the detoxification ofthese venoms .
194
WALTER KOCHOLATY and BILLY D. ASHLEY
2. Immunization of rabbits with these detoxified venoms resulted in the production of antibodies which protected mice against the action of the unaltered venoms . 3. Four additional snake venoms were shown to be detoxified by photooxidation and a comparison of al] seven venoms was made with respect to their proteolytic, esterase, and phospholipase A activities and behavior during photooxidation . Pathologic findings
ADDENDUM
Grossly, the effects of repeated intramuscular injections of the photooxidized venoms of V. russellü and A . piscivorus was similar in that there was inflammation of the musculature varying from small focal lesions to abscesses 3 cm in dia. Microscopically, the findings for the Y. russellü, and A. piscivorus venoms were also similar. The reaction of the musculature surrounding the areas of inflammation or abscessation were of particular interest. Surrounding the areas of complete necrosis of muscle there were zones of inflammatory cell infiltrations consisting mostly of plasma cells, eosinophiles and small round cells. Many multinucleate giant cells were also present . The giant cells appeared to be formed by the syncytium of the nuclei of degenerate perimysial tissue as well as nuclei of muscle cells. Finely granular mineralization was seen throughout the necrotic areas. In the lungs there was a medial hyperplasia of the muscular arteries and ballooning of endothelium was noted. A few sections of myocardium showed areas of degeneration and focal collections of inflammatory cells that may have been associated with intracardiac punctures. No other significant lesions were noted in the duodenum, pancreas, adrenals, liver, or kidney . This information was furnished by Col. M. A. Ross, Head, Department of Pathology. Acknowledgements-The authors acknowledge the technical assistance of C. R. Sxn:r us and T. A. Bn t naas. REFERENCES [1 ] Kocxoc.~rv, W., Detoxification of Crotalus atrox venomby photooxidation in thepresence of methylene blue, Toxicon, 3, 175,1966 . [2] Wsa,, L. and Mate, J., Photodynamic action of methylene blue on nicotine and its derivatives. Archs Biochem. Biophys., 29, 241, 1950. [3] WEn., L., Goxnox, W. G. and Bucter, A. R., Photooxidation of amino acids in the presence of methylene blue. Archs Blochem. Biophys., 33, 90, 1951 . [4] Ran, L. J. and Muavcx, H., A simple method of estimating fifty per cent end points. Am. J. Hyg., 21, 493,1938 . [S] Ca~sEta., D. H., GARVEY, J. S., CRS, N. E. and Sussnoxr, D. H., Methodsin Immunology, p. 118, W. A. Benjamin, New York, 1963 .