- Email: [email protected]
Collagenolytic activity of snake and spider venoms
Tourtroe, 1967, Vot . 4. M 231-253 . Perpmon Press Ltd ., Printed In Great Britain
COLLAGENOLYTIC ACTIVITY OF SNAKE AND SPIDER VENOMS B. KAsm and w. RAAB
Department of Medical Chemistry, University of Vienna, Vienna, Austria (Acceptedforpublication 10 August 1966) AbstractThe venoms of the snakes B. atrox, B. jararaca, C. durissus, A. piscivores, and the spiders P. fera, L. erythrognatha possess proteolytic activity which attacks gelatine, casein and azocoll. Denatured (heat, urea) collagen is digested too. However, native collagen is not attacked by the enzymes present in the venoms investigated . Following subcutaneous injection of A. piscivores venom, a statistically significant increase in urinary hydroxyproline excretion occurred . This finding indicates a degradation of collagen by animal venoms to vivo. The mechanism of this action remains unknown. INTRODUCTION
proteolytic enzymes may contribute to the severe local effects following the bites of certain venomous animals the occurrence of true collagenases in venoms has stimulated considerable interest . In vitro experiments have revealed the digestion by snake venoms of not only gelatine and casein [1] but also of azocoll [2]. The digestion of azocoll, however, did not ascertain a degradation of native collagen [3-6]. Thus, snake venoms were examined for their ability to produce softening of guinea pig muscle in vitro. In this study [5] all Viperidae venoms showed a collagenolytic action ; the highest activities were found in the venom of Bitis lachesis [2]. As the method for determination of collagenase activity [5] is subject to various misinterpretations, we have reinvestigated the problem ofcollagen digestion by animal venoms . In vitro, the actions of several snake and spider venoms on azocoll, gelatine and different collagen preparations were determined. In vitro experiments were designed to investigate the hydroxyproline excretion in rat urine following injections of crotalid venom. Recent observations have confirmed the correlation between hydroxyprofne excretion and collagen degradation [7-10]. SINCE
1 . Venoms
MATERIALS AND METHODS
Four snake venoms (Bothrops atrox (L .), Bothropsjararaca (Wied), Crotales durissus (L.), Agkistrodon piscivores (Lacepede), and 2 spider venoms (Phoneutria fera Perty, Lycosa erythrognatha Lucas) were investigated in vitro. Their action on different substrates was compared with crystalline trypsin and pepsin .
2. Substrates The collagenolytic action of the venoms was determined on azocoll, gelatine, casein and on three collagen preparations : 251
252
E. KAISER and W. RAAB
(a) Preparation A. Collagen fibers of rat tail tendons were separated from fat, muscle and loose connective tissues, rinsed in cold saline (0-15 M), cut into small pieces (2-3 cm) and stored in saline at 4°. Collagen fibers from bovine Achilles tendon were prepared in a similar manner. These preparations were used for qualitative experiments only.
(b) Preparation B. For quantitative experiments, the fibers of preparation A were cut into smaller pieces, homogenized in iced saline for 30 sec, washed with ethanol-ether for 3 min and air dried. The procedure did not exceed 10 min [1l].
(c) Preparation C. Preparation B was kept in 10 per cent saline at 4° for 2 weeks, with occasional changes of solutions. It was then washed and suspended in 0-05 M Na,HPO, at 4° for 3 days, washed again and treated successively with acetone, and ethanol, and ether, each at 4° for 20 hr. Finally, the powder was air dried [5, 111. (d) Denatured collagen preparations. Denaturation of collagen (preparations B or C) was accomplished by heat (65° for 20 min) or by incubation with urea (100 mg preparation BorCin5ml4M,5Mor6Murea at37'for24hr). 3. Quantitative determination of collagenolytic activity. The quantitative determinations were performed according to the method of SHERRY et al. [12] : venom was added to 50 mg of preparations B or C suspended in 4 ml buffer. After incubation 3 hr at 37°, 5 ml 10 per cent saline was added and the mixture filtered. In the filtrate, nitrogen was determined according to Kjeldahl . Hydroxyproline determinations were performed [13].
4. Hydroxyproline determination in urine following injection of snake venom. Male albino rats (150-180 g) were kept in metabolic cages. Twenty-four hr urine specimens were collected. The animals were fed a hydroxyproline-free diet. In 50 rats the normal value for hydroxyproline excretion was determined. Fifty rats were injected subcutaneously with 15 mg per kg of Agkistrodon piscivorus Lacepede venom in saline . Total urinary hydroxyproline was determined in the following manner : 2"0 ml of urine were hydrolysed with 1-0 ml 10 N HCl at 120° for 3 hr. Urinary pigments were adsorbed on charcoal . After filtration the pH of the filtrate was adjusted to 7-0 and a total vol. of 8-0 ml obtained by adding distilled water. From this solution, 2-0 ml were used for the hydroxyproline determination [14]. RESULTS
(1) Digestion ofazocoll, gelatine and casein
All venoms examined digested azocoll, gelatine and casein . The venom of Bothrops jararaca showed the highest activity at pH 8-0, and the venom of Lycosa erythrognatha at pH 9" 0 (Fig. 1).
(2) Digestion ofcollagen preparations A, B and C None of the venoms had lytic action on collagen fibers or collagen powder, not even after incubation periods up to 3 days . In this respect, no difference was found between the animal venoms and crystalline trypsin. However, denatured collagen preparations were digested by trypsin as well as by the venoms . (3) Hydroxyproline excretion in urinefollowing injection ofsnake venoms The normal value of hydroxyproline excretion in rat urine amounts to 0-56 ± 0-2 mg
Collagenolytic Activity of Snake and Spider Venoms
253
100 90 e0 70 60 50 40 30 20 10
Fia . 1 .
PROTEOLYTIC ACTIVITY OF THE vENOMs OF Bothrops jararaca natha (B) AoAnasr AzocoLL.
(A) AND Lycosa erythrog-
per 24 hr. In 24-hr urine specimens following injection of A. piscivores venom, a statistically significant increase of hydroxyproline excretion was observed (0"147 ± 0"58 mg per 24 hr ; P < 0-001) . On the second day following injection of snake venom, only a slight increase in hydroxyproline excretion was observed (Table 1).
âN 20
18 18 F2
T
10
0 $ 08 a x0-6 04
02
t
Venom (s .o)
Day
TABLE 1 . HYDRoxypRoLnvs EccRmoN nv 24-hr uRII4E spEECna Ns OF THE RAT AFTER mIECIYON OF THE VENOM OF Agkinrodon p1scivorus.
DISCUSSION
Several authors have investigated the digestive action of different proteolytic enzymes on collagen . A relatively high resistance of collagen against proteases has been found [15]. Digestion of collagen occured with pepsin at strongly acid pH [16], with beef spleen cathepsin at pH 2-0-4-5 [12] and with bacterial (clostridial) collagenase [3-6, 7]. None of the snake and spider venoms examined digested native collagen, but azocoll, gelatine and casein were easily attacked . Collagen, denatured by heat or urea was also
254
E. KAISER and W. RAAB
digested by the venoms. These findings support the results of other investigators [1l, 17, Raab, in press] . Digestion of extremely fine shredded collagen fibers by trypsin [15] was not observed in our experiments. Hydroxyproline excretion in urine has proved to be a good indicator for collagen metabolism . An increased degradation of collagen in vivo causes an increase in hydroxyproline excretion in urine. Following injection of snake-venom, a statistically significant increase of hydroxyproline in rat urine occurred. This finding indicates a degradation of collagen in vivo by the action of the venoms studied. Histologic investigations revealed the occurrence of severe vascular changes in dermal connective tissues following injection of animal venoms [18, 19] ; the changes often result in necrosis. Collagen digestion by proteolytic enzymes of the organism increases hydroxyproline excretion in urine. As has been demonstrated following experimental burns in rats, an increased hydroxyproline excretion occurs concomitantly with resorption [Raab, in press]. Following subcutaneous injection of snake venoms the increase in hydroxyproline excretion is found before resorption of necrotic tissue is started. Furthermore, necrosis is never of the same degree as noted in the experimental burns. Inflammatory tissues of the rat contain enzymes which digest collagen [20], but the activity of these enzymes appears far too weak to account for the significant increase in hydroxyproline excretion. A minor contributory effect, however, cannot be excluded. Hydroxyproline-containing collagen precursors are present in fibroblasts and possibly in mast-cells [21]. Mast cell degranulation might provoke a mobilization of such hydroxyproline containing substances. Following anaphylactic or anaphylactoid shock, only a small increase in urinary hydroxyproline excretion occurs. From this finding it can be excluded that mast-cell degranulation following injection of snake venoms [18, 22, Raab, in press] contributes significantly to the observed increase in urinary hydroxyproline output . As other mechanisms for the increase in urinary hydroxyproline can be ruled out, the observed effect may be attributed to a degradation of collagen in vivo under the influence ofanimal venoms . The mechanism leading to the degradation of the collagen is unknown.
REFERENCES
[ll Knrs&x, E. and Micas H.: Die Biochemle der tierischen C(Jte. Wien : Franz Deuticke, 259 p., 1958. Ill OIIUSrENaN, P. A., South African snake venoms and antivenoms. S. Afr. Inst. Med. An., Johannesburg, 1955.
[3] BmwEts., E., Proteolytic enzymes of Cl. welchil. Biochem. J. 46, 589, 1950. [4] Bmwmt, E. and vAN HSYmr4om, W. E., The biochemistry of the gas gangrene toxins. 5. The k-toxin
(collagenase) of Cl. welchil. Biochem. J. 42,140, 1948. [51 OARï.EY, C. L., WARRAcr, H. C. and vAN HEYNn1GEN, W. E., The collagenase (k-toxin) of Cl. welchil type A. J. Path . Bact. 58, 229,1946 . [6l OARLEY, C. L., WARRAcK, H. C. and WARREN, M. E., The kappa- and lambda-antigens of Clostridlam welchü. J. Path. Bact . 60, 495, 1948. [7l Dum, T. A. and HENNEmAN, P. H., Urinary hydroxyproline as an index of collagen turnover in bone. New Bogl. J. Med. 268,132,1963 . 181 C3usrAvsoN, K. H. : 71re Chemistry and Reactivity of Collagen. New York : Academic Press, 1956. 191 Kim, L., LAFFmrsr, F. W., PEARsm, O. H. and CuR'nss, P. H., Correlation of urinary hydroxyproline, serum alkaline phosphatase and skeletal calcium turnover. Metabolism 13, 272, 1964 . [10] SmEaoma, A., UDENFRnzbw, S., KAamt, H. and LEROY, E. C., Hydroxyproline and collagen metabolism. Ann. intern . Mad 63, 672, 1965 . [11] NEumANN, R. E. and Tnm ., A. A., Action of proteolytic enzymes on collagen. Proc . Soc. exp. Biol. Med. N. Y. 73, 409, 1950.
Collagenolytic Activity of Snake and Spider Venoms [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22]
255
TRou., W. and Ro88NBLum, E. D., °Collagenase" activity of cathepsins. Proc. Soc. exp. .Med. N. Y. 87,125, 1954 . Biol NEUMANN, R. E. and I.AoAN, M. A., The determination of hydroxyproline. J. biol. Clam. 184,299, 1950 . ST'BGFalANN, H., Mikrobestimmung von Hydroxyprolin mit Chloramin T und Dimethylaminobenzaldehyd. Z. physioL Chem . 311, 41, 1958. Simut, I. W., The action of trypsin and other proteases on collagen . Fnzymolo& 13, 293, 1949 . Samt, 1. W., The action of pepsin on collagen. Enzymologia 13, 288, 1949. AxgxoD, A. E. and MATT, C. J., Biochemical changes in thermally injured cutaneous tissue, susceptibility to proteolytic enzymes, and extractability of collagen. Proc. Soc. exp. Biol. Med. N. Y. 83,463, 1963. RAAB, W. and KAea, E., Die Wirkungen des Hautsekretes der Gelbbauchunke (Bombina variegata L.) auf das Hautbindegewebe von Meerschweinchen, Ratten und Mäusen. Arch. On. exp. Derrnat. 220, 374, 1964. RAAB, W. und KAtsm, E., Experimentelle Untersuchungen zur Frage der Mastzelldegranulierung durch Schlangengifte . Toxicon 3, 49, 1945 . BAz1N, S. et DBLAuNEY, A., Caractbres de cathepsines collagénolytiques présentes dans les tissues inflammés du rat. Ann. Inst. Pasteur 110, 192, 1966 . Sam, H. : 71ie Mast Cells. London : Butterworth, 1965. KAmEt, E. und RAAB, W., Histamin, 5-Hydroxytryptamin und mastzellersch6pfende Substanzen in tierischen Giften. Z. angew. Zool. 52, 1, 1965. SEmRRY, S.,
COLLAGENOLYTIC ACTIVITY OF SNAKE AND SPIDER VENOMS B. KAsm and w. RAAB
Department of Medical Chemistry, University of Vienna, Vienna, Austria (Acceptedforpublication 10 August 1966) AbstractThe venoms of the snakes B. atrox, B. jararaca, C. durissus, A. piscivores, and the spiders P. fera, L. erythrognatha possess proteolytic activity which attacks gelatine, casein and azocoll. Denatured (heat, urea) collagen is digested too. However, native collagen is not attacked by the enzymes present in the venoms investigated . Following subcutaneous injection of A. piscivores venom, a statistically significant increase in urinary hydroxyproline excretion occurred . This finding indicates a degradation of collagen by animal venoms to vivo. The mechanism of this action remains unknown. INTRODUCTION
proteolytic enzymes may contribute to the severe local effects following the bites of certain venomous animals the occurrence of true collagenases in venoms has stimulated considerable interest . In vitro experiments have revealed the digestion by snake venoms of not only gelatine and casein [1] but also of azocoll [2]. The digestion of azocoll, however, did not ascertain a degradation of native collagen [3-6]. Thus, snake venoms were examined for their ability to produce softening of guinea pig muscle in vitro. In this study [5] all Viperidae venoms showed a collagenolytic action ; the highest activities were found in the venom of Bitis lachesis [2]. As the method for determination of collagenase activity [5] is subject to various misinterpretations, we have reinvestigated the problem ofcollagen digestion by animal venoms . In vitro, the actions of several snake and spider venoms on azocoll, gelatine and different collagen preparations were determined. In vitro experiments were designed to investigate the hydroxyproline excretion in rat urine following injections of crotalid venom. Recent observations have confirmed the correlation between hydroxyprofne excretion and collagen degradation [7-10]. SINCE
1 . Venoms
MATERIALS AND METHODS
Four snake venoms (Bothrops atrox (L .), Bothropsjararaca (Wied), Crotales durissus (L.), Agkistrodon piscivores (Lacepede), and 2 spider venoms (Phoneutria fera Perty, Lycosa erythrognatha Lucas) were investigated in vitro. Their action on different substrates was compared with crystalline trypsin and pepsin .
2. Substrates The collagenolytic action of the venoms was determined on azocoll, gelatine, casein and on three collagen preparations : 251
252
E. KAISER and W. RAAB
(a) Preparation A. Collagen fibers of rat tail tendons were separated from fat, muscle and loose connective tissues, rinsed in cold saline (0-15 M), cut into small pieces (2-3 cm) and stored in saline at 4°. Collagen fibers from bovine Achilles tendon were prepared in a similar manner. These preparations were used for qualitative experiments only.
(b) Preparation B. For quantitative experiments, the fibers of preparation A were cut into smaller pieces, homogenized in iced saline for 30 sec, washed with ethanol-ether for 3 min and air dried. The procedure did not exceed 10 min [1l].
(c) Preparation C. Preparation B was kept in 10 per cent saline at 4° for 2 weeks, with occasional changes of solutions. It was then washed and suspended in 0-05 M Na,HPO, at 4° for 3 days, washed again and treated successively with acetone, and ethanol, and ether, each at 4° for 20 hr. Finally, the powder was air dried [5, 111. (d) Denatured collagen preparations. Denaturation of collagen (preparations B or C) was accomplished by heat (65° for 20 min) or by incubation with urea (100 mg preparation BorCin5ml4M,5Mor6Murea at37'for24hr). 3. Quantitative determination of collagenolytic activity. The quantitative determinations were performed according to the method of SHERRY et al. [12] : venom was added to 50 mg of preparations B or C suspended in 4 ml buffer. After incubation 3 hr at 37°, 5 ml 10 per cent saline was added and the mixture filtered. In the filtrate, nitrogen was determined according to Kjeldahl . Hydroxyproline determinations were performed [13].
4. Hydroxyproline determination in urine following injection of snake venom. Male albino rats (150-180 g) were kept in metabolic cages. Twenty-four hr urine specimens were collected. The animals were fed a hydroxyproline-free diet. In 50 rats the normal value for hydroxyproline excretion was determined. Fifty rats were injected subcutaneously with 15 mg per kg of Agkistrodon piscivorus Lacepede venom in saline . Total urinary hydroxyproline was determined in the following manner : 2"0 ml of urine were hydrolysed with 1-0 ml 10 N HCl at 120° for 3 hr. Urinary pigments were adsorbed on charcoal . After filtration the pH of the filtrate was adjusted to 7-0 and a total vol. of 8-0 ml obtained by adding distilled water. From this solution, 2-0 ml were used for the hydroxyproline determination [14]. RESULTS
(1) Digestion ofazocoll, gelatine and casein
All venoms examined digested azocoll, gelatine and casein . The venom of Bothrops jararaca showed the highest activity at pH 8-0, and the venom of Lycosa erythrognatha at pH 9" 0 (Fig. 1).
(2) Digestion ofcollagen preparations A, B and C None of the venoms had lytic action on collagen fibers or collagen powder, not even after incubation periods up to 3 days . In this respect, no difference was found between the animal venoms and crystalline trypsin. However, denatured collagen preparations were digested by trypsin as well as by the venoms . (3) Hydroxyproline excretion in urinefollowing injection ofsnake venoms The normal value of hydroxyproline excretion in rat urine amounts to 0-56 ± 0-2 mg
Collagenolytic Activity of Snake and Spider Venoms
253
100 90 e0 70 60 50 40 30 20 10
Fia . 1 .
PROTEOLYTIC ACTIVITY OF THE vENOMs OF Bothrops jararaca natha (B) AoAnasr AzocoLL.
(A) AND Lycosa erythrog-
per 24 hr. In 24-hr urine specimens following injection of A. piscivores venom, a statistically significant increase of hydroxyproline excretion was observed (0"147 ± 0"58 mg per 24 hr ; P < 0-001) . On the second day following injection of snake venom, only a slight increase in hydroxyproline excretion was observed (Table 1).
âN 20
18 18 F2
T
10
0 $ 08 a x0-6 04
02
t
Venom (s .o)
Day
TABLE 1 . HYDRoxypRoLnvs EccRmoN nv 24-hr uRII4E spEECna Ns OF THE RAT AFTER mIECIYON OF THE VENOM OF Agkinrodon p1scivorus.
DISCUSSION
Several authors have investigated the digestive action of different proteolytic enzymes on collagen . A relatively high resistance of collagen against proteases has been found [15]. Digestion of collagen occured with pepsin at strongly acid pH [16], with beef spleen cathepsin at pH 2-0-4-5 [12] and with bacterial (clostridial) collagenase [3-6, 7]. None of the snake and spider venoms examined digested native collagen, but azocoll, gelatine and casein were easily attacked . Collagen, denatured by heat or urea was also
254
E. KAISER and W. RAAB
digested by the venoms. These findings support the results of other investigators [1l, 17, Raab, in press] . Digestion of extremely fine shredded collagen fibers by trypsin [15] was not observed in our experiments. Hydroxyproline excretion in urine has proved to be a good indicator for collagen metabolism . An increased degradation of collagen in vivo causes an increase in hydroxyproline excretion in urine. Following injection of snake-venom, a statistically significant increase of hydroxyproline in rat urine occurred. This finding indicates a degradation of collagen in vivo by the action of the venoms studied. Histologic investigations revealed the occurrence of severe vascular changes in dermal connective tissues following injection of animal venoms [18, 19] ; the changes often result in necrosis. Collagen digestion by proteolytic enzymes of the organism increases hydroxyproline excretion in urine. As has been demonstrated following experimental burns in rats, an increased hydroxyproline excretion occurs concomitantly with resorption [Raab, in press]. Following subcutaneous injection of snake venoms the increase in hydroxyproline excretion is found before resorption of necrotic tissue is started. Furthermore, necrosis is never of the same degree as noted in the experimental burns. Inflammatory tissues of the rat contain enzymes which digest collagen [20], but the activity of these enzymes appears far too weak to account for the significant increase in hydroxyproline excretion. A minor contributory effect, however, cannot be excluded. Hydroxyproline-containing collagen precursors are present in fibroblasts and possibly in mast-cells [21]. Mast cell degranulation might provoke a mobilization of such hydroxyproline containing substances. Following anaphylactic or anaphylactoid shock, only a small increase in urinary hydroxyproline excretion occurs. From this finding it can be excluded that mast-cell degranulation following injection of snake venoms [18, 22, Raab, in press] contributes significantly to the observed increase in urinary hydroxyproline output . As other mechanisms for the increase in urinary hydroxyproline can be ruled out, the observed effect may be attributed to a degradation of collagen in vivo under the influence ofanimal venoms . The mechanism leading to the degradation of the collagen is unknown.
REFERENCES
[ll Knrs&x, E. and Micas H.: Die Biochemle der tierischen C(Jte. Wien : Franz Deuticke, 259 p., 1958. Ill OIIUSrENaN, P. A., South African snake venoms and antivenoms. S. Afr. Inst. Med. An., Johannesburg, 1955.
[3] BmwEts., E., Proteolytic enzymes of Cl. welchil. Biochem. J. 46, 589, 1950. [4] Bmwmt, E. and vAN HSYmr4om, W. E., The biochemistry of the gas gangrene toxins. 5. The k-toxin
(collagenase) of Cl. welchil. Biochem. J. 42,140, 1948. [51 OARï.EY, C. L., WARRAcr, H. C. and vAN HEYNn1GEN, W. E., The collagenase (k-toxin) of Cl. welchil type A. J. Path . Bact. 58, 229,1946 . [6l OARLEY, C. L., WARRAcK, H. C. and WARREN, M. E., The kappa- and lambda-antigens of Clostridlam welchü. J. Path. Bact . 60, 495, 1948. [7l Dum, T. A. and HENNEmAN, P. H., Urinary hydroxyproline as an index of collagen turnover in bone. New Bogl. J. Med. 268,132,1963 . 181 C3usrAvsoN, K. H. : 71re Chemistry and Reactivity of Collagen. New York : Academic Press, 1956. 191 Kim, L., LAFFmrsr, F. W., PEARsm, O. H. and CuR'nss, P. H., Correlation of urinary hydroxyproline, serum alkaline phosphatase and skeletal calcium turnover. Metabolism 13, 272, 1964 . [10] SmEaoma, A., UDENFRnzbw, S., KAamt, H. and LEROY, E. C., Hydroxyproline and collagen metabolism. Ann. intern . Mad 63, 672, 1965 . [11] NEumANN, R. E. and Tnm ., A. A., Action of proteolytic enzymes on collagen. Proc . Soc. exp. Biol. Med. N. Y. 73, 409, 1950.
Collagenolytic Activity of Snake and Spider Venoms [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22]
255
TRou., W. and Ro88NBLum, E. D., °Collagenase" activity of cathepsins. Proc. Soc. exp. .Med. N. Y. 87,125, 1954 . Biol NEUMANN, R. E. and I.AoAN, M. A., The determination of hydroxyproline. J. biol. Clam. 184,299, 1950 . ST'BGFalANN, H., Mikrobestimmung von Hydroxyprolin mit Chloramin T und Dimethylaminobenzaldehyd. Z. physioL Chem . 311, 41, 1958. Simut, I. W., The action of trypsin and other proteases on collagen . Fnzymolo& 13, 293, 1949 . Samt, 1. W., The action of pepsin on collagen. Enzymologia 13, 288, 1949. AxgxoD, A. E. and MATT, C. J., Biochemical changes in thermally injured cutaneous tissue, susceptibility to proteolytic enzymes, and extractability of collagen. Proc. Soc. exp. Biol. Med. N. Y. 83,463, 1963. RAAB, W. and KAea, E., Die Wirkungen des Hautsekretes der Gelbbauchunke (Bombina variegata L.) auf das Hautbindegewebe von Meerschweinchen, Ratten und Mäusen. Arch. On. exp. Derrnat. 220, 374, 1964. RAAB, W. und KAtsm, E., Experimentelle Untersuchungen zur Frage der Mastzelldegranulierung durch Schlangengifte . Toxicon 3, 49, 1945 . BAz1N, S. et DBLAuNEY, A., Caractbres de cathepsines collagénolytiques présentes dans les tissues inflammés du rat. Ann. Inst. Pasteur 110, 192, 1966 . Sam, H. : 71ie Mast Cells. London : Butterworth, 1965. KAmEt, E. und RAAB, W., Histamin, 5-Hydroxytryptamin und mastzellersch6pfende Substanzen in tierischen Giften. Z. angew. Zool. 52, 1, 1965. SEmRRY, S.,