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A method for estimating Crotalus atrox venom concentrations
Toxicon, 1967, Vol. 5. pp . 35-38. Pergamon Press Ltd., Printed in Great Britain
A METHOD FOR ESTIMATING CROTALUS ATROX VENOM CONCENTRATIONS BOB D. JOHNSON, H . L. STAHNKE and R. KOONCE Poisonous Animals Research Laboratory, Arizona State University, Tempe, Arizona, U.S.A . (Acceptedfor publication 18 January 1967)
Abstract-Venom from the rattlesnake, Crotalus atrox Baird and Girard 1853, was studied. The procedure for estimating raw venom concentration showed that this snake's venom concentration is not dependent upon its size . Intraspecific variation in venom complexity was shown on disc electrophoresis . INTRODUCTION
studies concerned with the secretions of rattlesnake venoms, raw venoms are infrequently used and the use of weighed dried venoms has predominated . Use of raw venoms of known concentrations might conserve time, if concentrations could be ascribed to a specific property or activity. Venom from the Western diamondback rattlesnake Crotalus atrox Baird and Girard 1853 was studied. We report here procedures for estimating the concentration of raw venom. Relationships existing between venom protein and paramecia lethality (cell lysing ability) are briefly examined . Disc electropherograms of lyophilized and raw venoms are compared . IN MosT
MATERIALS AND METHODS
Samples of raw venom from individual C. atrox snakes were extracted directly into large centrifuge tubes. These were immediately centrifuged to remove debris . Each sample was then divided into 0-05 ml aliquots . The tubes containing these aliquots were tightly plugged with cotton and stored at -20° until used. The raw venom pool was prepared by combining an equal number of aliquots from each snake . All protein estimations were performed by the method Of ITZHAM and GILL [1]. Prior to adding 1 ml of reagent 1 (0-21 per cent CuSO4 - 5H 9 0 in 30 per cent NaOH) or 1 ml of reagent 2 (30 per cent NaOH), 0-05 ml of pooled raw venom was diluted to 20 ml (1 : 400 dilution) with distilled water. 1 : 800 and 1 : 1600 dilutions permitted a linear curve to be plotted. In order to calculate the concentration of venom, the O.D.D (optical density difference) values from this line were compared to O.D .D values obtained by using lyophilized samples with known concentrations from the pooled venom. Individual snake venom concentrations were determined by comparing their 1 : 400,1 : 800 and 1 :1600 O.D .D values to those obtained with the venom pool. The calculated concentrations of the (0-05 ml) raw venom samples were confirmed by weighing after lyophilization. 35
36
BOB D. JOHNSON, H. L. STAHNKE and R. KOONCE
Paramecium multimicronucleatum Powers and Mitchell 1910 LD 6o determinations were performed according to procedures described by JOHNSON et al. [2]. These LD6o are in mg of venom per ml of sterile wheat broth. Disc electrophoresis was used to compare the protein complexity of lyophilized and raw venoms . An eight column electrophoresis apparatus [3] was used as previously outlined [4]. RESULTS
The O.D.D values of the raw venom (diluted 1 : 400) from the individual rattlesnakes and the venom pool are recorded in Table 1 . These estimations show that raw venom concentration varies from individual to individual in this species. No correlations between rattlesnake weight and venom concentration were observed. After lyophilization, 0-10 ml raw venom volumes were weighed; these values, as shown in Table l, were in close agreement with the calculated raw venom concentrations .
In general, cell lysing ability increased with venom protein content. Exceptions to this were noted however. The P. multimicronucleatum LD ba determinations ranged, from 0-39 mg/ml for snake No. 5 to 0-89 mg/ml for snake No. 6. The complexity of the C. atrox venom pool, as evidenced by disc electrophoresis, is shown in Fig. 1 . Electropherograms of raw and lyophilized venoms from this pool were very similar. The electropherograms of each individual snake venom could be easily distinguished from the others .
1 2 3 4 5 6 venom Pool
Snake
364 432 325 402 601 1505 -
Wt. of snake (9)
1-061/0-694 0717/0-694 090110-694 0652/0-694 0959/0-694 0-700/0-694 0-694/0-694
O.D.D (1 :400) venom pool cone.
O.D.D (1 :400) of individual cone.
= = = = =
=
1-53 1-03 1-30 094 1-38 1 .01 1 .00
= Coefficient 400 400 400 400 400 400 400 x x x x x x x
1-53 1-03 1-30 0-94 1-38 1-01 1-00
x x x x x x x
0528 me/ml 0528 0528 0528 0528 0528 0528
400 x Coefficient x 1 : 400 cone. of venom pool
T~ 1. Crotalus atrox vENOM CONCENTRATION DEIII~TIONS
= = = = = = =
3232 217-6 274-4 199-4 291-6 213-2 211-2
3215 21-3 27-10 19-59 29-09 21-22 21-10
Calculated = individual raw Wt. of0. 1 ml venom cone. raw venom after (Mg/ml) lyop~tion
w
ó
p0
c) n k c
BOB D. JOHNSON, H. L. STAHNKE and
38
R.
KOONCE
DISCUSSION
It seems possible from these data that envenomation by a smaller adult C. atrox rattlesnake might be as serious as envenomation by a larger one . The differences in volume injected might be compensated for by the differences in raw venom concentration . Further, the volume of venom injected by C. atrox snakes can, for many reasons, be quite variable . The most important consideration is the amount of venom injected, thus the concentration of the venom is very important . Different venom concentrations may depend upon such factors as a snake's diet, frequency of feeding, recent previous envenomations, and health . The use of raw venoms with known concentrations could eliminate the necessity of drying and weighing venoms . The variability of per cent protein per unit weight of venom must be considered however. El', cm values of lyophilized venoms from individual adult C. atrox snakes showed variations of approximately 5 per cent. These variations were due, in part, to the hygroscopic nature of the venoms during weighing . Because of the extremely variable and complex nature of venoms, their cell lysing ability cannot, in all instances, be correlated to total protein content . Disc electrophoresis showed that venom protein components of individual snakes are, at least quantitatively, quite different, and that lyophilization does not appreciably alter the electrophoretic patterns of the venom protein components. This similarity in lyophilized and raw venoms might further indicate the feasibility of using raw venoms . Acknowledgements-We thank M . DUFFY, R. RODGERs, work was supported by N.I .H. Grant No . NB 02330.
and
J. HOPPE
for their technical assistance. This
REFERENCES R. F. and GILL, D. M., A micro-biuret method for estimating proteins . Analyt. Biochem. 9, 401, 1964 . [2] JOHNSON, B. D., TULLAR, J. C. and STAHNKE, H. L., A quantitative protozoan bioassay method for determining venom potencies . Toxicon 3, 297, 1966 . [3] DAvis, B. J., Disc electrophoresis II . Method and application to serum proteins . Ann. N. Y. Acad. Sci. 121,404, 1964 . [41 STAHNKE, H. L. and JOHNSON, B. D. Tarabtula venom (Aphonepelma, sp . In: Animal Toxins (Ed. by RUSSELL, F. E. and SAUNDERS, P. R. Oxford . Pergamon Press, 1967 . [1]
ITzHAm,
A METHOD FOR ESTIMATING CROTALUS ATROX VENOM CONCENTRATIONS BOB D. JOHNSON, H . L. STAHNKE and R. KOONCE Poisonous Animals Research Laboratory, Arizona State University, Tempe, Arizona, U.S.A . (Acceptedfor publication 18 January 1967)
Abstract-Venom from the rattlesnake, Crotalus atrox Baird and Girard 1853, was studied. The procedure for estimating raw venom concentration showed that this snake's venom concentration is not dependent upon its size . Intraspecific variation in venom complexity was shown on disc electrophoresis . INTRODUCTION
studies concerned with the secretions of rattlesnake venoms, raw venoms are infrequently used and the use of weighed dried venoms has predominated . Use of raw venoms of known concentrations might conserve time, if concentrations could be ascribed to a specific property or activity. Venom from the Western diamondback rattlesnake Crotalus atrox Baird and Girard 1853 was studied. We report here procedures for estimating the concentration of raw venom. Relationships existing between venom protein and paramecia lethality (cell lysing ability) are briefly examined . Disc electropherograms of lyophilized and raw venoms are compared . IN MosT
MATERIALS AND METHODS
Samples of raw venom from individual C. atrox snakes were extracted directly into large centrifuge tubes. These were immediately centrifuged to remove debris . Each sample was then divided into 0-05 ml aliquots . The tubes containing these aliquots were tightly plugged with cotton and stored at -20° until used. The raw venom pool was prepared by combining an equal number of aliquots from each snake . All protein estimations were performed by the method Of ITZHAM and GILL [1]. Prior to adding 1 ml of reagent 1 (0-21 per cent CuSO4 - 5H 9 0 in 30 per cent NaOH) or 1 ml of reagent 2 (30 per cent NaOH), 0-05 ml of pooled raw venom was diluted to 20 ml (1 : 400 dilution) with distilled water. 1 : 800 and 1 : 1600 dilutions permitted a linear curve to be plotted. In order to calculate the concentration of venom, the O.D.D (optical density difference) values from this line were compared to O.D .D values obtained by using lyophilized samples with known concentrations from the pooled venom. Individual snake venom concentrations were determined by comparing their 1 : 400,1 : 800 and 1 :1600 O.D .D values to those obtained with the venom pool. The calculated concentrations of the (0-05 ml) raw venom samples were confirmed by weighing after lyophilization. 35
36
BOB D. JOHNSON, H. L. STAHNKE and R. KOONCE
Paramecium multimicronucleatum Powers and Mitchell 1910 LD 6o determinations were performed according to procedures described by JOHNSON et al. [2]. These LD6o are in mg of venom per ml of sterile wheat broth. Disc electrophoresis was used to compare the protein complexity of lyophilized and raw venoms . An eight column electrophoresis apparatus [3] was used as previously outlined [4]. RESULTS
The O.D.D values of the raw venom (diluted 1 : 400) from the individual rattlesnakes and the venom pool are recorded in Table 1 . These estimations show that raw venom concentration varies from individual to individual in this species. No correlations between rattlesnake weight and venom concentration were observed. After lyophilization, 0-10 ml raw venom volumes were weighed; these values, as shown in Table l, were in close agreement with the calculated raw venom concentrations .
In general, cell lysing ability increased with venom protein content. Exceptions to this were noted however. The P. multimicronucleatum LD ba determinations ranged, from 0-39 mg/ml for snake No. 5 to 0-89 mg/ml for snake No. 6. The complexity of the C. atrox venom pool, as evidenced by disc electrophoresis, is shown in Fig. 1 . Electropherograms of raw and lyophilized venoms from this pool were very similar. The electropherograms of each individual snake venom could be easily distinguished from the others .
1 2 3 4 5 6 venom Pool
Snake
364 432 325 402 601 1505 -
Wt. of snake (9)
1-061/0-694 0717/0-694 090110-694 0652/0-694 0959/0-694 0-700/0-694 0-694/0-694
O.D.D (1 :400) venom pool cone.
O.D.D (1 :400) of individual cone.
= = = = =
=
1-53 1-03 1-30 094 1-38 1 .01 1 .00
= Coefficient 400 400 400 400 400 400 400 x x x x x x x
1-53 1-03 1-30 0-94 1-38 1-01 1-00
x x x x x x x
0528 me/ml 0528 0528 0528 0528 0528 0528
400 x Coefficient x 1 : 400 cone. of venom pool
T~ 1. Crotalus atrox vENOM CONCENTRATION DEIII~TIONS
= = = = = = =
3232 217-6 274-4 199-4 291-6 213-2 211-2
3215 21-3 27-10 19-59 29-09 21-22 21-10
Calculated = individual raw Wt. of0. 1 ml venom cone. raw venom after (Mg/ml) lyop~tion
w
ó
p0
c) n k c
BOB D. JOHNSON, H. L. STAHNKE and
38
R.
KOONCE
DISCUSSION
It seems possible from these data that envenomation by a smaller adult C. atrox rattlesnake might be as serious as envenomation by a larger one . The differences in volume injected might be compensated for by the differences in raw venom concentration . Further, the volume of venom injected by C. atrox snakes can, for many reasons, be quite variable . The most important consideration is the amount of venom injected, thus the concentration of the venom is very important . Different venom concentrations may depend upon such factors as a snake's diet, frequency of feeding, recent previous envenomations, and health . The use of raw venoms with known concentrations could eliminate the necessity of drying and weighing venoms . The variability of per cent protein per unit weight of venom must be considered however. El', cm values of lyophilized venoms from individual adult C. atrox snakes showed variations of approximately 5 per cent. These variations were due, in part, to the hygroscopic nature of the venoms during weighing . Because of the extremely variable and complex nature of venoms, their cell lysing ability cannot, in all instances, be correlated to total protein content . Disc electrophoresis showed that venom protein components of individual snakes are, at least quantitatively, quite different, and that lyophilization does not appreciably alter the electrophoretic patterns of the venom protein components. This similarity in lyophilized and raw venoms might further indicate the feasibility of using raw venoms . Acknowledgements-We thank M . DUFFY, R. RODGERs, work was supported by N.I .H. Grant No . NB 02330.
and
J. HOPPE
for their technical assistance. This
REFERENCES R. F. and GILL, D. M., A micro-biuret method for estimating proteins . Analyt. Biochem. 9, 401, 1964 . [2] JOHNSON, B. D., TULLAR, J. C. and STAHNKE, H. L., A quantitative protozoan bioassay method for determining venom potencies . Toxicon 3, 297, 1966 . [3] DAvis, B. J., Disc electrophoresis II . Method and application to serum proteins . Ann. N. Y. Acad. Sci. 121,404, 1964 . [41 STAHNKE, H. L. and JOHNSON, B. D. Tarabtula venom (Aphonepelma, sp . In: Animal Toxins (Ed. by RUSSELL, F. E. and SAUNDERS, P. R. Oxford . Pergamon Press, 1967 . [1]
ITzHAm,