Geographic and ontogenic variation in venom of the western diamondback rattlesnake (Crotalus atrox)

Toxicon, Vol. 24, No. 1, pp. 7 1 - 8 0 , 1986. Printed in Great Britain.

0041-0101/86 $3.00+ .00 © 1986 Pergamon Press Ltd.

G E O G R A P H I C A N D O N T O G E N I C V A R I A T I O N IN V E N O M OF THE W E S T E R N D I A M O N D B A C K R A T T L E S N A K E (CROTALUS

A TR OX) SHERMAN A. MINTON a n d ScoTT A. WEINSTEIN* Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN 46223, U.S.A.

(Accepted for publication 30 May 1985) S. A. MINTONand S. A. WEINSTEXN.Geographic and ontogenic variation in venom of the western diamondback rattlesnake (Crotalus atrox). Toxicon 24, 71- 80, 1986. - - Venom samples from western diamondback rattlesnakes (Crotalus atrox) from 13 localities in the United States were tested for i.v. and s.c. lethality for mice, protease activity, hemorrhagic activity, and the presence of Mojave toxin. Electrophoresis on polyacrylamide gel was used to compare protein composition. The neutralizing effect of two commercial antivenoms was evaluated against selected samples of venom. Venom of young snakes from north Texas was compared with that of adults from the same locality. Venom samples from the southwest portion of the range showed highest lethality, those from the northeast portion lowest. This trend was reversed with respect to protease activity. Hemorrhagic activity showed little geographic variation, but northern samples tended to be slighly higher. Differences in venom protein composition were evident between snakes from the eastern and western portions of the range. Mojave toxin in small to trace amounts was detected in two Arizona venom samples and one from west Texas. Antivenoms were relatively ineffective in neutralizing lethality. Venom of young snakes from north Texas was much more lethal by s.c. injection than that of adult snakes from any part of the range, but very low in protease activity. Hemorrhagic activity was about equal to that of adult snakes from the same region. Fifteen months later, lethality had declined almost five-fold, and protease activity had approached adult levels. There was a distinct change in protein composition. Mojave toxin was not detected in venoms of the young snakes. INTRODUCTION THE WESTERN DIAMONDBACK RATTLESNAKE (Crotalus atrox) is one o f the largest o f rattlesnakes. W i d e l y d i s t r i b u t e d a n d o f t e n a b u n d a n t , it is the leading cause o f serious a n d fatal snakebites in the s o u t h w e s t e r n U n i t e d States a n d p r o b a b l y in n o r t h e r n Mexico. Its r a n g e extends f r o m central A r k a n s a s to s o u t h e a s t e r n C a l i f o r n i a t h r o u g h nearly all o f Texas to Zacatecas a n d n o r t h e r n parts o f the states o f Veracruz, Q u e r e t a r o , a n d S i n a l o a with isolated p o p u l a t i o n s in C h i a p a s a n d Oaxaca. While there is m u c h i n f o r m a t i o n o n the b i o c h e m i s t r y , toxicology a n d p h a r m a c o l o g y o f Crotalus atrox v e n o m (ELLIOTT, 1978; MEBS, 1978; LEE, 1979), there has b e e n n o investigation o f geographical v a r i a t i o n in its v e n o m despite the species' extensive d i s t r i b u t i o n . I n this p a p e r we c o m p a r e C. atrox v e n o m samples f r o m v a r i o u s parts o f the U.S. range with respect to lethality, h e m o r r h a g i c activity, protease activity, presence o f M o j a v e toxin, a n d capacity to be neutralized b y a n t i v e n o m . C o n s t r a i n t s o f time a n d difficulty in o b t a i n i n g collecting permits prevented o u r i n c l u d i n g M e x i c a n p o p u l a t i o n s in this study. *Present address: Department of Microbiology, Sackler Institute, New York University School of Medicine, 550 First Avenue, New York, NY 10016, U.S.A.

71

72

S . A . MINTON and S. A. WEINSTEIN

Iowa N e b r a s k a

Nevada

L/

Utah

Colorad

so

o

o\

Kansas

%

Ariz~n a el x

lssour

/

Oklahoma

t N ewl /

M e x i c

ho

e2

e8 Texas

/ (/ t j

FIG.

1. DISTRIBUTION OF Crotalus a t r o x IN THE WESTERN UNITED STATES LOCALITIES FOR SNAKE SAMPLES MENTIONED IN TEXT..

(broken line)

AND

(1) Maricopa Co., Arizona (MZ), 3 snakes, sample lyophilized. (2) Tucson, Arizona (TZ), 2 snakes, samples air-dried. (3) Portal, Arizona (PZ), 3 snakes, sample air-dried. (4) Presidio Co., Texas (PT), 1 snake, sample lyophilized. (5) Big Bend, Texas (BB), 8 snakes, sample air-dried, (6) Valverde Co., Texas (VV), 3 snakes, sample lyophilized. (7) Freer, Texas (FT), >50 snakes, sample lyophilized (8) Sweetwater, Texas (SW), >50 snakes, sample lyophilized. (9) Archer Co., Texas (AC), >100 snakes, sample lyophilized. (10) Young Co., Texas (YC), >50 snakes, sample lyophilized. (11) Clay Co., Texas (CC), >50 snakes, sample lyophilized. (12) Harmon Co., Oklahoma (HO), 1 snake, sample lyophilized. (13) Waynoka, Oklahoma (WO), >50 snakes, sample lyophilized. MATERIALS AND METHODS All venoms were collected by us or by reliable individuals known to us. Except for localities represented by one animal, all venom samples are pools of the venom of all snakes available from that locality. Venom from snakes less than about 60 cm in total length were not included in the general samples, but two samples from juvenile snakes were collected and examined separately. Localities of collection and other information are given in Fig. 1. Lethality was determined by i.v. and s.c. injection into male white mice of 20 - 25 g weight using groups of four animals at each dose level. The LDso was calculated by the Spearman-Karber method (WORLD HEALTH ORGANIZATION, 1981). Hemorrhagic activity was determined by a modification of the method of KONIX) et al. (1960). White rats weighing 300-350 g were anaesthetized with pentobarbital and injected intradermally with doses of venom ranging from 1 to 50/~g. Animals were sacrificed after 18 hr, skinned, and the diameter of the hemorrhagic areas on the inner surface of the skin was recorded. The minimum hemorrhagic dose (MHD) was defined as the least amount of venom producing a hemorrhagic zone approximately 5 mm in diameter. Protease activity was determined by use of a protein gel substrate (Bio-Rad Laboratories, Richmond, CA). Venom solutions from 0.125 mg/ml to 1.0 mg/ml were added to wells cut in the gel plate, and zones of clearing were measured after 18 hr. A trypsin solution (0.1 mg/ml) similarly diluted was used as a standard. In antivenom neutralization tests, venom solutions containing 30 to 40 s . c . LDso/mi were mixed with an equal volume of antivenom or 50°7o normal human gamma globulin and the mixture incubated for 30 min at 37°C. Following incubation, 0.1 ml doses (containing 1.5 or 2 LDso) were injected s.c. into 2 0 - 24 g male white mice, 4 animals per group. The number of survivors after 48 hr was recorded. The antivenoms used were recently manufactured packages of Antivenin Crotalidae Polyvalent (Wyeth, U.S.A.) and Suero Antiviperino (Laboratorios MYN, Mexico).

Variation in Crotalus atrox V e n o m

73

Selected venom a n d toxin samples were analyzed on sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) according to the m e t h o d of LAEMMLI (1970) using a 15 x 9 x 0.15 cm gel slab with a 10-20°/0 gradient. Samples (30/~g) were incubated at 100°C for 2 min in T r i s - H C I buffer, pH 6.8, containing sodium dodecyl sulfate, glycerol, a n d bromphenol blue. Electrophoresis was initiated at 50 V for 30 min, then continued at 100 V for approximately 4 hr until the dye neared the bottom o f the gel. Proteins were stained with 0. 1% Coomassie brilliant blue for 1 8 - 20 hr, followed by destalning in m e t h a n o l - acetic acid. Presence of the highly lethal polypeptide toxin known as Mojave toxin (BIEBER et al., 1975) was determined by use of an ELISA assay using a n t i t o x i n - enzyme conjugate. The antitoxin was produced against toxin isolated from venom of a Crotalus scutulatus known to have type A venom (GLENN and STRAIGHT, 1978). For further details of this assay see Mitcro~4 et al. (1984). A n additional assay for Mojave toxin was carried out using the Western transfer technique by the m e t h o d of BATTEIGER et al. (1982). In this technique, proteins resolved by polyacrylamide gel electrophoresis are electrophoretically transferred to nitrocellulose m e m b r a n e and antiserum added. Antibody-bound antigen bands are then developed with laSI-labelled staphylococcal protein A, a n d an autoradiogram is produced on photographic film. Sensitivity of this m e t h o d is comparable to that of ELISA.

RESULTS

Data on lethality by i.v. and s.c. routes of inoculation are shown in Table 1. While the mean s.c. LDso for all groups (17.0 m g / k g ) is close to values previously reported, the mean i.v. LDso (2.50 m g / k g ) is lower (GLENN and STRAIGHT, 1982). There is a trend toward higher lethality in western populations, the mean i.v. and s.c. LDso values for three Arizona samples being 2.4 and 13.4, while corresponding values for five north Texas and O k l a h o m a samples are 3.1 and 18.1 m g / k g . However, the most lethal C. atrox venoms are those produced by young snakes. A venom sample f r o m two north Texas snakes collected in March 1983 and probably young of the previous summer had a s.c. LDso of 2.68 m g / k g , about 6.6 times as toxic as v e n o m f r o m adult snakes f r o m the same area. Another juvenile o f about the same size but o f unknown provenance had v e n o m with a s.c. LDso o f 2.84 m g / k g . Fifteen months later, at an age o f about two years, the north Texas snakes produced v e n o m with a s.c. LDso o f 12.9 m g / k g , still appreciably more lethal than that o f adult snakes (Table 2). The adult sample with highest s.c. lethality came f r o m snakes

TABLE l. LETHAL TOXICITY, MINIMUM HEMORRHAGIC DOSE, AND PROTEASE ACTIVITY OF ADULT Crotalus atrox VENOM SAMPLESFROM VARIOUSLOCALITIES

Locality Big Bend, T X Valverde Co., T X Presidio Co., T X Freer, T X Sweetwater, TX Archer Co., TX Clay Co., T X Young Co., T X W a y n o k a , OK H a r m o n Co., OK Portal, A Z Tucson, A Z Maricopa Co., A Z Mean ± S.D.

i.v. 1.87 0.92 1.30 2.62 2.93 2.55 3.37 2.71 4.21 2.87 2.37 3.06 1.77 2.50 ± 0.84

s.c. 7.8 17.8 22.8 17.5 24.5 17.7 16.5 16.9 22.0 17.5 13.1 15.6 11.5 17.0 ± 4.4

s.c./i.v.

Minimum hemorrhagic dose ~g)*

Per cent protease activity t

4.2 19.3 17.5 6.7 8.4 6.9 4.9 6.2 5.2 6.1 5.5 5.1 6.5

6 . 2 5 - 6.25 1.2 - 3.1 n.d. 6.25 - 12.5 1.2 - 3.1 3.1 - 3.1 3.1 - 3.1 1.0 - 3.1 1.0 - 3.1 6 . 2 5 - 6.25 3.1 - 8.0 8.0 - 12.5 4.0 - 8.0

54-70 57-69 66 - 70 54 - 59 8 8 - 91 72-76 79-85 90-95 8 0 - 87 81-92 29-34 2 3 - 29 19-31

*Two determinations. tArea of clear zone produced by I0/~g v e n o m / a r e a of clear zone produced by l /~g trypsin × I00. Two determinations.

74

S. A . M I N T O N a n d S. A. W E I N S T E I N TABLE 2. LETHAL TOXICITY, MINIMUM HEMORRHAGIC DOSE, AND PRO'lEASE ACTIVITY OF JUVENILE Crotalus atrox VENOMS

Locality N o r t h Texas, 7 - 8 m o n t h s age N o r t h Texas 2 2 - 23 m o n t h s age U n k n o w n locality 7 - 8 m o n t h s age N o r t h Texas a d u l t snakes*

s.c. LDso ( m g / k g )

Minimum hemorrhagic dose ~ g ) *

Per cent protease activity*

2.68

3.12

17 - 22

12.86

1.56

7 0 - 78

2.84

n.d.

16 - 22

17.03

1.0 - 3.1

72 - 95

*Single d e t e r m i n a t i o n s . 1. T w o d e t e r m i n a t i o n s . *Data f r o m T a b l e 1.

*See T a b l e

collected in the Big Bend region of Texas, most of them around the base of the Rosillos Mountains. Two other samples from the Rio Grande valley of west Texas were aberrant in having s.c. LDsovalues that were 17 - 19 times the i.v. LDso, in contrast to 4 - 8 times for all other samples. One of these (VV) had the highest i.v. lethal potency of any sample examined. Variation in minimal hemorrhagic dose (MHD) showed no clear geographic trends. Five of six samples from north Texas and Oklahoma had MHD values of less than 5/~g, while three Arizona samples had MHD values of more than 5/~g. A south Texas sample (FT) also had a comparatively high MHD. Low MHD values (1.56-3.12 /~g) were characteristic of the juvenile snakes. Our minimal hemorrhagic doses were larger than those reported by OHSAKA et al. (1966), however, they used rabbits rather than rats for assay. BJARNASON and Tu (1978) reported isolation of five hemorrhagic toxins from C. atrox venom. Protease activity showed a tendency to decrease from east to west, with the samples tending to fall into three groups (Fig. 2). Greatest activity was seen in the two Oklahoma samples and three samples (CC, YC, SW) from north Texas. Least activity was shown by the three Arizona samples. Intermediate activity was seen in three Texas samples from the Rio Grande drainage (BB, PT, VV), a south Texas sample (FT), and a north Texas sample (AC). Venom of north Texas juvenile snakes collected in March 1983 had low protease activity, about 25°7o that of adult snakes from the same locality. In June 1984 venom of these snakes had protease activity only a little lower than that of adults. The high protease activity of C. atrox on casein substrates was reported by OSHIMA et al. (1969) and KOCHOLATY et al. (1971) and was associated with high bradykinin-releasing and clotting activity. ELLIOTT (1978) commented on the lack of correlation between hemorrhagic and protease activity. Results of antivenom neutralization tests showed a disconcerting failure of either Wyeth Polyvalent Crotalid Antivenin or the Mexican Suero Antiviperino to neutralize C. atrox venom at a dose of 2 s.c. LDso. When the dose was reduced to 1.5 LDso, there was some indication of protection, particularly with the Wyeth antivenom. There was no indication of geographic variation in the pattern of neutralization (Table 3). If results for all samples at the 1.5 LDso level are pooled, there are 13 survivors of 20 in the Wyeth

75

Variation in Crotalus otrox Venom

Iooo "I

.~

.F~ T

F . v , ptA

cw.sv

7

500 -

250 -

// 8_ e

F~v~ I 6

I 7

Diameter

I 8

I 9

of Clear

.wc I I0

ill

,,O0..,m,I

YS I 12

I ~ I

Z o n e in G e l

I 14

I 15

I 16

I 17

I 18

(mm)

FIG. 2. PROTEASE ACTIVITY OF ADULT C. o f r o x VENOMS (l m g / m l ) COMPARED WITH A SOLUTION OF TRYPSIN ( 0 . l mg) SIMILARLY DILUTED. Venom concentration = 10x trypsin concentration. Each point represents the mean of two determinations. Letters refer to the following samples: M = Maricopa Co., Arizona; Pz = Portal, Arizona; T = Tucson, Arizona; F = Freer, Texas; B = Big Bend, Texas; V = Valverde Co., Texas; Pt = Presidio Co., Texas; A = Archer Co., Texas; C = Clay Co., Texas; W = Waynoka, Oklahoma; H = H a r m o n Co., Oklahoma; S = Sweetwater, Texas; Y = Young Co., Texas.

TABLE 3. NEUTRALIZATION OF SELECT Crotalus atrox VENOM SAMPLES BY TWO ANTIVENOMS. CHALLENGE BY S.C. INJECTION

Venom sample and dose (units of LDso) Antiserum

Wyeth Antivenom MYN Antivenom H u m a n IgG

FT

AC

WO

MZ

PZ

2.0

1.5

2.0

1.5

2.0

1.5

2.0

1.5

2.0

1.5

4/4* 3/4 3/4

0/4 1/4 4/4

4/4 4/4 4/4

1/4 n.d. 4/4

4/4 4/4 4/4

2/4 4/4 3/4

4/4 4/4 4/4

3/4 3/4 3/4

1/4 2/4 4/4

1/4 3/4 4/4

*Number of deaths/number of animals injected. n.d. = not determined.

treated group and 5 of 16 in the MYN treated group. The X2 test indicates significant protection with Wyeth antivenom, but not with MYN antivenom Differences in SDS-PAGE patterns were found among all the eight samples examined (Figs. 3 and 4). Band patterns of the three Arizona Samples (MZ, PZ, TZ) were very similar and characterized by a particularly heavy band indicating a protein with a molecular weight of about 60,000. This band was present in other samples, but was less strongly defined. Samples from Oklahoma (WO), north Texas (AC), and south Texas (FT) showed great similarity with one another, but were distinctly different from Arizona samples. A sample from the upper Rio Grande valley (VV) showed the greatest number of bands, including some not found in other samples. Venom of snakes 7 - 8 months old lacked at least six bands seen in venoms of adult snakes, but had two bands lacking or

76

S.A. MINTONand S. A. WEINSTEIN

very weakly developed in adult venoms. These same snakes at an age of 2 2 - 23 months produced venom showing seven additional bands, but two bands present earlier were not detected (Fig. 4). Assay of all C. atrox samples for Mojave toxin using the ELISA technique showed its presence in very low concentration in two Arizona samples, MZ and PZ. With SDSPAGE these samples showed bands of the same molecular weight as isolated Mojave toxin (Fig. 4). These were not seen in other samples. Use of the Western transfer technique confirmed presence of the toxin in these samples and indicated a higher concentration in MZ. Additionally, a very weak reaction was seen with the BB sample (Fig. 5). Venom of north Texas juvenile snakes was negative for Mojave toxin by the ELISA and Western transfer techniques. DISCUSSION In this investigation of Crotalus atrox venom samples from 13 localities within the U.S. range of the species, two trends axe detectable. One is an increase in protease activity toward the northeastern portion of the range; the other an increase in lethality toward the southwestern portion. Hemorrhagic activity showed no clear-cut geographic trend, although the samples with lowest activity were from western and southern localities, while those with highest activity were from northern localities. Mojave toxin, in small to trace amounts, was detected in two of three Arizona samples and a sample from west Texas. The inverse relationship between protease activity and lethality is similar to that reported by GLENN et al. (1983) between populations of C. scutulatus with type A venom and those with type B venom. However, the high lethality of type A scutulatus venom apparently depends upon the presence of Mojave toxin, and we have little evidence that this is the case with C. atrox. Mojave toxin does seem to be present in most of the highly lethal samples, but only in extremely small amounts, moreover, it is absent in the most lethal sample, that of venom of juvenile snakes from north Texas. Mojave toxin may have been a component of the venom of the common ancestor of scutulatus and atrox with subsequent selection pressures tending to conserve it in scutulatus and eliminate it in atrox. An alternative hypothesis is that of occasional genetic exchange between scutulatus and atrox where their ranges overlap (JACOB, 1977). In all localities where atrox venom contained Mojave toxin, C. atrox is sympatric with a C. scutulatus population whose venom contains high concentrations of Mojave toxin (WEINSTEINet al., 1985). This study confirms earlier observations of marked differences between venoms of young and adult rattlesnakes (MINTON, 1967; FIERO et al., 1972; REID and THEAKSTON, 1978). Venom of young snakes from north Texas was much more lethal than that of adults from the same locality, but had only about one fourth the proteolytic activity. The minimum hemorrhagic dose was approximately equal to that of venom of adult snakes, and tests for Mojave toxin were negative. Venom of the same snakes collected 15 months later had about one-fifth the lethality of the earlier sample, but was still more toxic than venom of adult snakes, and had adult proteolytic activity. Polyacrylamide gel electrophoresis indicated changes in at least nine venom proteins, with loss of two juvenile bands and gain of seven adult bands. JOHNSON (1968) observed lower protein content in venom of juvenile C. atrox in comparison with adults and differences in disc electrophoresis patterns between juveniles and adults, as well as differences among adults. The geographical origin of his specimens was not given. Polyacrylamide gel electrophoresis also indicated differences between eastern and western populations of C. atrox. Western samples showed heavier bands in the 60,000

77

FT AC

WO

BB

TZ

VV

68

24 13

03 ¢0

'~" 00

,-j

-,3

i ~i~i!~iii iii!~i!ii I~ !~il

~ i

1

2

3

4

~8

34 ;.4

18 3

FIG.

3.

SDS-POLYACRYLAMIDE GEL PATTERNS OF SIX REPRESENTATIVE C. SAMPLES.

a t r o x ADULT VENOM

For letter designations of venoms see Fig. 1. N u m b e r s on right are molecular weights (in thousands) of protein standards. FIG. 4 . SDS-POLYACRYLAM1DE GEL PATTERNS COMPARING VENOMS OF C. a t r o x AGE

7 - 8 MONTHS

(J83) AND 22-- 23 MONTHS (J84) AND DEMONSTRATINGPRESENCE OF MOJAVETOXIN. N u m b e r s refer to the following: (1) Type A venom of C. scutulatus; (2) Mojave toxin isolated from this venom; (3) V e n o m of C. atrox, Portal, Arizona; (4) Venom of C. atrox, Maricopa Co., Arizona. Arrow shows Mojave toxin band. N u m b e r s on right are molecular weights of protein standards. Circles in lane 2 are an artefact.

78

FIG 5. AUTORADIOGRAPHRESULTINGFROM WESTERNTRANSFER OF VENOMS DEVELOPED WITH ANTIMOJAVE TOXIN AND txSI-PROTEIN m. Twenty microgram samples were electrophoresed on SDS-polyacrylamide gel and the resolved proteins transferred electrophoretically to nitrocellulose paper. This paper was developed with anti-Mojave toxin antiserum and ~25I-protein A. The t2sI was then detected by autoradiography. Venoms in numbered lanes are as follows: (1) C. scutulatus, Type A;(2) AC;(3) CC;(4) YC;(5) VV;(6) FT;(7) PZ;(8) MZ;(9) WO;(10) MO;(11) J83;(12) J84;(13) BB;(14)TZ. Arrows indicate Mojave toxin bands.

Variation in Crotalus atrox Venom

79

r e g i o n , lighter b a n d s in a p p r o x i m a t e l y t h e 50,000 r e g i o n , a n d fewer b a n d s in t h e r e g i o n b e l o w 13,000. These b a n d s m a y r e p r e s e n t p r o t e i n s a s s o c i a t e d with lethality a n d p r o t e a s e activity, w h i c h v a r y inversely f r o m west to east. O n l y m i n o r d i f f e r e n c e in p r o t e i n c o m p o s i t i o n were seen b e t w e e n v e n o m s o f O k l a h o m a a n d s o u t h T e x a s snakes. T h e e x t r a o r d i n a r i l y large n u m b e r o f p r o t e i n s in the VV s a m p l e f r o m west T e x a s is an a n o m a l y f o r w h i c h we have no e x p l a n a t i o n . T w o c o m m e r c i a l a n t i v e n o m s used clinically in areas where bites b y C. a t r o x are relatively f r e q u e n t failed to c o n f e r significant p r o t e c t i o n w h e n a v e n o m d o s e o f 2 LDso was i n c u b a t e d with 0.1 ml o f a n t i v e n o m a n d the m i x t u r e i n j e c t e d s.c. i n t o mice. W h e n the d o s e was r e d u c e d t o 1.5 LDso, the P o l y v a l e n t C r o t a l i d A n t i v e n i n ( W y e t h , U S A ) p r o v i d e d p a r t i a l b u t significant p r o t e c t i o n ; S u e r o A n t i v i p e r i n o ( L a b o r a t o r i e s M Y N , M e x i c o ) d i d n o t s h o w p r o t e c t i o n . C. a t r o x v e n o m is one o f f o u r v e n o m s u s e d to i m m u n i z e horses in p r o d u c t i o n o f the W y e t h a n t i v e n o m , a n d is also used in p r o d u c t i o n o f the M Y N a n t i v e n o m (CHIPPAUX a n d GYFFON, 1983). T h e low n e u t r a l i z i n g c a p a c i t y o f these a n t i v e n o m s implies t h a t at least s o m e o f t h e v e n o m c o m p o n e n t s r e s p o n s i b l e for s.c. l e t h a l i t y are p o o r antigens. This s h o u l d be t a k e n into a c c o u n t b y m a n u f a c t u r e r s o f a n t i v e n o m , as well as b y clinicians w h o t r e a t e n v e n o m a t i o n b y the western d i a m o n d b a c k rattlesnake. W e are well a w a r e t h a t n o t all o u r v e n o m s a m p l e s a r e e q u a l l y r e p r e s e n t a t i v e o f the p o p u l a t i o n s f r o m which t h e y c a m e . O n e O k l a h o m a a n d five T e x a s s a m p l e s were f r o m large n u m b e r s o f snakes t a k e n in spring at h i b e r n a t i n g sites. T h e r e m a i n i n g seven s a m p l e s were f r o m 1 - 8 r a n d o m l y collected snakes. A l t h o u g h snakes u n d e r 60 c m were e x c l u d e d f r o m the s a m p l e s , the BB s a m p l e i n c l u d e d t h r e e snakes in t h e 6 5 - 75 c m size g r o u p , a f a c t o r t h a t m a y e x p l a i n its high s.c. lethality. T h e small size o f the A r i z o n a s a m p l e s is u n f o r t u n a t e , h o w e v e r , their p r o p e r t i e s are c o n g r u e n t with o n e a n o t h e r , a n d we believe the o b s e r v e d diferences b e t w e e n t h e m a n d the e a s t e r n s a m p l e s are significant. Acknowledgments - - This work was supported by a grant from Wyeth Laboratories Inc. who also supplied a

sample of antivenin. Mexican antivenin was supplied by BROOKSM. DE CERVANTES,Queretaro, Mexico. For gifts of snakes and venom samples we thank CARLOSBONILLA,Colorado State University, DAVIDHARDY, Tucson, Arizona, MARVINHENRY, Archer City, Texas, VICTORHUTCHINSON,University of Oklahoma, FRED KRAUS, University of Michigan Museum of Zoology, ROBERTMORRISON,Shelbyville, Indiana, and JOHN C. PEgEZ, Texas A and I University. CHARLESE. WILDE, Indiana University School of Medicine, gave much helpful advice and loaned items of equipment. BERNICEELLISprovided helpful technical assistance. Figures were prepared with the aid of the Illustration Department, Indiana University Medical Center. Thanks to KAREN COFFMANfor typing the manuscript.

REFERENCES BATTEIGER,B., NEWHALL,W. J. V. and JONES, R. B. (1982) The use of Tween-20 as a blocking agent in the immunological detection of proteins transferred to nitrocellulose. J. immun. Meth. 55, 297. BEIBER, A. L., Tu, T. and Tu, A. T. (1975) Studies of an acidic cardiotoxin isolated from the venom of the Mojave rattlesnake (Crotalus scutulatus). Biochim. biophys. Acta 400, 178. BJARNASON,J. B. and Tu, A. T. (1978) Isolation and characterization of five hemorrhagic toxins from western diamondback rattlesnake (Crotalus atrox) venom and the role of zinc in hemorrhagic toxin e. Biochemistry 17, 3395. CHIPPAUX, J. P. and GOYFFON,M. (1983) Producers of antivenomous sera. Toxicon 21, 739. ELLIOTT, W. B. (1978) Chemistry and immunology of reptilian venoms. In: Biology o f the Reptilia, Vol. 8, Physiology, 8, p. 163 (GANS,C. and GANS, K. A., Eds). London: Academic Press. FIERO, M. K., SIEFERT,M. W., WEAVER,T. J. and BONILLA,C. A. (1972) Comparative study of juvenile and adult prairie rattlesnake (Crotalus viridis viridis) venoms. Toxicon 10, 81. GLENN, J. L. arid STRAIGHT,R. C. (1978) Mojave rattlesnake Crotalus scutulatus scutulatus venom: variation in toxicity with geographical origin. Toxicon 16, 81.

80

S . A . MINTON and S. A. WEINSTEIN

GLENN, J. L. and STRAIGHT, R. C. (1982) The rattlesnakes and their venom yield and lethal toxicity. In: Rattlesnake Venoms Their Action and Treatment, p. 3 (Tu, A. T., Ed.). New York: Marcel Dekker. GLENN, J. L., STRAIGHT, R. C., WOLFE, M. C. and HARDY, D. L. (1983) Geographical variation in Crotalus scutulatus scutulatus (Mojave rattlesnake) venom profiles. Toxicon 21, 119. JACOB, J. S. (1977) An evaluation of the possibility of hybridization between the rattlesnake Crotalus atrox and C. scutulatus in the southwestern United States. SWest. Nat. 22, 469. JOHNSON, B. D. (1968) Selected crotalidae venom properties as a source of taxonomic criteria. Toxicon 6, 5. KOCHOLATY, W. F., LEDFORD, E. B., DALY, J. G. and BILLINGS, T. A. (1971) Toxicity and some enzymatic properties and activities in the venoms of Crotalidae Elapidae, and Viperidae. Toxicon 9, 131. KONDO, H., KONDO, S,, IKEZAWA,H., MURATA,R. and OHSAKA,A. (1960) Studies on the quantitative method for determination of hemorrhagic activity of habu snake venom. Jpn. J. reed. Sci. Biol. 13, 43. LAEMML1, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, Lond. 227, 680. LEE, C. Y. fled.) (1979) Snake Venoms. Berlin: Springer Verlag. MEBS, D. (1978) Pharmacology of reptilian venoms. In: Biology o f the Reptilia Vol. 8, Physiology B, p. 437 (GANS, C. and GANS, K. A., Eds). London: Academic Press. MINTON, S. A. (1967) Observations on toxicity and antigenic makeup of venoms from juvenile snakes. In: Animal Toxins, p. 211 (RUSSELL, F. E. and SAUNDERS,P., Eds). Oxford: Pergamon Press. MINI'ON, S. A., WEINSTEIN, S. A. and WILDE, C. E. (1984) An enzyme-linked immunoassay for detection of North American pit viper venoms. J. Toxic. clin. Toxic. 22, 303. OHSAKA, A., OMORI-SATo-T., KONDO, H., KONDO, S. and MURATA, R. (1966) Biochemical and pathological aspects of hemorrhagic principles in snake venoms with special reference to habu (Trimeresurusflavoviridis) venom. Meres Inst. Butantan, Simp, Int. 33, 193. OSHIMA, G., SATO-OHMORI,T. and SUZUKI, T. (1969) Proteinase, arginine esterase hydrolase and a kininreleasing enzyme in snake venoms. Toxicon 7, 229. REID, H. A. and THEAKSTON, R. D. G. (1978) Changes in coagulation effects by venoms of Crotalus atrox as snakes age. Am. J. trop. Med, Hyg, 27, 1053. WEINSTEIN,S. A., MINTON,S. A. and WILDE,C. E. (1985) The distribution among ophidian venoms of a toxin isolated from the venom of the Mojave rattlesnake (Crotalus scutulatus scutulatus). Toxicon 23, (Tox 977). WORLD HEALTH ORGANIZATION (1981) Progress in the characterization of venoms and standardization of antivenoms, WHO Offset Pubs No. 58, p. 23.