- Email: [email protected]
A crotoxin homolog from the venom of the uracoan rattlesnake (Crotalus vegrandis)
T ~ n , Vol. 25, NO. 10, pp. 1113-1120, 1997. Primed in Great Britain.
0041-0101/87 $3.00+ .00 © 19ff~PergamonJournalsLtd.
A CROTOXIN HOMOLOG FROM THE VENOM OF THE URACOAN RATTLESNAKE (CROTALUS VEGRANDIS)
IVAN I. KAISER and STEVEN D. AIRD Department of Molecular Biology, University Station, Box 3944, University of Wyoming, Laramie, WY 82071, U.S.A.
(Accepted for publication 8 April 1987) I. I. KAXSERand S. D. AmD. A crotoxin homolog from the venom of the Uracoan rattlesnake
(Crotalus vegrandis). Toxicon 25, 1113 - 1120, 1987. - - A major protein toxin from the venom of Crotalus ve&randis was examined by gel filtration, anion-exchange chromatography, and SDS polyacrylamide gel electrophoresis. The toxin was separated into several isoforms by ion-exchange chromatography and spontaneously dissociated into free acidic and basic subunits, mimicking the behavior of crotoxin. Rabbit antisera raised against crotoxin reacted strongly in enzyme-linked immuno$orbent assays with the intact C. vegrandis toxin isoforms and their basic subunits, and formed precipitin lines of identity with intact crotoxin in double immunodiffusion gels. These results indicate that vegrandis toxin is strongly homologous with crotoxin from the venom of
Crotalus duriss~ terrOTcus.
CROTOXIN is a potent enzymatic neurotoxin first isolated from the venom of the South American rattlesnake Crotalus durissus terrificus (SLOTTA and FRAENKEL-CONRAT, 1938). It is a non-covalently linked, heterodimeric protein composed of a non-toxic acidic subunit and a toxic basic subunit. Phospholipase A2 activity is associated with only the basic subunit, although sequence studies on the acidic subunit indicate that it was derived from a phospholipase ancestral sequence (AIRD et al., 1985). For nearly 40 years crotoxin was believed to be the only crotalid neurotoxin. Recent immunological evidence suggests that crotoxin homologs are present in a largenumber of rattlesnake v e n o m s (GLENN and STRAIGHT, 1985; HENDERSON and BIEBER, 1986). Furthermore, it is now clear that Mojave toxin from C. s. scutulatus and concolor toxin from C. v. concolor are functionally and structurally very similar to crotoxin (HENDON, 1975; CATE and BIEBER, 1978; HENDON and BIEBER, 1982; POOL and BIEBER, 1981; AIRD and KAISER, 1985a; AmD et al., 1986). The Uracoan rattlesnake (Crotalus vegrandis) is restricted to a small region in northeastern Venezuela. Morphological characteristics suggest that it is a C. durissus derivative, and its closest congener is believed to be the South American rattlesnake (C. d. terrificus) (KLAUnER, 1972). Owing perhaps largely to its limited distribution, the venom of C. vegrandis has been little studied. In fact, two papers appear to comprise the entire literature (SCANNONE et al., 1978; GUBENSEK et al., 1978). Crotalus vegrandis breeds readily in captivity and its occurrence in both zoo and private collections has increased dramatically in the past several years. However, this species is not attractive for venom extraction for a variety of reasons. In captivity it reaches lengths of up to 1.2 m. Despite 1113
1114
IVAN I. KAISER and STEVEN D. AIRD
its length a n d b u l k , it possesses relatively small v e n o m glands a n d rarely yields m o r e t h a n 1 5 0 - 2 5 0 ~l o f v e n o m per extraction. I n a d d i t i o n , it possesses a n extremely surly d i s p o s i t i o n a n d r e s p o n d s violently w h e n a n y a t t e m p t is m a d e to restrain it, e n d a n g e r i n g b o t h the s n a k e a n d its h a n d l e r . A d u l t s m a y be s u b d u e d safely with a n i n h a l a b l e anesthetic (AIRD, 1986), b u t this is a t i m e - c o n s u m i n g practice w h e n large n u m b e r s o f a n i m a l s need to be milked. These b e h a v i o r a l a t t r i b u t e s m a y also c o n t r i b u t e to the p a u c i t y o f literature c o n c e r n i n g the v e n o m o f this snake. I n this m a n u s c r i p t we report o n the existence o f a c r o t o x i n h o m o l o g f r o m the v e n o m o f C. vegrandis. W e c o m p a r e its c h r o m a t o g r a p h i c , electrophoretic, a n d i m m u n o l o g i c a l b e h a v i o r a n d its toxicity, with those o f crotoxin. MATERIALS AND METHODS
Materials Venom was manually extracted from three anesthetized adult and eight juvenile Crotahts vefrandis maintained in our venom production facility (AI~.D, 1986), P e r ~ n j u j a t e d goat anti-rabbit and antimouse IgGs, (2,2'-azino-di-)-3-ethyibem~hiazoflne suifoni¢ acid (ABTS), and Tween-20 were obtained from Sisma. Gelatin was obtained from Bio Rad and bovine serum albumin Fraction V was purchased from Pentax.
Venom fractionation Crude venom (0.6 nd) that was fractionated initiallyby gel filtration was diluted to 4.5 ml with 0.1 M u~dium acetate (pH 4.0) buffer, centrifuged as ~ previomly (An~ and ICtus__l , 19858), and applied to a 2.5 x 94 cm Sephacryl S-200 column. Anion-er,chu~e e l w o m a t ~ y of the ~ p a l t o x i c ~ fraction of adult venom was carried out on a FPLC system ~harmacia) ~ a 0.5 × 5 em Mono Q column (Pharmacia) equilibrated in 50 mM Tris - HCI (pH 7,2)~ Crude adult veuom fractioaated at either pH 8.1 or 8.4 (20 mM Tris - HCI) save similar, but not identical absorbance profiles to that obtained at pH 7.2. Later experiments used 20 mM Tris-HCI (pH 8.4) when it appeared that higher pH slightly enhanced ~ of toxin isoforms. Juvenile venoms were fractionated on a Mono Q columnequilibrated in 20 mM Tris -- HCI (pH 8.4) and were qualitatively identical to adult venoms at that pH (compare Fil~ 5A and B), Flow rates were 1- 1.5 ml/ min and eiuent was monitored at 280 nm. Venom proteins were eiuted with a gradient of NaC1.
Electrophoresis and lethality as~ys SDS-PAGE employed 15% gels (AIRD el a[., 1988; PHARMACIA,1984) and lethality assays followed the method of AInD and KAISEr(1985b).
Elisa and immunod~ffusion Enzyme-linked immunosorhent assays and double immunodiffusionwere carried out as described ehewhere
(KAISEg et al., 1986). Ninety-six well. U bottomed microtiter plates (Dynatech lmmulon lI) were used in the ELISA. Indirect double antibody sandwich ELISA, were run in 96-well microfiter ~__t~ coated with rabbit polyclonal antibody (1 j~8/ml) raised atlaimt intact erotoxin ~ et al., 1986) and affinity purified on a column of immobilized crotoxin, prepared by the general procedure of Jotlt~rONE and Ttloa~ (l~2). After blocking with 1% gelatin and washing with "wash" buffer ( p h o n e buffered saline- 0.$efe Tween-20), $ ~ of each Mono Q fraction was mixed with 96 ~1 of phosphate buffered saline- 1~ bovine ~rum ~ - 0 . 5 % Tween-20 (diluting buffer) and incubated for 2 hr at room temperature. Wells were washed with "wash" buffer, then incubated for 2 hr with a purified, non-neutralizing mouse monoclonal antibody fine 2 at 5/~g/ml raised against the basic subunit of crotoxin (Kaiser and Middlebrook, in preparation). After washing, the wells were treated with goat anti-mouse IgG horseradish peroxidase conjullate and washed allain. Peroxida~ substrate (ABTS) at 1 mg/ml dissolved in 50 mM citrate (pH 4.0) containing0.03% H A was permitted to react for 7.5 rain. Color development was stopped by the addition of an equal volume of 10~ SDS and reading at 415 nm in a Titertek Muitiskan MC (Flow Laboratories). RESULTS
Chromatography P e a k 3 f r o m gel f i l t r a t i o n represented f r o m 41 t o 4 4 ~ o f the p r o t e i n recovered f r o m the c o l u m n (Fig. 1) in d i f f e r e n t r u n s , a n d c o n t a i n e d a p o t e n t t o x i n t h a t eluted i n the same
Crotoxin Homolog from C. vegrondis Venom
1115
3
2
I °-2-
5
d-
°o
6
2O
3O
~o
4O
Rt~e Arft~
FIG. 1. GEL FILTRATION OF CRUDE VENOM POOLED FROM THREE ADULT SEPHACRYL S-200.
Crotalus vegrondis ON
Crude venom (0.6 ml; ,~0.2 rag) was passed over a 2.5 x 94 cm column equilibrated with 0.1 M sodium acetate O H 4.0). Protein concentrations were determined by absorbance at 280 nm. T u b e s 4 2 - 4 8 , which constituted , ~ 4 1 % o f the material applied, were pooled for subsequent anion exchange chromatography.
2
3
!ino
I0.0
20.0
30.0
40.0
FIG. 2. F P L C
ANION EXCHANGE COLUMN CHROMATOGRAPHY OF POOLED FRACTIONS FROM THE SEPHACRYL S-200 run. Peak 3 from Fig. 1 was pooled, dialyzed against deionized water and lyophilized. A b o u t 8 m g was redissolved in 5 ml o f 50 m M Tris - H C I ( p H 7.2) and applied to a M o n o Q column and eluted with a linear gradient o f N a C I ( . . . . . . ) in the above b u f f e r . T h e three main peaks were pooled
separately, dialyzed against deionized water and lyophilized.
position as crotoxin and related crotalid presynaptic neurotoxins (AIRD and KAISER, 1985a). When tubes 4 2 - 4 8 (peak 3) were pooled, dialyzed against deionized water, redissolved in 50 m M T r i s - H C I (pH 7.2) and fractionated on M o n o Q, three discrete peaks were resolved (Fig. 2). These were pooled separately, dialyzed and lyophilized. W h e n an aliquot o f each peak was diluted and re-run on M o n o Q, extensive dissociation occurred with peaks 2 and 3. Peak 1 dissociation was less extreme. V e n o m from each o f the three adult snakes used to m a k e the pooled sample was collected and fractionated
1116
IVAN 1. KAISER and STEVEN D. AIRD
FIG. 3. OUCHTERLONY AGAR GEL-DIFFUSION PLATE.
Center wellcontains intact crotoxin rabbit antiserum. Peripheral wells: (1) buffer only; (2)intact crotoxin; (3) peak 1, Fig. 2; (4) peak 2, Fig. 2; (5) peak 3, Fig. 2; (6) intact crotoxin. All samples were at concentration of 0.125 mg/ml. directly on Mono Q. Each individual venom contained all three peaks observed in the pooled sample (Fig. 2).
Toxicity assays Toxicity assays were performed on each of the three individual peaks from Fig. 2. Peaks 2 and 3 showed LDs0-vaiues similar to that of crotoxin (,x,0.05 tq~/g in mice). After 24 hr post injection we observed no deaths with peak 1; 1/3, 2/5, and 4/4 for peak 2 at 0.00, 0.06, and 0.08/ag/g, respectively; and 0/3, 4/5, and 5/5 for peak 3 at 0.04,O.06, and 0.08/ag/g, respectively. Peak 1 may be contaminated with a non-toxic protein that does not dissociate upon rechromatography and which reduces the toxicity (higher LD~o). As will be shown later, SDS-PAGE also suggests the presence of a contaminant. Immunological assays To determine the antigenic relatedness of the three peaks to each other and to crotoxin, we ran double immunodiffusion gels, using antisera raised against intact crotoxin. Crotoxin antiserum produced lines of identity with crotoxin and with all three toxin peaks (Fig. 3). Precipitin band intensity of peak 1 was weaker than that shown by intact crotoxin or by peaks 2 and 3. In results not shown, all three peaks recovered from Mono Q were used as plate coating antisens (each at 1 /~g/ml), along with crotoxin, and then reacted with serially diluted rabbit antisera raised against crotoxin (KAISER et al., 1986). All four rows of wells coated with the four different antigens reacted in a similar manner, showing a parallel decrease in absorbance at greater antiserum dilutions. At dilutions where absorbance decreases linearly with dilution, peak 1 absorbances were about 2 0 ~ lower than absorbances for p~_.ks 2 and 3. When intact crotoxin was replaced with its basic and acidic subnnlts in two subseqent assays, rabbit antisera raised against the crotoxin subunits cross-reacted equally with the three vegrandis isotoxins. That is, a b s o r b a c e s diminished proportionally to antiserum dilution, and the diminution of abmrbance for the three vegrandis isotoxins paralleled that of the crotoxin subunits. This indicated that
Crotoxin Homolog from C. vegrandisVenom
1117
FIG. 4. SDS-PAGE OFCROTOXIN,ITSSUBU~ITSANDTHETHREEDIFFERENTPEAKSFROMFIG. 2 ON 15 °7o GELS.
Early and later eluting peaks of crotoxin refer to their positions of elution from a DEAE-Sephacel column (see AN~.Dand KAXSER,1985a). Well: (1 and 12) molecular weight markers, lysozyme (14,400), trypsin inhibitor (21,500), carbonic anhydrase (31,000), ovalbumin (45,000), bovine serum albumin (66,200), and phosphorylase b (92,500); (2) early eluting crotoxin peak; (3) later eluting crotoxin peak; (4) basic subunit from early eluting peak; (5) repurified basic subunit from early eluting peak; (6) basic subunit from later eluting peak; (7) acidic subunit from early eiuting peak; (8) acidic subunit from later eluting peak; (9) peak l, Fig. 2; (10) peak 2, Fig. 2; (1 l) peak 3, Fig. 2. Approximately 20 ~g of protein was applied per well. the vegrandis isotoxins contain moieties that are immunologically identical to the subunits o f crotoxin.
SDS-PAGE S D S - P A G E in the absence o f sulfhydryl reducing agents dissociates the non-covalently associated subunits o f crotoxin and resolves the two subunits (AIRD and KAISER, 1985a). The three neurotoxin peaks f r o m C. vegrandis v e n o m (Fig. 2) dissociated into subunits with mobilities identical to the basic and acidic subunits o f crotoxin (Fig. 4). There appear to be at least two different forms o f the acidic subunit in the three isotoxins f r o m C. vegrandis. These co-migrate with the two different forms o f acidic subunit seen in crotoxin (Fig. 4; wells 2, 3, 7 and 8). It is not clear why the acidic subunits of crotoxin differ in their mobility in the presence o f SDS, since both should have similar charge/mass ratios. When reducing agent is added to the samples before electrophoresis, all basic subunits migrate with identical mobilities. Acidic subunits disappear, because o f disulfide
I V A N I. K A I S E R and S T E V E N D. A I R D
II 18
1A,
o
~o
I -1Bri tl
~)
~
~
5o
o
;)
~o
;o
~
, 0
5o
FIG. 5. ABSORBANCE ELUTION PROFILE (2~0 n m ) OF VENOM FROM AN INDIVIDUAL JUVENILE (A) AND ADULT (B) C. yeS/~n~/.s (SOLID LINE) COUPLED WITH A DOUBLE ANTIBODY SANDWICH E L I S A RESPONSIVE TO CROTOXIN-RELATED PROTEINS (DASHEDLINE - - 4 1 5 rim).
Each run consisted of 25 ~1 of crude venom dissolved in 1.0 ml of 20ram T r i s - H C ! (pH 8.4), f'dtered, and chromatographed using an FPLC system and a Mono Q column. Aliquots (5 ~1) from each fraction were assayed for immnrlological identity with crotoxin. NaCI concentration of the etuting buffers are represented by the dotted line, with the final concentration at tube 48 corresponding to 0.76 M.
bond reduction and dissociation of the three small peptides (BREITHAUPT et al., 1974; AIRY et al., 1985). In addition to examining the chromatographic profiles of individual and pooled, adult venoms from C. vegrandis, we fractionated eight individual crude venoms from juvenile snakes on Mono Q. In all cases we observed multiple components that eluted as had the neurotoxin isomers in adult venoms. This may be seen in Fig. 5, where a juvenile (A) and adult venom sample (13) were fractionated under identical conditions. Aliquots of fractions from both venoms also reacted positively and in a similar manner in double antibody sandwich ELISA using a combination of rabbit polyclonal and mouse monoclonal antibodies (Fig. 5). A peak eluting in the void volumes, comprised at least partially of the basic subunit of vegrandis toxin, also reacted strongly in the ELISA. Chromatography of either vegrandis toxin or crotoxin under conditions described for Fig. 5, but including 6 M urea, results in essentially complete dissociation of the toxin, with the basic subunit again eluting in the intial buffer. Recovery of this material from the crotoxin run, and chromatography on a cation exchange column (Mono S; 50 mM Hepes (pH 8), 6 M urea, and elution with a NaC1 gradient), produced a peak eluting at around 0.4 M NaCI. This eleetrophoresed on SDS-PAGE as basic subunit (slot 5, Fig. 4). DISCUSSION
The gel filtration prof'fle r ~ o r t e d here for C. vegrand/x venom on S~imcryl S-200 0Fig. 1) is very similar to that obtained by SCANNO~ et al., (1978) using Sephadex O-100. In
Crotoxin Homolog from C. vegrandis Venom
1119
both cases peak 3 appeared equivalent. The former authors reported that peak 3 was the only fraction recovered that had a more toxic i.v. LD~oin mice than crude venom. Based on its LDso and position o f elution, it was suggested that C. vegrandis v e n o m contained crotoxin. In our hands the principal toxic component also eluted f r o m the gel filtration column in the same manner as crotoxin. During F P L C , vegrandis toxin f r o m b o t h individual juvenile and adults exhibited several isoforms, which is also characteristic of crotoxin a n d Mojave toxin (A1RD and KAISER, 1985a: RAEL et al., 1986: FAURE and BON, 1986). Further, vegrandis toxin spontaneously dissociated into subunits, as was noted earlier with crotoxin f r o m certain lots of C. d. terrificus venom. We confirmed that the ultraviolet absorbing material eluting in the void volume f r o m the M o n o Q column (Fig. 5) was the basic subunit of vegrandis toxin by its cross-reaction with crotoxin antiserum in addition to its chromatographic and electrophoretic properties. Double immunodiffusion assays with the three separable isoforms o f vegrandis toxin f r o m M o n o Q (Fig. 2) demonstrated immunological identity between the isotoxins and with crotoxin. In addition, toxicity determinations confirm that two of the three peaks had LDs0-values similar to that of crotoxin. S D S - P A G E behaviour o f vegrandis toxin is likewise consistent with other crotalid heterodimeric presynaptic neurotoxins (AIRD and KAISER, 1985a). The weaker reactivity o f peak 1, on Ouchterlony gels (Fig. 3), its reduced toxicity and the appearance of a contaminant running near the leading edge of its basic subunit (Fig. 4; well 9) in S D S - P A G E , suggest the presence of a contaminating protein in peak 1 isolated f r o m the M o n o Q column. This contaminant m a y be a non-toxic homodimeric phospholipase As such as that f r o m C. a t r o x (RANDOLPHand HEINRIKSON, 1982) or C. a d a m a n t e u s (HEINRIKSON et al., 1977). The non-toxic phospholipases elute f r o m M o n o Q close to the presynaptic neurotoxins, but they do not dissociate spontaneously as do the heterodimeric phospholipases. Furthermore, when examined on SDS-PAGE, non-toxic phospholipases appear as a single band with a mobility very close to that of the basic subunit f r o m crotalid presynaptic neurotoxins (AIRD and KA1SER, unpublished data). In s u m m a r y , we conclude that juvenile and adult C. vegrandis v e n o m contains a toxic protein that is structurally and antigenically similar to the m a j o r neurotoxins isolated f r o m C. d. terrificus, C. s. s c u t u l a t u s and C. v. concolor. It also appears likely that this toxin is the product of a duplicated locus as has been suggested for crotoxin (FAURE and BON, 1986). Acknowledgements -- The authors thank CORRtNES. SEEBARTfor valuable technical assistance. This work was
supported in part by the U. S. Army Medical Acquisition Activity, Contract No. DAMD 17-86-C-6061. REFERENCES AmD, S. D. (1986) Methoxyflurane anesthesia in Crotalus: comparisons with other gas anesthetics. Herpet. Rev. 17, 82. AIRD, S. D. and KAISER,I. 1. (1985a) Comparative studies on three rattlesnake toxins. Toxicon 23, 361. AmD, S. D. and KAtSER,I. I. (1985b) Toxicity assays. Toxicon 23, 11. AmD, S. D., KmSER, 1. I., LEwts, R. V. and KRUGCEL,W. G. (1985) Rattlesnake presynaptic neurotoxins: primary structure and evolutionary origin of the acidic subunit. Biochemistry 24, 7054. AmP, S. D., KAtSER,I. I., LEWIS,R. V. and KRUC,GEL, W. G. (1986) A complete amino acid sequence for the basic subunit of crotoxin. Archs Biochem. Biophys. 249, 296. AmD, S. D., C. S. SEEaARTand I. I. KAISER.(1988) Preliminary fractionation and characterization of the venom of the Great Basin Rattlesnake (Crotalus viriais lutosus). Herlmtologica (in press). BREtTHAUPT,H., RUBSAMEN,K. and HAB~RMANTq,E. (1974) Biochemistry and pharmacology of the crotoxin complex. Fur. J. Biochem. 49, 333.
1120
IVAN I. KAISER and STEVEN D. AIRD
CATE, R. L. and BIEBER, A. L. (1978) Purification and characterization of Mojave (Crotalus scutulatus) toxin and its subunits. Archs Biochem. Biophys. 189, 397. FAURE, G. and BON, C. (1986) Several crotoxin isoforms are present in individual venoms from Crotalus durissus terrificus. Abstracts, Seventh European Symposium on Animal, Plant and Microbial Toxins, Prague, CSSR, August 18 - 22. GLENN, J. L. and STRAIOHT, R. C. (1985) Distribution of proteins immunologically similar to Mojave toxin among species of Crotalus and Sistrurus. Toxicon 23, 28. GUBENSEK, F., TURK, V. and DRUJAN, B. (1978) Proteolytic and clotting enzymes in some Venezuelan snake venoms. Period. Biol. 80, (Suppl. I), 101. HEINRIKSON, R. L., KRUEGER, E. T. and KEIM, P. S. (1977) Amino acid sequence of phospholipase A2-a from the venom of Crotalus adamanteus. A new classification of phospholipases A2 based upon structural determinants. J. biol. Chem. 252, 4913. HENDERSON, J. T. and BZEBER, A. L. (1986) Antigenic relationships between Mojave toxin subunits, Mojave toxin and some crotalid venoms. Toxicon 24, 473. HENDON, R. A. (1975) Preliminary studies on the toxin in the venom of Crotalus scutulatus (Mojave rattlesnake). Toxicon 13, 477. HENDON, R. A. and BIEBER, A. L. (1982) Presynaptic toxins from rattlesnake venoms. In: Rattlesnake Venoms: Their Actions and Treatment, p. 211 (Tu, A. T., Ed.). New York: Marcel Dekker. JOHNSTONE, A. and THORPE, R. (1982) lmmunochemistry in Practice, p. 202. Oxford: Biackwell Scientific. KAISER, I. I., MIDDLEnROOK, J. L., CRUMRINE, M. N. and STEVENSON, W. W. (1986) Cross-reactivity and neutralization lay rabbit antisera raised against crotoxin, its subunits and two related toxins. Toxicon 2,1, 669. KLAUBER, L. M. (1972) Rattlesnakes. Their Habits, Life Histories, and lnfluence on Mankind, 2nd Edn. Berkeley: University of California Press. PHARMACIA (1984) Polyacrylamide Gel Electrophoresis, Laboratory Techniques (revised edition), p. 28. Uppsala, Sweden: Pharmacia Fine chemicals. POOL, W. R. and BmBER, A. L. (1981) Fractionation of midget faded rattlesnake (Crotalus viridis concolor) venom: lethal fractions and enzymatic activies. Toxicon 19, 517. RAEL, E. D., SALO, R. J. and ZEPEDA, H. (1986) Monoclomal antibodies to Mojave toxin and use for isolation of cross-reacting proteins in Crotalus venoms. Toxicon, 24, 661. RANDOLPH, A. and HEINRIKSON, R. L. (1982) Crotalus atrox phospholipase A2. Amino acid sequence and studies on the function of the NH2-terminal region. J. biol. Chem. 257, 2155. SCANNONE, H. R., RODRIGUEZ,O. G. and LANCINI, A. R. (1978) Enzymatic activities and other characteristics of Crotalus vegrandis snake venom. In: Toxins: Animal, Plant and Microbial, p. 223 (ROSENm~RG, P., Ed.). New York: Pergamon. SLOTTA, K. H. and FRAEN~L-CON~T, H. (1938) Schlangengifte IlI. Mitt. Reiningung und kristallisation des klapperschlangen-giftes. Ber. dt. chem. Ges. 71, 1076.
0041-0101/87 $3.00+ .00 © 19ff~PergamonJournalsLtd.
A CROTOXIN HOMOLOG FROM THE VENOM OF THE URACOAN RATTLESNAKE (CROTALUS VEGRANDIS)
IVAN I. KAISER and STEVEN D. AIRD Department of Molecular Biology, University Station, Box 3944, University of Wyoming, Laramie, WY 82071, U.S.A.
(Accepted for publication 8 April 1987) I. I. KAXSERand S. D. AmD. A crotoxin homolog from the venom of the Uracoan rattlesnake
(Crotalus vegrandis). Toxicon 25, 1113 - 1120, 1987. - - A major protein toxin from the venom of Crotalus ve&randis was examined by gel filtration, anion-exchange chromatography, and SDS polyacrylamide gel electrophoresis. The toxin was separated into several isoforms by ion-exchange chromatography and spontaneously dissociated into free acidic and basic subunits, mimicking the behavior of crotoxin. Rabbit antisera raised against crotoxin reacted strongly in enzyme-linked immuno$orbent assays with the intact C. vegrandis toxin isoforms and their basic subunits, and formed precipitin lines of identity with intact crotoxin in double immunodiffusion gels. These results indicate that vegrandis toxin is strongly homologous with crotoxin from the venom of
Crotalus duriss~ terrOTcus.
CROTOXIN is a potent enzymatic neurotoxin first isolated from the venom of the South American rattlesnake Crotalus durissus terrificus (SLOTTA and FRAENKEL-CONRAT, 1938). It is a non-covalently linked, heterodimeric protein composed of a non-toxic acidic subunit and a toxic basic subunit. Phospholipase A2 activity is associated with only the basic subunit, although sequence studies on the acidic subunit indicate that it was derived from a phospholipase ancestral sequence (AIRD et al., 1985). For nearly 40 years crotoxin was believed to be the only crotalid neurotoxin. Recent immunological evidence suggests that crotoxin homologs are present in a largenumber of rattlesnake v e n o m s (GLENN and STRAIGHT, 1985; HENDERSON and BIEBER, 1986). Furthermore, it is now clear that Mojave toxin from C. s. scutulatus and concolor toxin from C. v. concolor are functionally and structurally very similar to crotoxin (HENDON, 1975; CATE and BIEBER, 1978; HENDON and BIEBER, 1982; POOL and BIEBER, 1981; AIRD and KAISER, 1985a; AmD et al., 1986). The Uracoan rattlesnake (Crotalus vegrandis) is restricted to a small region in northeastern Venezuela. Morphological characteristics suggest that it is a C. durissus derivative, and its closest congener is believed to be the South American rattlesnake (C. d. terrificus) (KLAUnER, 1972). Owing perhaps largely to its limited distribution, the venom of C. vegrandis has been little studied. In fact, two papers appear to comprise the entire literature (SCANNONE et al., 1978; GUBENSEK et al., 1978). Crotalus vegrandis breeds readily in captivity and its occurrence in both zoo and private collections has increased dramatically in the past several years. However, this species is not attractive for venom extraction for a variety of reasons. In captivity it reaches lengths of up to 1.2 m. Despite 1113
1114
IVAN I. KAISER and STEVEN D. AIRD
its length a n d b u l k , it possesses relatively small v e n o m glands a n d rarely yields m o r e t h a n 1 5 0 - 2 5 0 ~l o f v e n o m per extraction. I n a d d i t i o n , it possesses a n extremely surly d i s p o s i t i o n a n d r e s p o n d s violently w h e n a n y a t t e m p t is m a d e to restrain it, e n d a n g e r i n g b o t h the s n a k e a n d its h a n d l e r . A d u l t s m a y be s u b d u e d safely with a n i n h a l a b l e anesthetic (AIRD, 1986), b u t this is a t i m e - c o n s u m i n g practice w h e n large n u m b e r s o f a n i m a l s need to be milked. These b e h a v i o r a l a t t r i b u t e s m a y also c o n t r i b u t e to the p a u c i t y o f literature c o n c e r n i n g the v e n o m o f this snake. I n this m a n u s c r i p t we report o n the existence o f a c r o t o x i n h o m o l o g f r o m the v e n o m o f C. vegrandis. W e c o m p a r e its c h r o m a t o g r a p h i c , electrophoretic, a n d i m m u n o l o g i c a l b e h a v i o r a n d its toxicity, with those o f crotoxin. MATERIALS AND METHODS
Materials Venom was manually extracted from three anesthetized adult and eight juvenile Crotahts vefrandis maintained in our venom production facility (AI~.D, 1986), P e r ~ n j u j a t e d goat anti-rabbit and antimouse IgGs, (2,2'-azino-di-)-3-ethyibem~hiazoflne suifoni¢ acid (ABTS), and Tween-20 were obtained from Sisma. Gelatin was obtained from Bio Rad and bovine serum albumin Fraction V was purchased from Pentax.
Venom fractionation Crude venom (0.6 nd) that was fractionated initiallyby gel filtration was diluted to 4.5 ml with 0.1 M u~dium acetate (pH 4.0) buffer, centrifuged as ~ previomly (An~ and ICtus__l , 19858), and applied to a 2.5 x 94 cm Sephacryl S-200 column. Anion-er,chu~e e l w o m a t ~ y of the ~ p a l t o x i c ~ fraction of adult venom was carried out on a FPLC system ~harmacia) ~ a 0.5 × 5 em Mono Q column (Pharmacia) equilibrated in 50 mM Tris - HCI (pH 7,2)~ Crude adult veuom fractioaated at either pH 8.1 or 8.4 (20 mM Tris - HCI) save similar, but not identical absorbance profiles to that obtained at pH 7.2. Later experiments used 20 mM Tris-HCI (pH 8.4) when it appeared that higher pH slightly enhanced ~ of toxin isoforms. Juvenile venoms were fractionated on a Mono Q columnequilibrated in 20 mM Tris -- HCI (pH 8.4) and were qualitatively identical to adult venoms at that pH (compare Fil~ 5A and B), Flow rates were 1- 1.5 ml/ min and eiuent was monitored at 280 nm. Venom proteins were eiuted with a gradient of NaC1.
Electrophoresis and lethality as~ys SDS-PAGE employed 15% gels (AIRD el a[., 1988; PHARMACIA,1984) and lethality assays followed the method of AInD and KAISEr(1985b).
Elisa and immunod~ffusion Enzyme-linked immunosorhent assays and double immunodiffusionwere carried out as described ehewhere
(KAISEg et al., 1986). Ninety-six well. U bottomed microtiter plates (Dynatech lmmulon lI) were used in the ELISA. Indirect double antibody sandwich ELISA, were run in 96-well microfiter ~__t~ coated with rabbit polyclonal antibody (1 j~8/ml) raised atlaimt intact erotoxin ~ et al., 1986) and affinity purified on a column of immobilized crotoxin, prepared by the general procedure of Jotlt~rONE and Ttloa~ (l~2). After blocking with 1% gelatin and washing with "wash" buffer ( p h o n e buffered saline- 0.$efe Tween-20), $ ~ of each Mono Q fraction was mixed with 96 ~1 of phosphate buffered saline- 1~ bovine ~rum ~ - 0 . 5 % Tween-20 (diluting buffer) and incubated for 2 hr at room temperature. Wells were washed with "wash" buffer, then incubated for 2 hr with a purified, non-neutralizing mouse monoclonal antibody fine 2 at 5/~g/ml raised against the basic subunit of crotoxin (Kaiser and Middlebrook, in preparation). After washing, the wells were treated with goat anti-mouse IgG horseradish peroxidase conjullate and washed allain. Peroxida~ substrate (ABTS) at 1 mg/ml dissolved in 50 mM citrate (pH 4.0) containing0.03% H A was permitted to react for 7.5 rain. Color development was stopped by the addition of an equal volume of 10~ SDS and reading at 415 nm in a Titertek Muitiskan MC (Flow Laboratories). RESULTS
Chromatography P e a k 3 f r o m gel f i l t r a t i o n represented f r o m 41 t o 4 4 ~ o f the p r o t e i n recovered f r o m the c o l u m n (Fig. 1) in d i f f e r e n t r u n s , a n d c o n t a i n e d a p o t e n t t o x i n t h a t eluted i n the same
Crotoxin Homolog from C. vegrondis Venom
1115
3
2
I °-2-
5
d-
°o
6
2O
3O
~o
4O
Rt~e Arft~
FIG. 1. GEL FILTRATION OF CRUDE VENOM POOLED FROM THREE ADULT SEPHACRYL S-200.
Crotalus vegrondis ON
Crude venom (0.6 ml; ,~0.2 rag) was passed over a 2.5 x 94 cm column equilibrated with 0.1 M sodium acetate O H 4.0). Protein concentrations were determined by absorbance at 280 nm. T u b e s 4 2 - 4 8 , which constituted , ~ 4 1 % o f the material applied, were pooled for subsequent anion exchange chromatography.
2
3
!ino
I0.0
20.0
30.0
40.0
FIG. 2. F P L C
ANION EXCHANGE COLUMN CHROMATOGRAPHY OF POOLED FRACTIONS FROM THE SEPHACRYL S-200 run. Peak 3 from Fig. 1 was pooled, dialyzed against deionized water and lyophilized. A b o u t 8 m g was redissolved in 5 ml o f 50 m M Tris - H C I ( p H 7.2) and applied to a M o n o Q column and eluted with a linear gradient o f N a C I ( . . . . . . ) in the above b u f f e r . T h e three main peaks were pooled
separately, dialyzed against deionized water and lyophilized.
position as crotoxin and related crotalid presynaptic neurotoxins (AIRD and KAISER, 1985a). When tubes 4 2 - 4 8 (peak 3) were pooled, dialyzed against deionized water, redissolved in 50 m M T r i s - H C I (pH 7.2) and fractionated on M o n o Q, three discrete peaks were resolved (Fig. 2). These were pooled separately, dialyzed and lyophilized. W h e n an aliquot o f each peak was diluted and re-run on M o n o Q, extensive dissociation occurred with peaks 2 and 3. Peak 1 dissociation was less extreme. V e n o m from each o f the three adult snakes used to m a k e the pooled sample was collected and fractionated
1116
IVAN 1. KAISER and STEVEN D. AIRD
FIG. 3. OUCHTERLONY AGAR GEL-DIFFUSION PLATE.
Center wellcontains intact crotoxin rabbit antiserum. Peripheral wells: (1) buffer only; (2)intact crotoxin; (3) peak 1, Fig. 2; (4) peak 2, Fig. 2; (5) peak 3, Fig. 2; (6) intact crotoxin. All samples were at concentration of 0.125 mg/ml. directly on Mono Q. Each individual venom contained all three peaks observed in the pooled sample (Fig. 2).
Toxicity assays Toxicity assays were performed on each of the three individual peaks from Fig. 2. Peaks 2 and 3 showed LDs0-vaiues similar to that of crotoxin (,x,0.05 tq~/g in mice). After 24 hr post injection we observed no deaths with peak 1; 1/3, 2/5, and 4/4 for peak 2 at 0.00, 0.06, and 0.08/ag/g, respectively; and 0/3, 4/5, and 5/5 for peak 3 at 0.04,O.06, and 0.08/ag/g, respectively. Peak 1 may be contaminated with a non-toxic protein that does not dissociate upon rechromatography and which reduces the toxicity (higher LD~o). As will be shown later, SDS-PAGE also suggests the presence of a contaminant. Immunological assays To determine the antigenic relatedness of the three peaks to each other and to crotoxin, we ran double immunodiffusion gels, using antisera raised against intact crotoxin. Crotoxin antiserum produced lines of identity with crotoxin and with all three toxin peaks (Fig. 3). Precipitin band intensity of peak 1 was weaker than that shown by intact crotoxin or by peaks 2 and 3. In results not shown, all three peaks recovered from Mono Q were used as plate coating antisens (each at 1 /~g/ml), along with crotoxin, and then reacted with serially diluted rabbit antisera raised against crotoxin (KAISER et al., 1986). All four rows of wells coated with the four different antigens reacted in a similar manner, showing a parallel decrease in absorbance at greater antiserum dilutions. At dilutions where absorbance decreases linearly with dilution, peak 1 absorbances were about 2 0 ~ lower than absorbances for p~_.ks 2 and 3. When intact crotoxin was replaced with its basic and acidic subnnlts in two subseqent assays, rabbit antisera raised against the crotoxin subunits cross-reacted equally with the three vegrandis isotoxins. That is, a b s o r b a c e s diminished proportionally to antiserum dilution, and the diminution of abmrbance for the three vegrandis isotoxins paralleled that of the crotoxin subunits. This indicated that
Crotoxin Homolog from C. vegrandisVenom
1117
FIG. 4. SDS-PAGE OFCROTOXIN,ITSSUBU~ITSANDTHETHREEDIFFERENTPEAKSFROMFIG. 2 ON 15 °7o GELS.
Early and later eluting peaks of crotoxin refer to their positions of elution from a DEAE-Sephacel column (see AN~.Dand KAXSER,1985a). Well: (1 and 12) molecular weight markers, lysozyme (14,400), trypsin inhibitor (21,500), carbonic anhydrase (31,000), ovalbumin (45,000), bovine serum albumin (66,200), and phosphorylase b (92,500); (2) early eluting crotoxin peak; (3) later eluting crotoxin peak; (4) basic subunit from early eluting peak; (5) repurified basic subunit from early eluting peak; (6) basic subunit from later eluting peak; (7) acidic subunit from early eiuting peak; (8) acidic subunit from later eluting peak; (9) peak l, Fig. 2; (10) peak 2, Fig. 2; (1 l) peak 3, Fig. 2. Approximately 20 ~g of protein was applied per well. the vegrandis isotoxins contain moieties that are immunologically identical to the subunits o f crotoxin.
SDS-PAGE S D S - P A G E in the absence o f sulfhydryl reducing agents dissociates the non-covalently associated subunits o f crotoxin and resolves the two subunits (AIRD and KAISER, 1985a). The three neurotoxin peaks f r o m C. vegrandis v e n o m (Fig. 2) dissociated into subunits with mobilities identical to the basic and acidic subunits o f crotoxin (Fig. 4). There appear to be at least two different forms o f the acidic subunit in the three isotoxins f r o m C. vegrandis. These co-migrate with the two different forms o f acidic subunit seen in crotoxin (Fig. 4; wells 2, 3, 7 and 8). It is not clear why the acidic subunits of crotoxin differ in their mobility in the presence o f SDS, since both should have similar charge/mass ratios. When reducing agent is added to the samples before electrophoresis, all basic subunits migrate with identical mobilities. Acidic subunits disappear, because o f disulfide
I V A N I. K A I S E R and S T E V E N D. A I R D
II 18
1A,
o
~o
I -1Bri tl
~)
~
~
5o
o
;)
~o
;o
~
, 0
5o
FIG. 5. ABSORBANCE ELUTION PROFILE (2~0 n m ) OF VENOM FROM AN INDIVIDUAL JUVENILE (A) AND ADULT (B) C. yeS/~n~/.s (SOLID LINE) COUPLED WITH A DOUBLE ANTIBODY SANDWICH E L I S A RESPONSIVE TO CROTOXIN-RELATED PROTEINS (DASHEDLINE - - 4 1 5 rim).
Each run consisted of 25 ~1 of crude venom dissolved in 1.0 ml of 20ram T r i s - H C ! (pH 8.4), f'dtered, and chromatographed using an FPLC system and a Mono Q column. Aliquots (5 ~1) from each fraction were assayed for immnrlological identity with crotoxin. NaCI concentration of the etuting buffers are represented by the dotted line, with the final concentration at tube 48 corresponding to 0.76 M.
bond reduction and dissociation of the three small peptides (BREITHAUPT et al., 1974; AIRY et al., 1985). In addition to examining the chromatographic profiles of individual and pooled, adult venoms from C. vegrandis, we fractionated eight individual crude venoms from juvenile snakes on Mono Q. In all cases we observed multiple components that eluted as had the neurotoxin isomers in adult venoms. This may be seen in Fig. 5, where a juvenile (A) and adult venom sample (13) were fractionated under identical conditions. Aliquots of fractions from both venoms also reacted positively and in a similar manner in double antibody sandwich ELISA using a combination of rabbit polyclonal and mouse monoclonal antibodies (Fig. 5). A peak eluting in the void volumes, comprised at least partially of the basic subunit of vegrandis toxin, also reacted strongly in the ELISA. Chromatography of either vegrandis toxin or crotoxin under conditions described for Fig. 5, but including 6 M urea, results in essentially complete dissociation of the toxin, with the basic subunit again eluting in the intial buffer. Recovery of this material from the crotoxin run, and chromatography on a cation exchange column (Mono S; 50 mM Hepes (pH 8), 6 M urea, and elution with a NaC1 gradient), produced a peak eluting at around 0.4 M NaCI. This eleetrophoresed on SDS-PAGE as basic subunit (slot 5, Fig. 4). DISCUSSION
The gel filtration prof'fle r ~ o r t e d here for C. vegrand/x venom on S~imcryl S-200 0Fig. 1) is very similar to that obtained by SCANNO~ et al., (1978) using Sephadex O-100. In
Crotoxin Homolog from C. vegrandis Venom
1119
both cases peak 3 appeared equivalent. The former authors reported that peak 3 was the only fraction recovered that had a more toxic i.v. LD~oin mice than crude venom. Based on its LDso and position o f elution, it was suggested that C. vegrandis v e n o m contained crotoxin. In our hands the principal toxic component also eluted f r o m the gel filtration column in the same manner as crotoxin. During F P L C , vegrandis toxin f r o m b o t h individual juvenile and adults exhibited several isoforms, which is also characteristic of crotoxin a n d Mojave toxin (A1RD and KAISER, 1985a: RAEL et al., 1986: FAURE and BON, 1986). Further, vegrandis toxin spontaneously dissociated into subunits, as was noted earlier with crotoxin f r o m certain lots of C. d. terrificus venom. We confirmed that the ultraviolet absorbing material eluting in the void volume f r o m the M o n o Q column (Fig. 5) was the basic subunit of vegrandis toxin by its cross-reaction with crotoxin antiserum in addition to its chromatographic and electrophoretic properties. Double immunodiffusion assays with the three separable isoforms o f vegrandis toxin f r o m M o n o Q (Fig. 2) demonstrated immunological identity between the isotoxins and with crotoxin. In addition, toxicity determinations confirm that two of the three peaks had LDs0-values similar to that of crotoxin. S D S - P A G E behaviour o f vegrandis toxin is likewise consistent with other crotalid heterodimeric presynaptic neurotoxins (AIRD and KAISER, 1985a). The weaker reactivity o f peak 1, on Ouchterlony gels (Fig. 3), its reduced toxicity and the appearance of a contaminant running near the leading edge of its basic subunit (Fig. 4; well 9) in S D S - P A G E , suggest the presence of a contaminating protein in peak 1 isolated f r o m the M o n o Q column. This contaminant m a y be a non-toxic homodimeric phospholipase As such as that f r o m C. a t r o x (RANDOLPHand HEINRIKSON, 1982) or C. a d a m a n t e u s (HEINRIKSON et al., 1977). The non-toxic phospholipases elute f r o m M o n o Q close to the presynaptic neurotoxins, but they do not dissociate spontaneously as do the heterodimeric phospholipases. Furthermore, when examined on SDS-PAGE, non-toxic phospholipases appear as a single band with a mobility very close to that of the basic subunit f r o m crotalid presynaptic neurotoxins (AIRD and KA1SER, unpublished data). In s u m m a r y , we conclude that juvenile and adult C. vegrandis v e n o m contains a toxic protein that is structurally and antigenically similar to the m a j o r neurotoxins isolated f r o m C. d. terrificus, C. s. s c u t u l a t u s and C. v. concolor. It also appears likely that this toxin is the product of a duplicated locus as has been suggested for crotoxin (FAURE and BON, 1986). Acknowledgements -- The authors thank CORRtNES. SEEBARTfor valuable technical assistance. This work was
supported in part by the U. S. Army Medical Acquisition Activity, Contract No. DAMD 17-86-C-6061. REFERENCES AmD, S. D. (1986) Methoxyflurane anesthesia in Crotalus: comparisons with other gas anesthetics. Herpet. Rev. 17, 82. AIRD, S. D. and KAISER,I. 1. (1985a) Comparative studies on three rattlesnake toxins. Toxicon 23, 361. AmD, S. D. and KAtSER,I. I. (1985b) Toxicity assays. Toxicon 23, 11. AmD, S. D., KmSER, 1. I., LEwts, R. V. and KRUGCEL,W. G. (1985) Rattlesnake presynaptic neurotoxins: primary structure and evolutionary origin of the acidic subunit. Biochemistry 24, 7054. AmP, S. D., KAtSER,I. I., LEWIS,R. V. and KRUC,GEL, W. G. (1986) A complete amino acid sequence for the basic subunit of crotoxin. Archs Biochem. Biophys. 249, 296. AmD, S. D., C. S. SEEaARTand I. I. KAISER.(1988) Preliminary fractionation and characterization of the venom of the Great Basin Rattlesnake (Crotalus viriais lutosus). Herlmtologica (in press). BREtTHAUPT,H., RUBSAMEN,K. and HAB~RMANTq,E. (1974) Biochemistry and pharmacology of the crotoxin complex. Fur. J. Biochem. 49, 333.
1120
IVAN I. KAISER and STEVEN D. AIRD
CATE, R. L. and BIEBER, A. L. (1978) Purification and characterization of Mojave (Crotalus scutulatus) toxin and its subunits. Archs Biochem. Biophys. 189, 397. FAURE, G. and BON, C. (1986) Several crotoxin isoforms are present in individual venoms from Crotalus durissus terrificus. Abstracts, Seventh European Symposium on Animal, Plant and Microbial Toxins, Prague, CSSR, August 18 - 22. GLENN, J. L. and STRAIOHT, R. C. (1985) Distribution of proteins immunologically similar to Mojave toxin among species of Crotalus and Sistrurus. Toxicon 23, 28. GUBENSEK, F., TURK, V. and DRUJAN, B. (1978) Proteolytic and clotting enzymes in some Venezuelan snake venoms. Period. Biol. 80, (Suppl. I), 101. HEINRIKSON, R. L., KRUEGER, E. T. and KEIM, P. S. (1977) Amino acid sequence of phospholipase A2-a from the venom of Crotalus adamanteus. A new classification of phospholipases A2 based upon structural determinants. J. biol. Chem. 252, 4913. HENDERSON, J. T. and BZEBER, A. L. (1986) Antigenic relationships between Mojave toxin subunits, Mojave toxin and some crotalid venoms. Toxicon 24, 473. HENDON, R. A. (1975) Preliminary studies on the toxin in the venom of Crotalus scutulatus (Mojave rattlesnake). Toxicon 13, 477. HENDON, R. A. and BIEBER, A. L. (1982) Presynaptic toxins from rattlesnake venoms. In: Rattlesnake Venoms: Their Actions and Treatment, p. 211 (Tu, A. T., Ed.). New York: Marcel Dekker. JOHNSTONE, A. and THORPE, R. (1982) lmmunochemistry in Practice, p. 202. Oxford: Biackwell Scientific. KAISER, I. I., MIDDLEnROOK, J. L., CRUMRINE, M. N. and STEVENSON, W. W. (1986) Cross-reactivity and neutralization lay rabbit antisera raised against crotoxin, its subunits and two related toxins. Toxicon 2,1, 669. KLAUBER, L. M. (1972) Rattlesnakes. Their Habits, Life Histories, and lnfluence on Mankind, 2nd Edn. Berkeley: University of California Press. PHARMACIA (1984) Polyacrylamide Gel Electrophoresis, Laboratory Techniques (revised edition), p. 28. Uppsala, Sweden: Pharmacia Fine chemicals. POOL, W. R. and BmBER, A. L. (1981) Fractionation of midget faded rattlesnake (Crotalus viridis concolor) venom: lethal fractions and enzymatic activies. Toxicon 19, 517. RAEL, E. D., SALO, R. J. and ZEPEDA, H. (1986) Monoclomal antibodies to Mojave toxin and use for isolation of cross-reacting proteins in Crotalus venoms. Toxicon, 24, 661. RANDOLPH, A. and HEINRIKSON, R. L. (1982) Crotalus atrox phospholipase A2. Amino acid sequence and studies on the function of the NH2-terminal region. J. biol. Chem. 257, 2155. SCANNONE, H. R., RODRIGUEZ,O. G. and LANCINI, A. R. (1978) Enzymatic activities and other characteristics of Crotalus vegrandis snake venom. In: Toxins: Animal, Plant and Microbial, p. 223 (ROSENm~RG, P., Ed.). New York: Pergamon. SLOTTA, K. H. and FRAEN~L-CON~T, H. (1938) Schlangengifte IlI. Mitt. Reiningung und kristallisation des klapperschlangen-giftes. Ber. dt. chem. Ges. 71, 1076.