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Pharmacological and biochemical properties of a venom gland extract from the snake Thelotornis kirtlandi
Taxidon. Vol. IF, pp. 533-342. ~ Perpsmon 1978.
Press Ltd.
0041-0I01/78J1101-0333502 .00/0
Printed in Great Britain .
PHARMACOLOGICAL AND BIOCHEMICAL PROPERTIES OF A VENOM GLAND EXTRACT FROM THE SNAKE THELOTORNIS KIRTLANDI FRANTISEK KORNALIK, EVA Tr~IàORSKÁ
and
DIETRICH MEes*
Institute of Pathophysiology Charles University, Prague, CSSR ; "Institut fGr Rahtsmedizin, Frankfurt/Main, Westtun Germany (Accepted jar publication 28 February 1978)
Twsorestc~ and D. Mess. Pharmacological and biochemical properties of a venom gland extract from the snake 7nelotarnis kirtltvrdi. Toxkon 16 . 533-542, 1978 .-The coagulant activity and sours pharmacological and biochemical properties of a venom gland extract of the opistoglyphous snake 7&elotornis klrtlattdi were studied. An attempt was made to isolate the coagulating component from other enzymes. Its cool . wt is approximately 85,000 and its isoelectric point was at pH 523 . The coagulant activity, detectable only in one fraction, decreased considerably in the course of separation and lyophili7ation . Disc electrophoresis on polyacrylamide gel, of the isolated procoagulant and the raw gland extract, was performed. The raw gland extract coagulates citrated plasma iadependerttly of Ca't ions, platelets and phospholipids. It has no thrombin-like activity, directly converts prothmmbin to thrombin and in viva has a strong deßbrinating capacity . 7]titlotornis vtatom gland extract is stable when heated below SS°C for 10 min and retains its coagulant activity in the range of pH ó~0-8~2. T}fe LDeo on albino mice by i .v. mute is 023 mg/kg. Haemotrhagic activity is comparable to that of Bothrops Jar»rara. Neither phospholipase A nor i-amino acid oxidase activity were detatod . Phartrtaoological and biochemical properties of Thelotornis kirtlandi venom gland extract wen compared with some venoms of soknoglyphous snakes. F. KORNALIK, E.
INTRODUCTION
the founders of modern herpetology professor R. MERTENS died in 1975 from a rare bite by the opistoglyphous snake Thelotornls kirtlattdi . The predominant symptoms of the poisoning were severe haemorrhagic diathesis associated with total defibrination due to consumption coagulopathy . Lung oedema and renal failure were the actual cause of death on the 18th day after the bite. Total defibrination, lasting 8 days despite fibrinogen transfusion, was the main haematologic symptom . With some exceptions there is very little known about venoms of opistoglyphous snakes . Most of the papers contain a description of clinical symptoms ~BEIRAN ánd CURRIE, 1%7 ; FITZSIAtONS and SMITH, 1958 ; MACKAY et al., 1969 ; MA7ELL et al., 1973) . We tried therefore to analyse the venom gland extract of four specimens of Thelotornis kirtlotrdl. Our aim was to describe the coagulant activity and to assay some pharmacological and biochemical properties. ONE of
Preparation oJ'extracr
MATERIALS AND METHODS
Both venom glands from 4 specimen of Thelotorafs kirtlondf were ground and eluted with 4 parts (w/v) of saline . After 1 hr of stirring at room temperature the clear supernatant was freeze-dried and kept at 4°C before use. In all assays the freeze-dried gland extract was dissolved in distilled water and the conrentration of the solutions expressed in protein content per ml assessed using the method of LowaY er . al. (1951) . Other venoms or activators of known activity were often run simultaneously for comparison . Clotting activity in vitro was determined according to the tahnique of Owxex (1949) in a water bath 533
536
FRANTISEK KORNALIK, EVA TÁBORSKÁ and DIETRICH MEBS
at 37°C. The substrates and enzymes used were as follows; (a) Platelet rich plasma and platelet poor plasma -normal human citrated blood was centrifuged (at room temperature) at 200 g for I S min in order to obtain platelet rich plasma and at ]A00 g for 20 min to obtain platelet poor plasma . (b) Prothrombin free plasma -normal human oxalated plasma was absorbed twice on BaSO . (100 mg of BaSO . per I ml of plasma). (c) Bovine fibrinogen (Imuna n.p ., Sarisské Michalany, batch No. 01-0576) was rendered devoid of traces of Prothrombin by repeated absorption on BaSO,. (d) Prothrombin prepared according to the method of lt~aw.~t.r, and Scr~rua~ (1969) was obtained from Dr . S. Iwanaga of Ohsaka University and used in a concentration of 1 mglml in 0" 1 M Tris-HCI buffer, pH 7"3. (e) Thrombin-Topostasin Roche (SA F. Hoffmann--La Roche dt Co . AG, Basel, Schweiz) batch No . 8854191 X. (f) Prothrombin activator was prepared according to procedure of Mtu.Ex and COPELAND (I%5). (g) Ecarin-a snake coagulase isolated from F_clris carüratrrs venom was prepared as previously described (KORNAt.íK, 1971). (h) All other snake venorns used were collected from snakes kept in our institute in Prague . Prothrombin salvation (KORNALIK and BIAMBi+CK, 1975). To 1 ml of Prothrombin solution 0~1 ml of activators (77relotornls gland extract, Ecarin, Prothrombin activator) were added and test tubes placed in a water bath at 37 °C. At various intervals (0-24 hr) samples of 0~ l ml were withdrawn and added to 0'2 ml of 0"5~ fibrinogen solution and clotting time recorded . The ensuing thrombin activity expressed in NIH U per ml was calculated from a standard curve. Dejibrfrration in viva . White Wistar rats (180-200 g) were injected with different doses of gland extract or Ecarin . At various times samples of blood were withdrawn from the abdominal aorta while the animals were anesthetized with ether. Citrate was added to a final ratio of 9 blood : I citrate . Fibrinogen concentration was assessed in plasma samples according to the method of BLOMBi~CK and Bt-oMancx (1956) . For each time interval and each defibrinating substance batches of 10 rats were used . S.D . in every experiment was less than t 5 %. Isobtion of procoagrrlarrt . Gel filtration of 77relotornis gland extract on a Sephadex G-150 column (80 x. 1 "S cm) was carried out with O~OS M Tris-HCI buffer, pH 7"5. Fractions of G ml were collected. Clotting activity was tested on human citrated plasma and the isolated procoagulant freeze-0ried . MolecWar weight ojisolat~d procoagularrt was determined on a Sephadex G-100 column (100 ~~ I~5 cm) using 0"05 M Tris-HCI buffer, pH 7-0, with 0"S M NaCI as eluant . As reference proteins a-bungarotoxin (mol . wt 8,000) prepared by Dr. Mebs, cytochrome c (mol . wt 13,000), a-chymotrypsinogen (mol . wt 25 .000) and ovalbumin, (mol . wt 45,000) were used (Sews Feinbiochemical, Heidelberg, GFR) . Isoelectrlc point of isolated procoagulant was determined by isoekctric focusing performed in a 110 ml LKB column (LKB Productor AB, Bromma, Sweden), 2~ ampholine, pH gradient 3~5 to 100, 9t10 V for 24 hr at 18°C . Disc electrophoresis (Dwrs, 1964 ; OeNSrErN, 1964 ; SULITZEANU !t a/., 19tí7) was performed on 7~5 and 3~5~ polyacrylamide gel solution, using a pH 8" 3 Tris-glycine buffer system . A current of 4 mA was applied to each tube for 70--80 min at room temperature. Samples were applied in amounts of 200 Ng . Staining was with amidoblack in 7"5 % acetic acid . Heat stability. The gland extract in concentration of 2 pg/ml was heated to various temperature fur 10 min. After cooling the clotting activity of each sample was assessed as described. pH stability. The samples of gland extract (2 pgJml) were adjusted to required pH with 001 M HCl or 0"Ol M NaOH . After 20 min the pH was adjusted to the starting value and clotting time was assessed, Torlclry. Groups of five albino female mice (20 g) were injected i.v . with different concentrations of gland extract. The t.Dso was determined after 24 hr and statistically evaluated by the probit method . Naemorrhagic activity . Rabbit red blood cells labeled with °lchromitun were administerod together with l % Geigy blue into rabbits ear veins. Immediately afterwards O~l ml of various concentrations of the samples to be tested were injected s.c . into the depilated rabbits' back . One hour later the ° 1 Cr activity of skin samples, with haemorrhagic spots around the venom injection site, was measured on a scintillation counter with Nal (Tl) hollow crystal at I ~25 kV . Haemorrhagic activity was expressed as the amount of extravascular blood. ~-Amino acid oxidase activity. (L-Amino acid-0x~xireductase EC 1 .4.3.2) was determined aaording to the method Of KACHMAR and BOMER (1953) . L-Amino acid oxidase activity was expressed in umoles of pyruvate released from the substrate t.-kucine. Phosphodlesterase artivlty (orthophosphate-0iester-phosphohydrolase EC 3.1 .4 .1) was assessed by the method Of SINSHEIMER and KOERNER (1952) . The amount of p-nitrophenol released from the substrate is directly proportional to the enzyme activity. Plrospholipase activity (phosphatide-aryl-hydrolase EC 3 .1 .1 .4) was tested using egg-yolk lecithin as substrate (KORNALIK and MASTER, 1964). Lysolecithin released as a result of the enzyme activity was assessed by means of haemolytic effect on washed htunan red blood cells. The phospholipase activity wss expressed as pg/ml of the sample causing SO°ó haernolysis. RESU LTS
Coagulant activity of Thelotornis kirtlandi venom gland extract was studied first in a
T. b~irllaadi Vroom Gland Extrael
537
screening test using various substrates . The clotting activity is independent of the presence of platelets. The gland extract does not convert purified fibrinogen and therefore no thrombin-like activity can be attributed to it. The coagulant activity is, however, dependent on the presence of the prothrombin complex, which is absorbed on barium sulphate (Table 1). It seems obvious that the gland extract directly converts prothrombin to thrombin without other plasma cofactors being necessary. TABLE l . DEPENDENCE OF Thelolornlr kirrlarrdi cnACULANr ACTIVITY ON THE PRESENCE OF PLATELETS AND ON THE PRO THAOMHIN COMPLEX. COMPARISON WITH ECARIN, SIMPLASTIN AND THROMa1N
7ïrelorornls
Enzyme Scann Simplastin Thrombin seconds > 120 > 120 12
Substrate Fibrinogen > 120 Platelet rich 25 30 12 13 plasma Platelet poor ?, 22 14 13 plasma IiaSO, absorb . 42 > 120 13 plasma 55 System : 0~1 ml substrate + O~l ml saline + 0~1 ml enzyme . Results arc means of 10 experiments using 3 different samples of substrates with S.D . of z 2 sa .
To confirm this assumption the extract was allowed to react on purified prothrombin (0~I ~ solution = 2000 NTH U per 1 ml). At various time intervals the ensuing thrombinactivity was tested on O~S;ó bovine fibrinogen. Results are expressed in NIH thrombin units (Fig. 1 l. It is evident that the gland extract converts prothrombin directly to thrombin roughly in the same manner as Ecarin or prothrombin activator. zooo
System : i ml II+0 .3 saline " 0 .3 oetivotor TK
isoo
EC iooo
AC
0
Intubation time,
hr
FIG. I . CONVERSION OF PROTHROMHIN aY TÎltIOIOrRIS klrrlmál VENOM GLAND I7CTRACT (TIC), ECARIN (EC) OR PHYSIOLOGICAL ACTIVATOR OF PROTHROMDIN CAC) .
To 1 ml of prothrombin solution 0~1 ml of activators were added and tat tuba plead is a water bath at 37 ° C. At various intervals (0-24 hr) samples of 0" 1 ml were withdrawn and added to 0~3 ml of 0~3 ~ó fibrinogen solution and clotting time recorded . The ensuing thrombin acti~7ty expressed is NIH U per ml was calculated from a standard curve. In 8 eaperiatettts S.D . _ - 22 NIH U thrombin .
S38
FRANTISEK KORNALIK, EVA TABORSKÁ and DIETRICH MEBS
Defibrinating activity of the gland extract in vivo was tested on white Wistar rats and compared with that of Ecarin . A dose of 10 ltg of extract per kg or 151íg of Ecarin per kg was injected i.v. and in time intervals from 10 min to 72 hr the fibrinogen level in the plasma was assessed. Fibrinogen disappeared in the case of the gland extract within l0 min and after Ecarin injection in one hr. Afibrinogenemia lasted for 24 hr ; fibrinogen reached normal levels on the 3rd day after injection in both cases l,Fig. 2). Plasma samples collected at time intervals from 10 min to 12 hr in animals defibrinated with the gland extract exhibit strong haemolysis .
ioo
aO ó ;! 0~ a
Li o
1io
20
30
~ z
~ '
F-ié -I
FIG . 2 . DEPIBtIINATtNO ACTIVITY OF THE GLAND EXTRACT AND ECAAtN Îp V1r0 . White rats were injected with 10 ~ of the Mand extract per kg or with 1S 4íg of Ecarin per kg . At intervals fibrinogen (Fbg) concentration was assessed in plasma samples and expressed in of notanal fibrinogen level . For each time interval and each deftbrinating substance batches of 10 rats were used . S .D . in every experiment was less than -~ S "; fibrinogen .
An attempt has been made to isolate the procoagulant fraction from the crude gland extract by means of gel filtration on a Sephadex G-150 column . Activity appeared in tubes 25-35. Molecular weight of the isolated coagulane, assessed on Sephadex G-100 using various reference proteins was estimated as approximately 85,000 (Fig. 3). Isoelcctric point was at pH 5"25 by the electrofocusing method (Fig. 4). Homogeneity of the isolated procoagulant was analysed by means of disc electrophoresis on a polyacrylamide gel and compared with that of crude venom gland extract. Besides the main band (very likely the coagulane) two fainter bands were detected . The procoagulant lost its activity in the course of separation and lyophilization . Both coagulant activity and toxicity decreased 200 fold during these procedures . The in vivo defibrinating potency, however, decreased only ten fold in comparison with crude venom gland extract (Table 2). We decided therefore to analyse further only the crude Thelotorrris venom gland extract . Heated to 55°C or above for 10 min the extract lost most of its coagulant activity . The pH optimum was between 6~0 and 8"2. Lethality was assessed by i .v. injection of various concentrations of the gland extract in white mice and compared with two samples of Echis carinates venom. The LDso of the Thelotornis (0"25 mgJkg, S.D. = i 0" 13) was comparable to that more potent sample of Echis rarinatus venom (1~3 mg/kg, S. D . _ -i- 0" 14), while the less potent Echis carinates venom had an t.Dsu of 6" 5 mgJkg (S.D. _ ± 1 "3).
T. klrtlmgd! Venom Gland Extract
539
zz zo
i6
TMlotornie -enzyme
Iz 40
4 .2
44
46
4.9
S.0
loq mol. wt FiG. 3. MOLECULAR WEIGHT DETERMINATION OF iiIllOt0171lS kirtlaed! COAGULATIt~~ ENZYME vstNG SEPHADEX G-10O, COLUMN í00 x l~S an, Ofi5 M Tws-HC1 HuPPEa pH 7~0wmI 0" SM
NaQ . n-bungarotoxin (8000), cytochrome c (13,000), a~llymotrypsinogen (23,000) and ovalbumin (45,000) wen used as rrferrnt proteins . Molecular weight of 85,000 was assessed for 77telotorn/s procoagalmrt .
Tube
No.
IiETERIüNAT10N OF THE 180ELLCTRIC POINT (PI) OF Î%KIOIOI71lJ k/rl/má! COAOULATING ENZYME HY 190ELECTAIC FOC'U9IN0 PEAFOIt!(ED IN A ~ tO R)l C.'Oit .UMN (LKH), 2 ~ AlllHOiINE, pH GRADIENT 3~sao-o, 90o v porl 2a hr Ar ls°c. FIG .
4.
Haemorrhagic activity of the gland extract was compared to that of two other venoms, known to have a relatively high concentration of haemorrhagins. The activity was similar to that of Bothrops jararaca (Table 3). This activity of the gland extract is not as high as one would expect based upon the clinical symptoms described following Thelotorttls kirtlarldl bite (blebs-personal communication) . t-Amino acid oxidase activity of the gland extract was compared with samples of white and yellow venom of two specimens of Echis carirtatus . No L-amino acid oxidase activity could be detected in Tleelotorn/s kirtla~Idi. Phosphodiesterase activity was cam-
54 0
FRANTISEK KORNALIK, EVA TÁBORSKÁ and DIETRICH MEES TABLE 2. COMPARISON OF COAGULATING ACTIVITY, DEFIBRINAT INO POTENCY AND rRru" ~ ITY OF TItCIOtOrlflJ VENOM GLAND EXTRACT AND130LATED PROCOAGULANT Crude venom Isolated Gland extract procoagulant Clotting time 20 xc 2 Itglml 400IIg/ml Deßbrination in vivo S Etg/kg SO l+Ylkg LDeo als mg/kg 40 mg/kg Cotloentration of gland extract and isolated procoagulant expressed in pg/ml of protein required for clotting of normal human citrated plasma . Deflbrination assessed in rats 24 hr aRer i .v . igjection . LD,o on albino mice by i.v . route, as described in methods . TABLE 3 . HAEMORRHAGiC ACTIVr1Y OF 77ulatornts kirtland! EXPRE~ AS THE AMOUNT OF EXTAAVASCULAa RED BLOOD CELLS AT THE SITE OF INTRACUTANEOIJS INJECTION OF THE SAMPLE . COMPARISON IS MADE WrrH TWO OTHER VENOMS Amount of Exttavascular toxin (mg) red blood cells Toxin (ml) Agkistrodan rhodbstonta 0"2 0"248 Bothropsjttraraca a2 0" t l0
Thelatorntskirtiattdt
o" 1s
o"1l0
pared to that of three other venoms and is comparable to that of Agkistrodon rhodostoma venom . Using egg-yolk lecithin as substrate, and rat red blood cells, no phospholipase A activity could be detected in the gland extract even when high doses were used . DISCUSSION The results of the screening tuts using various substrates indicated the absence of any thrombin-like action of Thelotornis kirtlandi. Its action does not require platelets or CaJ+ indicating that other factors of the prothrombin complex, dependent on phospholipids and Cas+ (factor X and factor VII) were not involved in the prothrombin conversion caused by Thelotornis kirtlandi. In another series of experiments (not described in this paper) u mixture of normal and factor X deficient plasma was used as substrate for the gland extract . It was shown that gland extract does not activate factor X to factor Xa . Using purified prothrombin as substrate we conßrmed that gland extract converts prothrombin to thrombin in the same way as a physiological activator or Ecarin does. It is very likely that this coagulant is responsible for a rapid defibrination, due to consumption coagulopathy, in vivo . A striking Ending, however, was that fibrinogen disappeared in the case of the gland extract within 10 min after injection . Complete defibrination is not likely to be accomplished that fast to vivo . The fibrinogen assay method requires at least 30 min to perform . During this time in vitro defibrination of plasma contintus. The same of course applies for Frcarin, in which case a full defibrination, under the identical experimental conditions, is achieved in approximately 1 hr . This indicates that the gland extract is one of the fastest acting defibrinating substances ever described in snake venoms. The isolation method used for the coagulant enryme proved to be inconvenient because the separation was not satisfactory and the partially purifted substance lost most of its activity . Since the crude venom gland extract was lyophilized the same as the isolated fraction it is very likely that the loss of the specific activity of the coagulane was caused by the subsequent separation procedure. A decreased activity in the individual fraction
T. kirtlandi Venom Gland Extract
341
separated by various techniques from snake venoms is not unusual (MAS~rFJt and RAO, 1961 ; MoROZ et a1., 1966). It is assumed that for some of the specific activities a combined action of cofactors is necessary. The decrease could also be due to structural changes in the molecule during separation or to ionic changes in the eluents. Thelotoriris klrtlundi dissolved in an opalescent white solution without any trace of yellow coloration. A parallel between the flavin adenin dinucleotide content, yellow color and L-amino acid oxidase activity is well known and applies also for the gland extract (KORNALIK and MASTER, 1964). One of the most common enrymes in snake venoms is phospholipase Az. There is practically no phospholipase activity in the gland extract. In regard to the severe haemolytis, occurring even after sublethal doses, this finding indicates that the haemolytis was mainly due to the very rapidly developing consumption coagulopathy. Direct haemolytic effect of the gland extract could be ruled out in experiments in vitro using human and rat red blood cells. In two of the 26 samples of Echis carinatus venoms tested some years ago (TÁHORSKÁ, 1971) a total absence of phospholipase A could be detected . One of them, devoid incidentally of L-amino acid oxidase activity as well, was tested simultaneously with the gland extract. The intraspecies variability in the composition of individual snake venoms is a well known fact which, however, can be detected only by collecting venoms from individual specimens (.1Ih11NEZ-PORRAS, 1964 ; ScHeNBSxc, 1963). In our case the starting raw material was prepared from the glands of four snakes . The absence of the two enrymes in the extract can therefore hardly be explained as individual variability in the composition of the tested venoms and is probably a general attribute of the Thelotornis gland extract. The extract from the venom gland of the opistoglyphous snake Thelotornis kirtlandi differs in its biochemical and pharmacological properties from other snake venoms only in two respects : ( I ) The absence of two common enrymes-phospholipase A and L-amino acid oxidase and (2) the presence of a very powerful specific prothrombin converting coagulase . It should be stressed that in our studies a crude gland extract was used and the specific activities were calculated in relation to the amount of protein. It is obvious that the specific activity of the venom produced by the gland and injected in a bite would be much higher . REFERENCES
D. and Cttxwe, G. (I%7) Snake bite due to Thelotornis kirtlardi. Cent. .tfr. J. Med. 13, 137. Bi otitai+cu, B. and Bw~takcK, M . (1956) Purification of human and bovine ßbrinogen . Ark. Xatri l0, 415. Dwvts, H. J. (1964) Disc electrophoresis-11 . Method and application to serum proteins . Ann. N.Y. Acad. Scí. 121, 404. FITZSIMOnS, D. C. and SMITH, H. M . (1938) Another rear-fanged South African snake lethal to humans . Herpetologica 14, 198 . INGWALL, J. S. and SCHERAGw, H. A. (1%9) Purification and properties of bovine prothrombin, Biochemistry S, 1860 . JIMENÉZ-PoxxAS, J. M. (1964) Intraspaific variations in composition of venom of the jumping viper, Bothrop.r ttttmmifera . TJxiCOR 3, 187. KACHMAR, J . L. and B01'ex, P. D. (1953) Kinetic analysis of enzyme tractions--II. The potassium activation and calcium inhibition of pyruvic phosphoesterase . J . biol. Chem. 200, 669. K(1xNAL1K, F, and MwsTex, R . W. P. (1964) A comparative examination of yellow and white venoms of Vipers ammodytes. Toxicorr 2, 109 Koxxwr.lx, F (1971) Fibrinolytische Proteasen sus SchlanQerlgiften . Folio haemat. 95, 193. Koxlvwr.iK, F. and Bt .oMSXcr<, B (1975) Prothrombin activation induced by Ecarirt--a prothrombin ocmverting enzyme from Echis carluatus venom Thromb. Res . 6, 53 . Lowx}', O. H., ROSEHROUGH, N. J., FARR, A. L . and RwNnwLr-, R. J. (1931) Protein measurement with the Folin-phenol reagent. J. biol. Chem . 193, 263. Mwctcws~, N., FERGUSOIT, J. C., BACKSHAWE, A., Foxxesren, A. T. T. and McNtcot, G. P. (1969) The venom of the boomslang (Dispholidus t~pus) : in vivo and in vitro studies . 7ytromb . Diath. Haetnorrh . 21, 234. HEIRAN,
542
FRANTISEK KORNALIK, EVA TÁBORSKÁ and DIETRICH MEBS
Mestfat, R. W. P. and Rt+o, S. S. (1%1) IdentiSeation of enzyme and toxins in venons of indien cobra and Russell's Vips after starch rel electrophoresis . J. biol. Client. 236, 1986 . MArtn.t., ß., NYrux, D., Wtsttrtt:nn, B, and W*r~~ ....^N, S. (1973) Consumption coarulopathy caused by a boomalant bite. A cafe report. 77trotnb. Ree. 3, 173. Mtt.z~t, K. D. and Corewvn, W. H. (196s) Human thrombin : isolation and stabllity. Erpl . ~~lolec . Putlt. 4, 431. Moitoz, Ca., De Via, A. and St:u, M. (1966) Isolation and characterization of neurotoxin from Vlperu palsstinae venom. Blochirn. biopJtys. Acte 124, 136. Ontva~nv, L. (1964) Disc eleetrophotssis-I. Hackrround and theory . AndN. Y. Aced. Sci.121, 32l . Owaetv, P. A. (1949) A quantitative one-state method for the assay of prothrombin . Scand. J. clip . Lab . Invest. 1, 181. 5cttttrteatw, S. (1%3) immttaolotical (Ouchterlony method) identiScation of intrasubspecies qualitative differences in snake vettorn ootnposition. Toxican 2, 67 . $ITIal~R, R. L. and Koeantete, J. F. (19s2) A purification of venom phosphodiesterase . J. biol . Chem . 199, 293. Suu~tzsertu, D., St .evtx, M. and JerHestt ~ *, E. (1%7) Simplified technique for preparative disc electrophotesis--lI . Further improvements in apparatus and some details of performance . Analyt. Biochem. n, s7 . T~(aops[~(, E. (1971) Inttaapaxes variability of the venom of Echis carinates. Physiologic bohemoslov . 20, 307.
Press Ltd.
0041-0I01/78J1101-0333502 .00/0
Printed in Great Britain .
PHARMACOLOGICAL AND BIOCHEMICAL PROPERTIES OF A VENOM GLAND EXTRACT FROM THE SNAKE THELOTORNIS KIRTLANDI FRANTISEK KORNALIK, EVA Tr~IàORSKÁ
and
DIETRICH MEes*
Institute of Pathophysiology Charles University, Prague, CSSR ; "Institut fGr Rahtsmedizin, Frankfurt/Main, Westtun Germany (Accepted jar publication 28 February 1978)
Twsorestc~ and D. Mess. Pharmacological and biochemical properties of a venom gland extract from the snake 7nelotarnis kirtltvrdi. Toxkon 16 . 533-542, 1978 .-The coagulant activity and sours pharmacological and biochemical properties of a venom gland extract of the opistoglyphous snake 7&elotornis klrtlattdi were studied. An attempt was made to isolate the coagulating component from other enzymes. Its cool . wt is approximately 85,000 and its isoelectric point was at pH 523 . The coagulant activity, detectable only in one fraction, decreased considerably in the course of separation and lyophili7ation . Disc electrophoresis on polyacrylamide gel, of the isolated procoagulant and the raw gland extract, was performed. The raw gland extract coagulates citrated plasma iadependerttly of Ca't ions, platelets and phospholipids. It has no thrombin-like activity, directly converts prothmmbin to thrombin and in viva has a strong deßbrinating capacity . 7]titlotornis vtatom gland extract is stable when heated below SS°C for 10 min and retains its coagulant activity in the range of pH ó~0-8~2. T}fe LDeo on albino mice by i .v. mute is 023 mg/kg. Haemotrhagic activity is comparable to that of Bothrops Jar»rara. Neither phospholipase A nor i-amino acid oxidase activity were detatod . Phartrtaoological and biochemical properties of Thelotornis kirtlandi venom gland extract wen compared with some venoms of soknoglyphous snakes. F. KORNALIK, E.
INTRODUCTION
the founders of modern herpetology professor R. MERTENS died in 1975 from a rare bite by the opistoglyphous snake Thelotornls kirtlattdi . The predominant symptoms of the poisoning were severe haemorrhagic diathesis associated with total defibrination due to consumption coagulopathy . Lung oedema and renal failure were the actual cause of death on the 18th day after the bite. Total defibrination, lasting 8 days despite fibrinogen transfusion, was the main haematologic symptom . With some exceptions there is very little known about venoms of opistoglyphous snakes . Most of the papers contain a description of clinical symptoms ~BEIRAN ánd CURRIE, 1%7 ; FITZSIAtONS and SMITH, 1958 ; MACKAY et al., 1969 ; MA7ELL et al., 1973) . We tried therefore to analyse the venom gland extract of four specimens of Thelotornis kirtlotrdl. Our aim was to describe the coagulant activity and to assay some pharmacological and biochemical properties. ONE of
Preparation oJ'extracr
MATERIALS AND METHODS
Both venom glands from 4 specimen of Thelotorafs kirtlondf were ground and eluted with 4 parts (w/v) of saline . After 1 hr of stirring at room temperature the clear supernatant was freeze-dried and kept at 4°C before use. In all assays the freeze-dried gland extract was dissolved in distilled water and the conrentration of the solutions expressed in protein content per ml assessed using the method of LowaY er . al. (1951) . Other venoms or activators of known activity were often run simultaneously for comparison . Clotting activity in vitro was determined according to the tahnique of Owxex (1949) in a water bath 533
536
FRANTISEK KORNALIK, EVA TÁBORSKÁ and DIETRICH MEBS
at 37°C. The substrates and enzymes used were as follows; (a) Platelet rich plasma and platelet poor plasma -normal human citrated blood was centrifuged (at room temperature) at 200 g for I S min in order to obtain platelet rich plasma and at ]A00 g for 20 min to obtain platelet poor plasma . (b) Prothrombin free plasma -normal human oxalated plasma was absorbed twice on BaSO . (100 mg of BaSO . per I ml of plasma). (c) Bovine fibrinogen (Imuna n.p ., Sarisské Michalany, batch No. 01-0576) was rendered devoid of traces of Prothrombin by repeated absorption on BaSO,. (d) Prothrombin prepared according to the method of lt~aw.~t.r, and Scr~rua~ (1969) was obtained from Dr . S. Iwanaga of Ohsaka University and used in a concentration of 1 mglml in 0" 1 M Tris-HCI buffer, pH 7"3. (e) Thrombin-Topostasin Roche (SA F. Hoffmann--La Roche dt Co . AG, Basel, Schweiz) batch No . 8854191 X. (f) Prothrombin activator was prepared according to procedure of Mtu.Ex and COPELAND (I%5). (g) Ecarin-a snake coagulase isolated from F_clris carüratrrs venom was prepared as previously described (KORNAt.íK, 1971). (h) All other snake venorns used were collected from snakes kept in our institute in Prague . Prothrombin salvation (KORNALIK and BIAMBi+CK, 1975). To 1 ml of Prothrombin solution 0~1 ml of activators (77relotornls gland extract, Ecarin, Prothrombin activator) were added and test tubes placed in a water bath at 37 °C. At various intervals (0-24 hr) samples of 0~ l ml were withdrawn and added to 0'2 ml of 0"5~ fibrinogen solution and clotting time recorded . The ensuing thrombin activity expressed in NIH U per ml was calculated from a standard curve. Dejibrfrration in viva . White Wistar rats (180-200 g) were injected with different doses of gland extract or Ecarin . At various times samples of blood were withdrawn from the abdominal aorta while the animals were anesthetized with ether. Citrate was added to a final ratio of 9 blood : I citrate . Fibrinogen concentration was assessed in plasma samples according to the method of BLOMBi~CK and Bt-oMancx (1956) . For each time interval and each defibrinating substance batches of 10 rats were used . S.D . in every experiment was less than t 5 %. Isobtion of procoagrrlarrt . Gel filtration of 77relotornis gland extract on a Sephadex G-150 column (80 x. 1 "S cm) was carried out with O~OS M Tris-HCI buffer, pH 7"5. Fractions of G ml were collected. Clotting activity was tested on human citrated plasma and the isolated procoagulant freeze-0ried . MolecWar weight ojisolat~d procoagularrt was determined on a Sephadex G-100 column (100 ~~ I~5 cm) using 0"05 M Tris-HCI buffer, pH 7-0, with 0"S M NaCI as eluant . As reference proteins a-bungarotoxin (mol . wt 8,000) prepared by Dr. Mebs, cytochrome c (mol . wt 13,000), a-chymotrypsinogen (mol . wt 25 .000) and ovalbumin, (mol . wt 45,000) were used (Sews Feinbiochemical, Heidelberg, GFR) . Isoelectrlc point of isolated procoagulant was determined by isoekctric focusing performed in a 110 ml LKB column (LKB Productor AB, Bromma, Sweden), 2~ ampholine, pH gradient 3~5 to 100, 9t10 V for 24 hr at 18°C . Disc electrophoresis (Dwrs, 1964 ; OeNSrErN, 1964 ; SULITZEANU !t a/., 19tí7) was performed on 7~5 and 3~5~ polyacrylamide gel solution, using a pH 8" 3 Tris-glycine buffer system . A current of 4 mA was applied to each tube for 70--80 min at room temperature. Samples were applied in amounts of 200 Ng . Staining was with amidoblack in 7"5 % acetic acid . Heat stability. The gland extract in concentration of 2 pg/ml was heated to various temperature fur 10 min. After cooling the clotting activity of each sample was assessed as described. pH stability. The samples of gland extract (2 pgJml) were adjusted to required pH with 001 M HCl or 0"Ol M NaOH . After 20 min the pH was adjusted to the starting value and clotting time was assessed, Torlclry. Groups of five albino female mice (20 g) were injected i.v . with different concentrations of gland extract. The t.Dso was determined after 24 hr and statistically evaluated by the probit method . Naemorrhagic activity . Rabbit red blood cells labeled with °lchromitun were administerod together with l % Geigy blue into rabbits ear veins. Immediately afterwards O~l ml of various concentrations of the samples to be tested were injected s.c . into the depilated rabbits' back . One hour later the ° 1 Cr activity of skin samples, with haemorrhagic spots around the venom injection site, was measured on a scintillation counter with Nal (Tl) hollow crystal at I ~25 kV . Haemorrhagic activity was expressed as the amount of extravascular blood. ~-Amino acid oxidase activity. (L-Amino acid-0x~xireductase EC 1 .4.3.2) was determined aaording to the method Of KACHMAR and BOMER (1953) . L-Amino acid oxidase activity was expressed in umoles of pyruvate released from the substrate t.-kucine. Phosphodlesterase artivlty (orthophosphate-0iester-phosphohydrolase EC 3.1 .4 .1) was assessed by the method Of SINSHEIMER and KOERNER (1952) . The amount of p-nitrophenol released from the substrate is directly proportional to the enzyme activity. Plrospholipase activity (phosphatide-aryl-hydrolase EC 3 .1 .1 .4) was tested using egg-yolk lecithin as substrate (KORNALIK and MASTER, 1964). Lysolecithin released as a result of the enzyme activity was assessed by means of haemolytic effect on washed htunan red blood cells. The phospholipase activity wss expressed as pg/ml of the sample causing SO°ó haernolysis. RESU LTS
Coagulant activity of Thelotornis kirtlandi venom gland extract was studied first in a
T. b~irllaadi Vroom Gland Extrael
537
screening test using various substrates . The clotting activity is independent of the presence of platelets. The gland extract does not convert purified fibrinogen and therefore no thrombin-like activity can be attributed to it. The coagulant activity is, however, dependent on the presence of the prothrombin complex, which is absorbed on barium sulphate (Table 1). It seems obvious that the gland extract directly converts prothrombin to thrombin without other plasma cofactors being necessary. TABLE l . DEPENDENCE OF Thelolornlr kirrlarrdi cnACULANr ACTIVITY ON THE PRESENCE OF PLATELETS AND ON THE PRO THAOMHIN COMPLEX. COMPARISON WITH ECARIN, SIMPLASTIN AND THROMa1N
7ïrelorornls
Enzyme Scann Simplastin Thrombin seconds > 120 > 120 12
Substrate Fibrinogen > 120 Platelet rich 25 30 12 13 plasma Platelet poor ?, 22 14 13 plasma IiaSO, absorb . 42 > 120 13 plasma 55 System : 0~1 ml substrate + O~l ml saline + 0~1 ml enzyme . Results arc means of 10 experiments using 3 different samples of substrates with S.D . of z 2 sa .
To confirm this assumption the extract was allowed to react on purified prothrombin (0~I ~ solution = 2000 NTH U per 1 ml). At various time intervals the ensuing thrombinactivity was tested on O~S;ó bovine fibrinogen. Results are expressed in NIH thrombin units (Fig. 1 l. It is evident that the gland extract converts prothrombin directly to thrombin roughly in the same manner as Ecarin or prothrombin activator. zooo
System : i ml II+0 .3 saline " 0 .3 oetivotor TK
isoo
EC iooo
AC
0
Intubation time,
hr
FIG. I . CONVERSION OF PROTHROMHIN aY TÎltIOIOrRIS klrrlmál VENOM GLAND I7CTRACT (TIC), ECARIN (EC) OR PHYSIOLOGICAL ACTIVATOR OF PROTHROMDIN CAC) .
To 1 ml of prothrombin solution 0~1 ml of activators were added and tat tuba plead is a water bath at 37 ° C. At various intervals (0-24 hr) samples of 0" 1 ml were withdrawn and added to 0~3 ml of 0~3 ~ó fibrinogen solution and clotting time recorded . The ensuing thrombin acti~7ty expressed is NIH U per ml was calculated from a standard curve. In 8 eaperiatettts S.D . _ - 22 NIH U thrombin .
S38
FRANTISEK KORNALIK, EVA TABORSKÁ and DIETRICH MEBS
Defibrinating activity of the gland extract in vivo was tested on white Wistar rats and compared with that of Ecarin . A dose of 10 ltg of extract per kg or 151íg of Ecarin per kg was injected i.v. and in time intervals from 10 min to 72 hr the fibrinogen level in the plasma was assessed. Fibrinogen disappeared in the case of the gland extract within l0 min and after Ecarin injection in one hr. Afibrinogenemia lasted for 24 hr ; fibrinogen reached normal levels on the 3rd day after injection in both cases l,Fig. 2). Plasma samples collected at time intervals from 10 min to 12 hr in animals defibrinated with the gland extract exhibit strong haemolysis .
ioo
aO ó ;! 0~ a
Li o
1io
20
30
~ z
~ '
F-ié -I
FIG . 2 . DEPIBtIINATtNO ACTIVITY OF THE GLAND EXTRACT AND ECAAtN Îp V1r0 . White rats were injected with 10 ~ of the Mand extract per kg or with 1S 4íg of Ecarin per kg . At intervals fibrinogen (Fbg) concentration was assessed in plasma samples and expressed in of notanal fibrinogen level . For each time interval and each deftbrinating substance batches of 10 rats were used . S .D . in every experiment was less than -~ S "; fibrinogen .
An attempt has been made to isolate the procoagulant fraction from the crude gland extract by means of gel filtration on a Sephadex G-150 column . Activity appeared in tubes 25-35. Molecular weight of the isolated coagulane, assessed on Sephadex G-100 using various reference proteins was estimated as approximately 85,000 (Fig. 3). Isoelcctric point was at pH 5"25 by the electrofocusing method (Fig. 4). Homogeneity of the isolated procoagulant was analysed by means of disc electrophoresis on a polyacrylamide gel and compared with that of crude venom gland extract. Besides the main band (very likely the coagulane) two fainter bands were detected . The procoagulant lost its activity in the course of separation and lyophilization . Both coagulant activity and toxicity decreased 200 fold during these procedures . The in vivo defibrinating potency, however, decreased only ten fold in comparison with crude venom gland extract (Table 2). We decided therefore to analyse further only the crude Thelotorrris venom gland extract . Heated to 55°C or above for 10 min the extract lost most of its coagulant activity . The pH optimum was between 6~0 and 8"2. Lethality was assessed by i .v. injection of various concentrations of the gland extract in white mice and compared with two samples of Echis carinates venom. The LDso of the Thelotornis (0"25 mgJkg, S.D. = i 0" 13) was comparable to that more potent sample of Echis rarinatus venom (1~3 mg/kg, S. D . _ -i- 0" 14), while the less potent Echis carinates venom had an t.Dsu of 6" 5 mgJkg (S.D. _ ± 1 "3).
T. klrtlmgd! Venom Gland Extract
539
zz zo
i6
TMlotornie -enzyme
Iz 40
4 .2
44
46
4.9
S.0
loq mol. wt FiG. 3. MOLECULAR WEIGHT DETERMINATION OF iiIllOt0171lS kirtlaed! COAGULATIt~~ ENZYME vstNG SEPHADEX G-10O, COLUMN í00 x l~S an, Ofi5 M Tws-HC1 HuPPEa pH 7~0wmI 0" SM
NaQ . n-bungarotoxin (8000), cytochrome c (13,000), a~llymotrypsinogen (23,000) and ovalbumin (45,000) wen used as rrferrnt proteins . Molecular weight of 85,000 was assessed for 77telotorn/s procoagalmrt .
Tube
No.
IiETERIüNAT10N OF THE 180ELLCTRIC POINT (PI) OF Î%KIOIOI71lJ k/rl/má! COAOULATING ENZYME HY 190ELECTAIC FOC'U9IN0 PEAFOIt!(ED IN A ~ tO R)l C.'Oit .UMN (LKH), 2 ~ AlllHOiINE, pH GRADIENT 3~sao-o, 90o v porl 2a hr Ar ls°c. FIG .
4.
Haemorrhagic activity of the gland extract was compared to that of two other venoms, known to have a relatively high concentration of haemorrhagins. The activity was similar to that of Bothrops jararaca (Table 3). This activity of the gland extract is not as high as one would expect based upon the clinical symptoms described following Thelotorttls kirtlarldl bite (blebs-personal communication) . t-Amino acid oxidase activity of the gland extract was compared with samples of white and yellow venom of two specimens of Echis carirtatus . No L-amino acid oxidase activity could be detected in Tleelotorn/s kirtla~Idi. Phosphodiesterase activity was cam-
54 0
FRANTISEK KORNALIK, EVA TÁBORSKÁ and DIETRICH MEES TABLE 2. COMPARISON OF COAGULATING ACTIVITY, DEFIBRINAT INO POTENCY AND rRru" ~ ITY OF TItCIOtOrlflJ VENOM GLAND EXTRACT AND130LATED PROCOAGULANT Crude venom Isolated Gland extract procoagulant Clotting time 20 xc 2 Itglml 400IIg/ml Deßbrination in vivo S Etg/kg SO l+Ylkg LDeo als mg/kg 40 mg/kg Cotloentration of gland extract and isolated procoagulant expressed in pg/ml of protein required for clotting of normal human citrated plasma . Deflbrination assessed in rats 24 hr aRer i .v . igjection . LD,o on albino mice by i.v . route, as described in methods . TABLE 3 . HAEMORRHAGiC ACTIVr1Y OF 77ulatornts kirtland! EXPRE~ AS THE AMOUNT OF EXTAAVASCULAa RED BLOOD CELLS AT THE SITE OF INTRACUTANEOIJS INJECTION OF THE SAMPLE . COMPARISON IS MADE WrrH TWO OTHER VENOMS Amount of Exttavascular toxin (mg) red blood cells Toxin (ml) Agkistrodan rhodbstonta 0"2 0"248 Bothropsjttraraca a2 0" t l0
Thelatorntskirtiattdt
o" 1s
o"1l0
pared to that of three other venoms and is comparable to that of Agkistrodon rhodostoma venom . Using egg-yolk lecithin as substrate, and rat red blood cells, no phospholipase A activity could be detected in the gland extract even when high doses were used . DISCUSSION The results of the screening tuts using various substrates indicated the absence of any thrombin-like action of Thelotornis kirtlandi. Its action does not require platelets or CaJ+ indicating that other factors of the prothrombin complex, dependent on phospholipids and Cas+ (factor X and factor VII) were not involved in the prothrombin conversion caused by Thelotornis kirtlandi. In another series of experiments (not described in this paper) u mixture of normal and factor X deficient plasma was used as substrate for the gland extract . It was shown that gland extract does not activate factor X to factor Xa . Using purified prothrombin as substrate we conßrmed that gland extract converts prothrombin to thrombin in the same way as a physiological activator or Ecarin does. It is very likely that this coagulant is responsible for a rapid defibrination, due to consumption coagulopathy, in vivo . A striking Ending, however, was that fibrinogen disappeared in the case of the gland extract within 10 min after injection . Complete defibrination is not likely to be accomplished that fast to vivo . The fibrinogen assay method requires at least 30 min to perform . During this time in vitro defibrination of plasma contintus. The same of course applies for Frcarin, in which case a full defibrination, under the identical experimental conditions, is achieved in approximately 1 hr . This indicates that the gland extract is one of the fastest acting defibrinating substances ever described in snake venoms. The isolation method used for the coagulant enryme proved to be inconvenient because the separation was not satisfactory and the partially purifted substance lost most of its activity . Since the crude venom gland extract was lyophilized the same as the isolated fraction it is very likely that the loss of the specific activity of the coagulane was caused by the subsequent separation procedure. A decreased activity in the individual fraction
T. kirtlandi Venom Gland Extract
341
separated by various techniques from snake venoms is not unusual (MAS~rFJt and RAO, 1961 ; MoROZ et a1., 1966). It is assumed that for some of the specific activities a combined action of cofactors is necessary. The decrease could also be due to structural changes in the molecule during separation or to ionic changes in the eluents. Thelotoriris klrtlundi dissolved in an opalescent white solution without any trace of yellow coloration. A parallel between the flavin adenin dinucleotide content, yellow color and L-amino acid oxidase activity is well known and applies also for the gland extract (KORNALIK and MASTER, 1964). One of the most common enrymes in snake venoms is phospholipase Az. There is practically no phospholipase activity in the gland extract. In regard to the severe haemolytis, occurring even after sublethal doses, this finding indicates that the haemolytis was mainly due to the very rapidly developing consumption coagulopathy. Direct haemolytic effect of the gland extract could be ruled out in experiments in vitro using human and rat red blood cells. In two of the 26 samples of Echis carinatus venoms tested some years ago (TÁHORSKÁ, 1971) a total absence of phospholipase A could be detected . One of them, devoid incidentally of L-amino acid oxidase activity as well, was tested simultaneously with the gland extract. The intraspecies variability in the composition of individual snake venoms is a well known fact which, however, can be detected only by collecting venoms from individual specimens (.1Ih11NEZ-PORRAS, 1964 ; ScHeNBSxc, 1963). In our case the starting raw material was prepared from the glands of four snakes . The absence of the two enrymes in the extract can therefore hardly be explained as individual variability in the composition of the tested venoms and is probably a general attribute of the Thelotornis gland extract. The extract from the venom gland of the opistoglyphous snake Thelotornis kirtlandi differs in its biochemical and pharmacological properties from other snake venoms only in two respects : ( I ) The absence of two common enrymes-phospholipase A and L-amino acid oxidase and (2) the presence of a very powerful specific prothrombin converting coagulase . It should be stressed that in our studies a crude gland extract was used and the specific activities were calculated in relation to the amount of protein. It is obvious that the specific activity of the venom produced by the gland and injected in a bite would be much higher . REFERENCES
D. and Cttxwe, G. (I%7) Snake bite due to Thelotornis kirtlardi. Cent. .tfr. J. Med. 13, 137. Bi otitai+cu, B. and Bw~takcK, M . (1956) Purification of human and bovine ßbrinogen . Ark. Xatri l0, 415. Dwvts, H. J. (1964) Disc electrophoresis-11 . Method and application to serum proteins . Ann. N.Y. Acad. Scí. 121, 404. FITZSIMOnS, D. C. and SMITH, H. M . (1938) Another rear-fanged South African snake lethal to humans . Herpetologica 14, 198 . INGWALL, J. S. and SCHERAGw, H. A. (1%9) Purification and properties of bovine prothrombin, Biochemistry S, 1860 . JIMENÉZ-PoxxAS, J. M. (1964) Intraspaific variations in composition of venom of the jumping viper, Bothrop.r ttttmmifera . TJxiCOR 3, 187. KACHMAR, J . L. and B01'ex, P. D. (1953) Kinetic analysis of enzyme tractions--II. The potassium activation and calcium inhibition of pyruvic phosphoesterase . J . biol. Chem. 200, 669. K(1xNAL1K, F, and MwsTex, R . W. P. (1964) A comparative examination of yellow and white venoms of Vipers ammodytes. Toxicorr 2, 109 Koxxwr.lx, F (1971) Fibrinolytische Proteasen sus SchlanQerlgiften . Folio haemat. 95, 193. Koxlvwr.iK, F. and Bt .oMSXcr<, B (1975) Prothrombin activation induced by Ecarirt--a prothrombin ocmverting enzyme from Echis carluatus venom Thromb. Res . 6, 53 . Lowx}', O. H., ROSEHROUGH, N. J., FARR, A. L . and RwNnwLr-, R. J. (1931) Protein measurement with the Folin-phenol reagent. J. biol. Chem . 193, 263. Mwctcws~, N., FERGUSOIT, J. C., BACKSHAWE, A., Foxxesren, A. T. T. and McNtcot, G. P. (1969) The venom of the boomslang (Dispholidus t~pus) : in vivo and in vitro studies . 7ytromb . Diath. Haetnorrh . 21, 234. HEIRAN,
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FRANTISEK KORNALIK, EVA TÁBORSKÁ and DIETRICH MEBS
Mestfat, R. W. P. and Rt+o, S. S. (1%1) IdentiSeation of enzyme and toxins in venons of indien cobra and Russell's Vips after starch rel electrophoresis . J. biol. Client. 236, 1986 . MArtn.t., ß., NYrux, D., Wtsttrtt:nn, B, and W*r~~ ....^N, S. (1973) Consumption coarulopathy caused by a boomalant bite. A cafe report. 77trotnb. Ree. 3, 173. Mtt.z~t, K. D. and Corewvn, W. H. (196s) Human thrombin : isolation and stabllity. Erpl . ~~lolec . Putlt. 4, 431. Moitoz, Ca., De Via, A. and St:u, M. (1966) Isolation and characterization of neurotoxin from Vlperu palsstinae venom. Blochirn. biopJtys. Acte 124, 136. Ontva~nv, L. (1964) Disc eleetrophotssis-I. Hackrround and theory . AndN. Y. Aced. Sci.121, 32l . Owaetv, P. A. (1949) A quantitative one-state method for the assay of prothrombin . Scand. J. clip . Lab . Invest. 1, 181. 5cttttrteatw, S. (1%3) immttaolotical (Ouchterlony method) identiScation of intrasubspecies qualitative differences in snake vettorn ootnposition. Toxican 2, 67 . $ITIal~R, R. L. and Koeantete, J. F. (19s2) A purification of venom phosphodiesterase . J. biol . Chem . 199, 293. Suu~tzsertu, D., St .evtx, M. and JerHestt ~ *, E. (1%7) Simplified technique for preparative disc electrophotesis--lI . Further improvements in apparatus and some details of performance . Analyt. Biochem. n, s7 . T~(aops[~(, E. (1971) Inttaapaxes variability of the venom of Echis carinates. Physiologic bohemoslov . 20, 307.