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Unveiling the nature of black (Dendroaspis polylepis) through venomics and immunoprofiling Identification of key toxin targets for antivenom development

Laustsen, Andreas Hougaard; Lomonte, Bruno; Lohse, Brian; Fernandez, Julian; Maria Gutierrez, Jose

Published in: Journal of Proteomics

Link to article, DOI: 10.1016/j.jprot.2015.02.002

Publication date: 2015

Document Version Peer reviewed version

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Citation (APA): Laustsen, A. H., Lomonte, B., Lohse, B., Fernandez, J., & Maria Gutierrez, J. (2015). Unveiling the nature of (Dendroaspis polylepis) venom through venomics and antivenom immunoprofiling: Identification of key toxin targets for antivenom development. Journal of Proteomics, 119, 126-142. https://doi.org/10.1016/j.jprot.2015.02.002

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1

2

3 Unveiling the nature of black mamba (Dendroaspis polylepis) venom

4 through venomics and antivenom immunoprofiling: Identification of key

5 toxin targets for antivenom development

6

7 Andreas H. Laustsen1, Bruno Lomonte2, Brian Lohse1, Julián Fernández2, José María

8 Gutiérrez2

9

10 1 Department of Drug Design and Pharmacology, Faculty of Health and Medical

11 Sciences, University of Copenhagen, Denmark

12 2 Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica,

13 San José, Costa Rica

14

15 Running title: Proteomics of mamba venom

16

17 Keywords: Dendroaspis polylepis; black mamba; venom: proteomics;

18 immunoprofiling;

19

20

21 Address for correspondence: 22 Dr José María Gutiérrez 23 Professor 24 Instituto Clodomiro Picado 25 Facultad de Microbiología 26 Universidad de Costa Rica 27 San José, COSTA RICA 28 [email protected] 29 30 31 Abstract

32 The venom proteome of the black mamba, Dendroaspis polylepis, from Eastern Africa,

33 was, for the first time, characterized. Forty-one different proteins and one nucleoside

34 were identified or assigned to protein families. The most abundant proteins were

35 Kunitz-type proteinase inhibitors, which include the unique mamba venom components

36 ‘’, and α- and other representatives of the three-finger toxin

37 family. In addition, the venom contains lower percentages of proteins from other

38 families, including metalloproteinase, hyaluronidase, prokineticin, nerve growth factor,

39 vascular endothelial growth factor, phospholipase A2, 5'-nucleotidase, and

40 phosphodiesterase. Assessment of acute toxicity revealed that the most lethal

41 components were α-neurotoxins and, to a lower extent, dendrotoxins. This venom also

42 contains a relatively high concentration of adenosine, which might contribute to toxicity

43 by influencing the toxin biodistribution. ELISA immunoprofiling and preclinical

44 assessment of neutralization showed that polyspecific antivenoms manufactured in

45 and India were effective in the neutralization of D. polylepis venom, albeit

46 showing different potencies. Antivenoms had higher titers against α-

47 neurotoxins than against dendrotoxins, and displayed high titers against less toxic

48 proteins of high molecular mass. Our results reveal the complexity of D. polylepis

49 venom, and provide information for the identification of its most relevant toxins to be

50 neutralized by antivenoms.

51

52 Biological significance

53 The black mamba, Dendroaspis polylepis, is one of the most feared in the world,

54 owing to the potency of its venom, the severity and rapid onset of clinical

55 manifestations of envenomings, and its ability to strike fast and repeatedly. The present 56 study reports the first proteomic analysis of this venom. Results revealed a complex

57 venom constituted predominantly by proteins belonging to the Kunitz-type proteinase

58 inhibitor family, which comprises the dendrotoxins, and to α-neurotoxins of the three-

59 finger toxin family. The proteins showing highest acute toxicity were α-neurotoxins,

60 which induce post-synaptic blockade of the neuromuscular junctions, followed by

61 dendrotoxins, which inhibit the voltage-dependent potassium channels. The

62 combination of these two types of toxins in the venom underscores the presence of a

63 dual strategy that results in a highly effective mechanism for prey subduction. This

64 complex toxic arsenal is likely to provide D. polylepis with high trophic versatility. The

65 rapid onset and severity of neurotoxic clinical manifestations in envenomings by D.

66 polylepis demand the rapid administration of effective and safe antivenoms. Preclinical

67 tests showed that an antivenom from South Africa and two antivenoms from India were

68 effective in the neutralization of this venom, albeit differing in their potency. Moreover,

69 ELISA immunoprofiling of these antivenoms against all venom fractions revealed that

70 antivenoms have higher titers against α-neurotoxins than against dendrotoxins, thus

71 underscoring the need to develop improved immunization strategies. The results of this

72 investigation identified the most relevant toxins present in D. polylepis venom, which

73 need to be targeted by antivenoms or other type of inhibitors.

74 75 1. Introduction

76 Dendroaspis polylepis (Black Mamba) is probably the most feared snake in Africa [1],

77 making it a popular icon in movies and even a “street name” for certain psychoactive

78 illegal drugs [2]. D. polylepis is an olive green snake (with a black mouth, hence the

79 name “black mamba”) from the four-membered genus Dendroaspis (family ).

80 It is distributed in Eastern and Southern Africa, with few records in Western Africa,

81 living in open bush country, and typically feeds on small mammals [3]. D. polylepis

82 specimens have been reported to reach 4.3 m, be able to lift a significant part of its body

83 off the ground enabling to strike the upper body of its victim, move at high speed, and

84 can strike repeatedly [1, 3]. Its fangs are between 3-6 mm, and 4-8 ml of venom can be

85 delivered by adult specimens [3].

86

87 D. polylepis is classified within Category 1 by the World Health Organization

88 (WHO) in 17 countries in sub-Saharan Africa. This category corresponds to of

89 ‘highest medical importance’, i.e. as ‘highly venomous snakes, which are common or

90 widespread and cause numerous , resulting in high levels or morbidity,

91 disability or mortality’ [4]. The venom from D. polylepis is known to be highly

92 neurotoxic. Early symptoms include paresthesia, a strange taste in the mouth, nausea,

93 retching, vomiting, abdominal pain, diarrhea, sweating, salivation, “gooseflesh”, and

94 conjunctival congestion. Onset of neurotoxicity may occur already after 15 min,

95 eliciting effects such as , diplopia, dysphagia, flaccid paralysis, slurred speech,

96 dyspnea due to respiratory muscle paralysis, and involuntary skeletal muscle

97 contractions or fasciculations [1,3]. These envenomings are not associated with

98 hemolysis or local signs, i.e. swelling, hemorrhage, or necrosis [1,3,5]. Bites from D.

99 polylepis are reported to have a very high fatality rate if the victim is not treated [1,6]. 100 In contrast, timely administration of effective antivenoms or implementation of

101 mechanical ventilation has been shown to prevent death from these envenomings

102 [1,3,7].

103

104 Antivenom is the only effective treatment against envenoming [4,8].

105 Currently, the following manufacturers produce polyspecific antivenoms claimed to be

106 effective against D. polylepis venom: Sanofi Pasteur, VINS Bioproducts Ltd., Serum

107 Institute of India Ltd., South African Vaccine Producers (SAIMR), Allegens Pharma

108 SAS, and Instituto Bioclon (information obtained from the websites of the

109 manufacturers). However, there are few studies on the preclinical efficacy of these

110 antivenoms against Dendroaspis sp .

111

112 The field of development and preclinical testing of antivenoms has entered a

113 new stage with the introduction of proteomic tools for the study of venoms (i.e.

114 venomics), together with the detailed toxicological characterization of venoms and their

115 components [9]. In addition, the analysis of immunological reactivity of antivenoms has

116 gained momentum with the use of proteomic methods to analyze which venom

117 components are recognized by antivenom , a field known as ‘antivenomics’

118 [10,11]. When antivenomics is combined with the preclinical analysis of the

119 neutralization of venoms by antivenoms, a complete picture of preclinical antivenom

120 efficacy is obtained (see for example [12]). This methodological platform allows for a

121 more rational, knowledge-based design of antivenoms.

122

123 Previous biochemical studies on the venom of D. polylepis have contributed to

124 the characterization of its most relevant toxins. Among them, dendrotoxins are unique 125 components of the genus Dendroaspis [13]. Dendrotoxins show homology with Kunitz-

126 type proteinase inhibitors and exert their pharmacological action by interacting with

127 voltage-dependent potassium channels [14,15]. As a consequence of this interaction,

128 dendrotoxins potentiate the effect of by facilitating the release of this

129 neurotransmitter at the presynaptic nerve terminal, thus provoking an excitatory effect

130 [14]. The three-dimensional structures of some dendrotoxins have been elucidated [16,

131 17], and few have been cloned or chemically synthesized, allowing for structure-

132 function studies [18-20].

133

134 In addition to dendrotoxins, Dendroaspis sp venoms contain proteins of the

135 three-finger toxin family, including α-neurotoxins that bind to the nicotinic cholinergic

136 receptor at the motor end-plate of muscle fibers [21], as well as the so-called muscarinic

137 toxins, which bind to muscarinic cholinergic receptors [22,23], and fasciculins, which

138 are acetylcholinesterase inhibitors found in the venom of D. angusticeps [24]. The rich

139 biochemical diversity of Dendroaspis sp venoms also includes calciceptine, a selective

140 inhibitor of L-type calcium channels [25], mamba intestinal toxins [26], and mambalgin,

141 which blocks the acid-sensing channels associated with pain [27-29]. A weak

142 phospholipase A2 activity has been described for D. polylepis venom [5].

143 Generally, most studies on D. polylepis venom have been focused on the

144 biochemical and pharmacological characterization of the toxins, and many have been

145 motivated by the prospect of employing the toxins as either probes for studies in

146 Neuroscience (short neurotoxins, muscarinic toxins, and dendrotoxins) or as lead

147 compounds for drug development efforts (e.g. [27]). Although there have been a few

148 previous attempts to generate an overall picture of the toxins present in the venom of D.

149 polylepis [30], and the toxicities of some venom fractions/toxins has been assessed 150 [31,32], a complete overview of the venom composition and the identity of individual

151 toxins coupled to their in vivo potency has not been established. Such combined

152 biochemical and toxicological profile is required in order to identify the most abundant

153 lethal and pharmacologically relevant components of this venom, i.e. to identify the

154 targets that need to be neutralized by antivenoms. In addition, it is necessary to

155 investigate the preclinical profile of antivenoms distributed in sub-Saharan Africa for

156 the treatment of envenomings by D. polylepis, and the ability of these antivenoms to

157 bind and neutralize its most relevant neurotoxins.

158 The main aim of the present study was three-fold: to perform, for the first time, a

159 proteomic characterization of D. polylepis venom, to identify all the neurotoxic

160 components of the venom, and to assess the ability of three selected commercial

161 antivenoms to recognize all venom fractions and to neutralize the toxicity of the whole

162 venom.

163

164 2. Materials and Methods

165 2.1 Snake venom

166 Dendroaspis polylepis venom was obtained from Latoxan SAS, Valence, France.

167 The venom is a pool obtained from several specimens collected in .

168

169 2.2 Venom separation by reverse-phase HPLC and SDS-PAGE

170 Following the ‘snake venomics’ analytical strategy [10], crude venom was

171 fractionated by a combination of RP-HPLC and SDS-PAGE separation steps. Venom (2

172 mg) was dissolved in 200 μL of water containing 0.1% trifluoroacetic acid (TFA;

173 solution A) and separated by RP-HPLC (Agilent 1200) on a C18 column (250 x 4.6 mm,

174 5 μm particle; Teknokroma). Elution was carried out at 1 mL/min by applying a 175 gradient towards solution B (acetonitrile, containing 0.1% TFA): 0% B for 5 min, 0–

176 15% B over 10 min, 15–45% B over 60 min, 45–70% B over 10 min, and 70% B over 9

177 min, as previously described [33]. Manually collected fractions were dried in a vacuum

178 centrifuge, redissolved in water, reduced with 5% -mercaptoethanol at 100 °C for 5

179 min, and further separated by SDS-PAGE in 12% gels (Bio-Rad). Proteins were stained

180 with colloidal Coomassie blue G-250, and gel images were acquired on a ChemiDoc®

181 recorder using ImageLab® software (Bio-Rad).

182

183 2.3 Protein identification by tandem mass spectrometry of tryptic peptides

184 Protein bands were excised from the polyacrylamide gels and subjected to

185 reduction (10 mM dithiothreitol), alkylation (50 mM iodoacetamide), and overnight in-

186 gel digestion with sequencing grade trypsin (Sigma), in 50 mM ammonium bicarbonate

187 at 37 °C. The resulting tryptic peptides were extracted with 50% acetonitrile containing

188 1% TFA, and analyzed by MALDI-TOF-TOF on an AB4800-Plus Proteomics Analyzer

189 (Applied Biosystems). Peptides were mixed with an equal volume of saturated α-cyano-

190 hydroxycinnamic acid (in 50% acetonitrile, 0.1% TFA), and spotted (1 μL) onto an

191 Opti-TOF 384-well plate, dried, and analyzed in positive reflector mode. TOF spectra

192 were acquired using 1500 shots and a laser intensity of 3000. The ten most intense

193 precursor ions were automatically selected and their TOF/TOF fragmentation spectra

194 were acquired using 500 shots at a laser intensity of 3900. External calibration in each

195 run was performed with CalMix® standards (ABSciex) spotted onto the same plate. For

196 protein identification, resulting spectra were searched against the UniProt/SwissProt

197 database using ProteinPilot® v.4 and the Paragon® algorithm (ABSciex) at ≥ 95%

198 confidence, or manually interpreted. Few peptide sequences with lower confidence

199 scores were manually searched using BLAST (http://blast.ncbi.nlm.nih.gov) for protein 200 similarity and protein family assignment.

201

202 RP-HPLC fractions corresponding to non-peptidic molecules, eluting in the

203 initial peaks of the chromatogram, were analyzed by ESI-MS/MS on a Q-Trap® 3200

204 instrument (Applied Biosystems). Samples (10 μL) were loaded into metal-coated

205 capillary tips (Proxeon) and directly infused into a nano-ESI source operated at 1300 V.

206 Ions were fragmented by collision-induced dissociation using the Enhanced Product Ion

207 tool with Q0 trapping. Settings for MS/MS analyses were: Q1, unit resolution; collision

208 energy, 25–45 eV; linear ion trap Q3 fill time, 250 ms; and Q3 scan rate, 1000 amu/s.

209 Resulting CID spectra were interpreted manually. nESI-MS performed in this

210 instrument was also used to determine the isotope-averaged mass of intact proteins in

211 selected peaks from the RP-HPLC separation. For this purpose, mass spectra were

212 acquired in Enhanced Multicharge mode in the m/z range 700-1700, and deconvoluted

213 with the aid of the Analyst® v.1.5 software (ABSciex).

214

215 2.4 Relative protein abundance estimations

216 The relative abundances of the venom proteins identified were estimated by

217 integrating the areas of their chromatographic peaks at 215 nm, using the ChemStation®

218 software (Agilent), which estimates peptide bond abundance [10]. For HPLC peaks

219 containing several electrophoretic bands, their percentage distributions were assigned by

220 densitometry, using ImageLab® (Bio-Rad). Finally, for electrophoretic bands in which

221 more than one protein was identified by the MALDI-TOF-TOF peptide analysis, their

222 percentage distributions were estimated on the basis of the corresponding intensities of

223 the intact protein ions, as observed in the nESI-MS analysis. Intensities lower than 10%

224 (relative to the major protein ions in such mixtures) were considered as traces. 225

226 2.5 Nucleoside and FAD analysis

227 The presence of selected nucleosides (adenosine, inosine, guanosine), and

228 flavine adenine dinucleotide (FAD) was determined by spiking a sample of 1 mg of

229 venom with 10 μg of each nucleoside or FAD, respectively, and separating it by

230 reverse-phase HPLC as described in section 2.2. If the nucleoside or FAD peak

231 coincided with a peak already present in a crude venom sample (as judged by the

232 increment in the height of the peak), and if this venom peak showed an ESI-MS

233 spectrum essentially identical to that of the nucleoside or FAD, the identity of venom

234 component was judged to be the same as the nucleoside or FAD. Further confirmation

235 of the molecular identity of adenosine was obtained by acquiring its collision-induced

236 dissociation MS/MS spectrum in positive mode, using the Enhanced Product Ion tool of

237 Analyst v1.5 in the QTrap3200 mass spectrometer, to show the expected reporter ion

238 transition 268 → 136. Nucleoside abundance was estimated by deriving un-spiked

239 nucleoside concentration from integrating the areas of both spiked and un-spiked

240 chromatographic peaks.

241

242 2.6 In vitro enzymatic activities

243 2.6.1. Phospholipase A2 activity

244 PLA2 activity was assayed on the monodisperse synthetic chromogenic substrate

245 4-nitro-3-octanoyloxybenzoic acid (NOBA) [34]. 25 μL containing various amounts of

246 venom were mixed with 200 μL of 10 mM Tris, 10 mM CaCl2, 0.1 M NaCl, pH 8.0, and

247 25 μL of NOBA to achieve a final substrate concentration of 0.32 mM. Plates were

248 incubated at 37 °C for 60 min, and absorbances were recorded at 405 nm in a microplate

249 reader. 250

251 2.6.2 Proteinase activity

252 Variable amounts of venom (10 to 40 μg) were added to 100 μL of azocasein (10

253 mg/mL in 50 mM Tris–HCl, 0.15 M NaCl, 5 mM CaCl2 buffer, pH 8.0), and incubated

254 for 90 min at 37 °C. The reaction was stopped by addition of 200 μL of 5%

255 trichloroacetic acid, and after centrifugation (5 min, 13,000 rpm), 150 μL of

256 supernatants were mixed with 100 μL of 0.5 M NaOH, and absorbances were recorded

257 at 450 nm. The absorbance of azocasein incubated with distilled water alone was used

258 as a blank, being subtracted from all readings [35].

259

260 2.7 Toxicological profiling

261 2.7.1

262 In vivo assays were conducted in CD-1 mice of both sexes, supplied by Instituto

263 Clodomiro Picado, following protocols approved by the Institutional Committee for the

264 Use and Care of Animals (CICUA), University of Costa Rica. Mice were housed in

265 cages for groups of 4–8, and were provided food and water ad libitum.

266

267 2.7.2 Toxicity of crude venom and isolated toxins

268 The lethality of the whole venom and fractions or isolated toxins was tested by

269 intravenous (i.v.) injection in groups of four mice (18–20 g body weight). Various

270 amounts of venom or fractions/toxins were dissolved in phosphate-buffered saline

271 (PBS; 0.12 M NaCl, 0.04 M sodium phosphate buffer, pH 7.2), and injected in the

272 caudal vein, using an injection volume of 100 µL. Then, deaths occurring within 24 h

® 273 were recorded. LD50 was calculated by probits [36], using the BioStat software

274 (AnalySoft). 275

276 The acute toxicity of venom fractions was initially screened by selecting a dose

277 based on fraction abundance in the venom and assuming a venom yield of 400 mg

278 (http://snakedatabase.org/pages/LD50.php#legendAndDefinitions), and 50 kg as the

279 weight of a human being. On this basis, a cutoff fraction dose (mg/kg) was selected and

280 tested. Fractions that were not lethal at this dose were considered as having insignificant

281 acute toxicity, whereas fractions which did kill mice at this level were further evaluated,

282 and precise LD50s were determined for them.

283

284 2.7.3 Determination of synergistic toxicity

285 The lethality of fraction 4 (short 1) in the presence of a constant dose

286 of fraction 8 ( 1) was tested by intravenous (i.v.) injection in groups of four

287 mice (18–20 g body weight). Various amounts of fraction 4 were dissolved in PBS

288 containing 500 µg/ml fraction 8, and injected in the caudal vein, using an injection

289 volume of 100 µL. Then, deaths occurring within 24 h were recorded. LD50 was

290 calculated by probits [36], using the BioStat® software (AnalySoft).

291

292 2.7.4 Hemorrhagic activity

293 Groups of three mice (18–20 g body weight) received an intradermal venom

294 injection (5 μg or 10 μg in 100 μL of PBS) in the abdominal region. After 2 h, animals

295 were euthanized by carbon dioxide inhalation, and their skin was removed to observe

296 the hemorrhagic areas.

297

298 2.8 Antivenoms 299 The following polyspecific antivenoms were used: (a) SAIMR (South African

300 Institute for Medical Research) Polyvalent from South African

301 Vaccine Producers (Pty) Ltd (batch number BC02645, expiry date 07/2016); (b) Snake

302 Venom Antivenom (Central Africa) from VINS Bioproducts Ltd (batch 12AS13002,

303 expiry date 04/2017); (c) Snake Venom Antivenom (African) from VINS Bioproducts

304 Ltd (batch 13022, expiry date 01/2018). In addition, the monospecific Micrurus

305 nigrocinctus Anticoral Antivenom from Instituto Clodomiro Picado (batch

306 5310713ACLQ, expiry date 07/2016) was used for comparison.

307

308 2.9 Immunoreactivity of antivenoms against venom fractions by ELISA

309 Wells in MaxiSorp plates (NUNC, Roskilde, Denmark) were coated overnight

310 with 0.6 μg of each HPLC venom fraction dissolved in 100 μL PBS. Wells were

311 blocked by adding 100 μL PBS containing 2% (w:v) bovine serum albumin (BSA,

312 Sigma) and mixed at room temperature for 1 h. Plates were washed five times with

313 PBS. A dilution of each antivenom in PBS + 2% BSA was prepared such that the

314 concentration of antivenom proteins was 86 μg/mL (as measured by their absorbance at

315 280 nm on a NanoDrop® 2000c instrument, Thermo Scientific). 100 μL of antivenom

316 solution were added to the each well in triplicates, and incubated for 2 h, followed by

317 five additional washings with PBS. Then, 100 µL of a 1:2000 dilution of conjugated

318 antibody (Sigma A6063, rabbit anti-horse IgG (whole molecule)-alkaline phosphatase in

319 PBS + 2% BSA) was added to each well. The plates were incubated for 2 h, and then

320 washed five times with FALC buffer (0.05 M Tris, 0.15 M NaCl, 20 M ZnCl2, 1 mM

321 MgCl2, pH 7.4). Color development was achieved by the addition of 100 µL p-

322 nitrophenyl phosphate (1 mg/mL in 9.7% v/v diethanolamine buffer, pH 9.8). The 323 absorbances at 405 nm were recorded (Multiskan FC, Thermo Scientific) at several time

324 intervals.

325

326 2.10 Neutralization studies with antivenoms

327 Mixtures containing a fixed amount of venom and several dilutions of antivenoms were

328 prepared using PBS as diluent, and incubated at 37 ºC for 30 min. Controls included

329 venom incubated with PBS instead of antivenom. Aliquots of 100 µL of the solutions,

330 containing 4 LD50s of venom (52 µg/) were then injected i.v. into groups of four

331 mice (18-20 g). Deaths occurring within 24 h were recorded for assessing the

332 neutralizing capacity of antivenoms. In cases where an antivenom did show ability to

333 neutralize D. polylepis venom, neutralization was expressed as the Median Effective

334 Dose (ED50) of antivenom, defined as the ratio mg venom/mL antivenom at which 50%

335 of the injected mice were protected. ED50s were estimated by probits, as described in

336 Section 2.6.2.

337

338 3.0 Results and Discussion

339

340 3.1 Venomics

341 In the present study, a proteomic characterization of D. polylepis venom was carried out

342 for the first time. The venom was resolved by RP-HPLC into 27 peaks, of which the

343 three first eluting from the column were non-peptidic. The other 24 peptidic peaks were

344 further resolved into 38 bands by SDS-PAGE separation (Fig.1). In-gel digestion and

345 MALDI-TOF-TOF analysis yielded a total of 41 different proteins, since several

346 proteins eluted simultaneously in RP-HPLC and had similar migration profiles in SDS-

347 PAGE. A previous study, based on capillary electrophoresis separation, detected 70 348 different peptides in this venom, although the majority of them were not identified [30].

349 In our study, some extent of higher order protein structure was observed for some peaks,

350 as exemplified by fraction 20. In the electrophoretic separation of this peak, two bands

351 were seen, but the upper band was shown to be a dimer of the lower band by further

352 mass spectrometry analysis.

353 Peaks 1 to 3 did not show proteins by gel electrophoresis and were therefore

354 analyzed by direct infusion using nESI-MS/MS. Of these, the prominent peak 2 showed

355 a molecular mass of 268 Da, and the nucleoside analysis by HPLC (Fig.3A) revealed its

356 identity as adenosine, which was further confirmed by the reporter ion transition 268 →

357 136 in its collision-induced dissociation MS/MS spectrum (Fig.3B). On the other hand,

358 all peptidic peaks were either identified as known proteins from D. polylepis, or

359 assigned to toxin families on the basis of sequence homology of their tryptic peptides

360 with proteins from other snake species (Table 1).

361 Furthermore, all peptidic peaks were tested for toxicity in a model, except for

362 peaks 26 and 27, which yielded very low amount of proteins and correspond to

363 metalloproteinases, which are likely to have limited toxicity (Table 2). The overall

364 protein composition of the venom of D. polylepis, expressed as percentage of total

365 protein and nucleoside content, is represented in Fig.2. In terms of abundance and

366 toxicity, two main families, Kunitz-type proteinase inhibitors, including dendrotoxins

367 (KUN; 61.1%), and three-finger toxins (3FTx; 31.0%) predominate in the venom of D.

368 polylepis, which also contains a fairly high amount of non-peptidic components (NP;

369 2.5%) of which the majority (2.4%) was shown to be adenosine (Fig.3). Dendrotoxins

370 are able to block subtypes of voltage-dependent potassium channels of the Kv1

371 subfamily in neurons [15,37,38], leading to facilitatory actions on neurotransmission 372 [39], resulting in their toxicity [31]. -neurotoxins present among the identified three-

373 finger toxins are able to bind to nicotinic receptors at the motor end-plate of muscle

374 fibers, thus generating a flaccid paralysis leading to respiratory failure and death

375 [21,23,40]. Despite the fact that D. polylepis venom is formed by an exceptionally low

376 number of predominant protein families, there is a high number of components within

377 each family, thus comprising a relatively complex venom. This may be a general theme

378 within elapids, which are known to have primarily neurotoxic venoms constituted by

379 neurotoxins of the three-finger family and, in some cases, by PLA2s [41-44]. Vonk et al.

380 [45] described the so far only genome of a , Ophiophagus hannah (king

381 ), and identified a high diversity within the family three-finger toxins in this

382 elapid, most likely obtained through gene duplication and accumulated mutations. In the

383 case of D. polylepis venom, the characteristic neurotoxins of the three-finger family

384 occur together with a unique type of neurotoxins, i.e. dendrotoxins, which bear

385 homology with Kunitz-type proteinase inhibitors.

386

387 Other protein types present in the venom of D. polylepis include members of the

388 metalloproteinase (MP), hyaluronidase (HYA), prokineticin (KTC), nerve growth factor

389 (NGF), vascular endothelial growth factor (VEGF), phospholipase A2 (PLA2), 5'-

390 nucleotidase (NUCL), and phosphodiesterase (PDE) families. A notorious finding was

391 the very low amount of PLA2, which is an abundant component in the majority of elapid

392 venom proteomes characterized so far [42,46-49]. This finding was further supported by

393 the in vitro PLA2 activity assay, revealing negligible activity of D. polylepis venom

394 (Fig.4A). Presence of proteinase activity was also assessed in vitro and found to be very

395 low (Fig.4B). This is in agreement with the proteomic analysis showing a presence of

396 only 3.2% metalloproteinases. In agreement with the low proteinase activity detected, 397 the venom of D. polylepis was devoid of hemorrhagic activity when tested in vivo at a

398 dose of 10 µg/mouse. Only a few, non-peptidic venom components remained

399 unidentified in D. polylepis venom, altogether representing 0.5% of the total content

400 (Table 1).

401

402 The results of toxicity testing of the fractions are shown in Table 2. For the study of

403 the lethal effect of venom fractions, the possible effect of the solvents used in RP-HPLC

404 separation, i.e. TFA and acetonitrile, on the various proteins was considered. To

405 approach this issue, venom was incubated with either PBS or 50% acetonitrile + 0.1%

406 TFA (solvent B), and the LD50 estimated afterwards. No significant variations were

407 observed between LD50s of these venom samples, hence evidencing that separation of

408 venom proteins by RP-HPLC does not affect their toxicity (see Table 2). When tested in

409 mice, only α-neurotoxins (present in peaks 4, 5, 6, 7, and 9) and dendrotoxins (present

410 in peaks 5 to 11) were found to be relevant for acute toxicity, i.e. inducing lethality

411 within 24 h. Our results underscore the fact that peaks containing α-neurotoxins are

412 largely responsible for the toxicity of this venom, with some dendrotoxins playing a

413 secondary role. In general terms, our findings on toxicity of D. polylepis venom

414 components agree with those described by Schweitz et al. [31]. One major deviation in

415 LD50 with previous studies was found for peak 8, containing dendrotoxin-I. A value of

416 38 mg/kg was previously reported for this toxin [50]; however, we estimated an LD50

417 value of around 5 mg/kg (see Table 2). A Toxicity Score is shown in Table 2, which

418 takes into account both potency and abundance. Toxins with a high score are of high

419 medical relevance, with the assumption that the acute toxicity in mice is indicative of

420 toxicity in human beings. These are abundant and/or very potent, while toxins with a

421 low score are less relevant for acute toxicity. 422

423 The responses in mice after injections of α-neurotoxins and dendrotoxins differed.

424 The former exerted an almost immediate effect resulting in flaccid paralysis and death

425 within 10-30 min when using supralethal doses. Administration of dendrotoxins, on the

426 other hand, led to a different pattern, characterized by an excitatory effect at all tested

427 toxin levels, resulting in death between 20 min to several hours when using high doses.

428 Also, while the in vivo response for neurotoxins was dose-dependent and highly

429 reproducible, in terms of lethality, dendrotoxins showed an erratic dose-dependent

430 response, with some mice dying at low doses, while other mice survived at high doses.

431 This pattern was especially evident in the case of peak 20 (dendrotoxin-B), which killed

432 two out of three mice at a dose of 1 mg/kg, yet only killed one out of four mice at the

433 highest dose tested (4.2 mg/kg). An LD50 could thus not be determined for this toxin.

434 One possibility for explaining this phenomenon could be an effect due to higher order

435 protein structure occurring at different concentrations, thus affecting the

436 pharmacokinetics and the pharmacodynamics of the toxin. However, the understanding

437 of this atypical phenomenon clearly demands further studies.

438 Mambalgins, which are members of the three-finger toxin family, were identified in

439 D. polylepis venom and constitute 1.4% of the venom proteins. Mambalgins are an

440 interesting group of proteins that have been studied for their ability to inhibit the Acid-

441 Sensing Ion Channel 1a, and are being further investigated as lead compounds for the

442 development of drugs for pain relief [27-29]. Despite having a similar structure as the

443 short-chain α-neurotoxins, these three-finger toxins are known not to be toxic, which

444 was confirmed in the current work (see Table 2).

445 An interesting question about the venom of D. polylepis from an adaptive

446 perspective is why it contains such a wealth of dendrotoxins, which are markedly less 447 lethal than the α-neurotoxins. Had the entire venom consisted of the most potent toxin,

448 short neurotoxin-1 (peak 4), the LD50-value would have been almost 10 times lower due

449 to the low abundance of this toxin in the venom. Dendrotoxins might show synergistic

450 effects with α-neurotoxins. If the toxic effects were not additive or synergistic, only the

451 effect of the toxin that had the highest potency (as a result of a combination of high

452 abundance, toxicity, and rapidity of action) would predominate, leaving the lethal

453 effects of other toxins masked. Fraction 4, containing short neurotoxin-1, fulfills the

454 criteria as the most potent toxin. If synergistic or additive effects were not present,

455 measurement of LD50 for the whole venom would have been about 2.5 mg/kg, given

456 that this toxin has an LD50 of 0.08 mg/kg, but it represents only 3.6% of venom proteins

457 Since the LD50 for the whole venom was determined to 0.68 mg/kg (see below), this

458 strongly suggests that other less toxic venom components contribute to overall lethality

459 in a synergistic fashion. The existence of synergism between venom components in D.

460 polylepis venom was demonstrated by Strydom [32]. In order to further investigate this

461 phenomenon, LD50 of fraction 4 (short neurotoxin-1) was determined in the presence of

462 a constant, sub-lethal dose of fraction 8 (dendrotoxin 1), the same dendrotoxin that was

463 examined by Strydom [32], in order to evaluate whether the toxicity of short neurotoxin

464 1 is potentiated by this dendrotoxin. In our results, however, we could not detect any

465 synergistic effects between these two toxins, given that the determined LD50 of 0.09

466 mg/kg (95% confidence limits: 0.01-0.18 mg/kg) does not differ significantly from the

467 LD50 of fraction 4 when administered alone (see Table 2). However, the presence of

468 synergistic effects between other toxins cannot be excluded.

469

470 Another possibility might be that, despite not being highly lethal, dendrotoxins are

471 likely to play a relevant role in prey capture by provoking an excitatory effect (observed 472 in our experiments) that clearly causes a transient immobilization of the prey, thus being

473 biologically meaningful. Finally, there might also exist an adaptive value in having a

474 more diverse and complex venom, since it not only makes it more versatile against a

475 variety of potential prey but also makes it difficult for prey and predators to develop

476 resistance towards the whole venom, in evolutionary terms. The biochemical and

477 pharmacological complexity of snake venoms, unveiled by proteomic and toxicological

478 studies, highlights a pattern of redundancy in toxic components and mechanisms, as to

479 ensure prey immobilization in a variety of ecological scenarios.

480

481 From our results, the relatively high abundance of adenosine in the venom of D.

482 polylepis was noteworthy. Alone, adenosine is not a particularly toxic compound, with

483 an LD50 of 500 mg/kg [51]. However, adenosine is known to exert vasodilation and to

484 activate neuronal adenosine A1 receptors, leading to suppression of acetylcholine

485 release from motor neurons and excitatory neurotransmitters from central sites [52].

486 Hence, the action of this nucleoside, in the context of the whole venom, may contribute

487 to overall toxicity by either modifying the biodistribution of neurotoxins or by

488 potentiating their activity, a hypothesis that could be further studied. It has similarly

489 been described that the lethality of toxin from the tarantula, Dugesiella hentzi, is

490 potentiated by adenosine triphosphate (ATP) present in the venom [53].

491 Toxicity in mice is not always translatable into toxicity in other types of prey or

492 human beings. However, in terms of therapeutic targets, based on their 'Toxicity Score',

493 the very likely most relevant toxins in D. polylepis venom that should be neutralized by

494 an effective antivenom are (in order of importance): short neurotoxin-1 (P01416), α-

495 elapitoxin Dpp2c (P01397), dendrotoxin-1 (P00979), and dendrotoxin-B (P00983). In

496 addition, it is also likely that muscarinic toxin-α (P80494) and a toxin similar to 497 dendrotoxin- from D. angusticeps (P00982) would be relevant to target. Thus,

498 antivenomic studies with D. polylepis venom should establish whether antivenoms are

499 able to bind these toxins.

500

501 3.2 Neutralization by antivenoms and profiling of their immunoreactivity against venom

502 fractions

503 Four different antivenoms were tested for their ability to neutralize whole D. polylepis

504 venom. Venom had an LD50 of 0.68 mg/kg, when using the i.v. route in mice. The two

505 Indian antivenoms and the SAIMR antivenom were effective in the neutralization of

506 lethality, albeit to a different extent. ED50s were (in mg venom neutralized per ml

507 antivenom): 0.76 mg/ml for VINS African (95% confidence limits: 0.57-1.32 mg/ml),

508 0.97 mg/ml for VINS Central Africa (95% confidence limits: 0.68-1.44 mg/ml), and

509 5.26 mg/ml for the SAIMR antivenom (95% confidence limits: 3.54-9.02 mg/ml). The

510 higher efficacy of the SAIMR antivenom could partially be explained by the fact that

511 the protein concentration in this antivenom was about three times higher than that of the

512 Indian antivenoms. However, in addition it seems that the SAIMR antivenom has a

513 higher titer of antibodies against relevant neurotoxins in D. polylepis venom. Our

514 findings imply that more vials are likely to be needed of the Indian antivenoms in order

515 to achieve the same neutralization capacity as the SAIMR antivenom. In contrast, the

516 monospecific Micrurus nigrocinctus antivenom was ineffective even at a

517 venom/antivenom ratio of 300 µg venom/mL antivenom (Table 3). This indicates that

518 antibodies raised against the α-neurotoxins present in this Central American elapid

519 venom [42] do not cross-neutralize D. polylepis neurotoxins.

520 521 In order to further investigate the different antivenoms, binding of F(ab’)2

522 antibody fragments, present in the antivenoms, to all peaks separated by RP-HPLC was

523 measured by ELISA (Fig.5). Generally, it was observed that SAIMR Polyvalent Snake

524 Antivenom showed a stronger toxin binding than the other antivenoms, especially

525 against all the toxicologically relevant -neurotoxins and dendrotoxins (Fig.5). It is

526 noteworthy that antivenoms showed higher titers against high molecular mass venom

527 components (metalloproteinases, nucleosidases, hyaluronidases, and

528 phosphodiesterases) than against low molecular mass neurotoxins.

529

530 A potential flaw of ELISA analysis of antivenoms is that different toxins may

531 show different binding efficiencies to ELISA plates, which may lead to lower detection

532 signals of IgGs binding to a toxin that does not bind well to the plate. Nevertheless,

533 assuming that binding is somewhat similar for all toxins in D. polylepis venom, there is

534 a trend towards more (or higher affinity) antibodies being present against larger toxins

535 (Fig.5). This agrees with previous antivenomic studies, which have shown that low

536 molecular mass toxins, particularly three finger toxins and PLA2s, are poorly

537 immunogenic [42,46]. This would have the implication that the immune response

538 evoked in the immunized animals during antivenom production is skewed towards

539 large, more immunogenic toxins, such as metalloproteinases. In the case of D. polylepis

540 this is unfavorable, since these larger toxins are of no medical concern due to their very

541 low relative abundance and low toxicity in this venom. Hence, having antibodies

542 directed against medically irrelevant toxins might dilute the concentration of relevant

543 antibodies, leading to a need for more antivenom vials during treatment. This, in turn,

544 may increase the likelihood of adverse reactions due to the need to administer higher

545 amounts of antivenom proteins for an effective neutralization. 546

547 Finally, ELISA results revealed a poor antibody response in all the antivenoms

548 when tested against dendrotoxins present in peaks 8 and 20 (Fig.5). This can be

549 explained on the following grounds: (a) Dendrotoxins are low molecular mass, poorly

550 immunogenic venom components. (b) The antivenoms tested are produced by

551 immunizing horses with mixtures of a large number of venoms; hence, dendrotoxins,

552 present only in Dendroaspis sp venoms, would constitute a low percentage of the

553 immunizing mixture. (c) In contrast with dendrotoxins, α-neurotoxins are present in

554 Dendroaspis and venoms, thus comprising a higher percentage of the immunizing

555 mixture than dendrotoxins. Further studies are necessary to assess the immune response

556 against dendrotoxins and its improvement, and to develop ways to enhance such

557 response. This could be approached by improved immunizing schemes or by the

558 generation of recombinant antibodies against these toxins.

559

560 4.0 Concluding remarks and outlook

561 In the present study, the venom of D. polylepis was, for the first time, subjected to a

562 thorough proteomics analysis, which revealed that the venom was dominated by Kunitz-

563 type proteinase inhibitors, including dendrotoxins (61.1% of the venom) and toxins of

564 the three-finger family (31.0% of the venom), and that these components were by far the

565 most toxic fractions of the venom. Also, the presence of adenosine was shown to be

566 relatively high (2.4%), suggesting that this nucleoside might play an auxiliary role in

567 . Additionally, other protein types comprise 5.4% of the venom. The

568 presence of multiple fractions having several neurotoxins in this venom underscores the

569 possibility of synergistic effects between different types of neurotoxins or the adaptation

570 of a versatile toxin arsenal for a variety of prey types. Neutralization studies showed 571 that polyspecific antivenoms manufactured in South Africa and India are effective for

572 neutralizing lethal activity of D. polylepis venom, albeit showing different ED50 values.

573 In contrast, an antivenom raised against M. nigrocinctus venom was ineffective for

574 neutralizing D. polylepis venom. The ELISA immunoprofiling of antivenoms with the

575 different fractions of D. polylepis venom revealed a higher antibody titer against high

576 molecular mass venom components, such as metalloproteinases, than against the

577 toxicologically relevant low molecular mass neurotoxins, in agreement with the low

578 immunogenicity of the latter. In particular, all antivenoms showed a low titer against

579 dendrotoxins, which are unique to mamba venoms. Hence, ELISA titers alone do not

580 show a good correlation with neutralization in the case of D. polylepis venom,

581 emphasizing the need to complement immunochemical analysis of antivenom with the

582 study of their neutralizing potency in mice and ultimately in humans.

583

584 Our proteomic and toxicological observations indicate that effective antivenom

585 against the venom of D. polylepis should contain neutralizing antibodies against the

586 following venom components, in descending order of relevance: short neurotoxin-1

587 (P01416), -elapitoxin Dpp2c (P01397), dendrotoxin-1 (P00979), dendrotoxin-B, D.

588 polylepis (P00983), and perhaps muscarinic toxin-(P80494) and a toxin similar to

589 dendrotoxin- from D. angusticeps (P00982). More studies within this field will pave

590 the way to identify the targets that are crucial in the development of more effective

591 antivenoms or other types of antitoxins.

592

593 Acknowledgments

594 The authors thank Dr Steven D. Aird (Okinawa Institute of Science and Technology,

595 Japan) for fruitful discussions about nucleosides present in snake venom. We thank 596 Mikael Engmark (Technical University of Denmark) for helping procure antivenoms

597 and choosing the focus of D. polylepis. We further thank Jens Kringelum (Technical

598 University of Denmark), Alexandra Bak Jakobsen (Denmark), and Christina Milbo

599 (Technical University of Denmark) for fruitful scientific discussions. We thank the

600 Department of Drug Design and Pharmacology, University of Copenhagen, and

601 Instituto Clodomiro Picado, Universidad de Costa Rica, for supporting the research.

602 Finally, we thank the following foundations for financial support: Drug Research

603 Academy (University of Copenhagen), Dansk Tennis Fond Oticon Fonden, Knud

604 Højgaards Fond, Rudolph Als Fondet, Henry Shaws Legat, Læge Johannes Nicolai

605 Krigsgaard of Hustru Else Krogsgaards Mindelegat for Medicinsk Forskning og

606 Medicinske Studenter ved Københavns Universitet, Lundbeckfonden, Torben of Alice

607 Frimodts Fond, Frants Allings Legat, Christian og Ottilia Brorsons Rejselegat for Yngre

608 Videnskabsmænd- og kvinder, and Fonden for Lægevidenskabens Fremme.

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763 764 Figure legends

765

766 Figure 1: Separation of D.polylepis (A) venom proteins by RP-HPLC (C), followed by

767 SDS-PAGE (B). Two mg of venom were fractionated on a C18 column and eluted with

768 an acetonitrile gradient (dashed line), as described in Materials and Methods. Peptidic

769 fractions were further separated by SDS-PAGE under reducing conditions. Molecular

770 weight markers (Mw) are indicated in kDa. Coomassie-stained bands were excised, in-

771 gel digested with trypsin, and subjected to MALDI-TOF/TOF analysis for assignment to

772 protein families, as shown in Table 1. Non-peptidic RP-HPLC peaks 1 to 3 were

773 analyzed by direct infusion nESI-MS in order to determine the isotope-averaged masses

774 of intact proteins.

775

776 Figure 2: Composition of D. polylepis venom according to protein families, expressed

777 as percentages of the total protein content. KUN: bovine pancreatic trypsin

778 inhibitor/Kunitz inhibitor; 3FTx: three-finger toxin; MP: metalloproteinase; VEGF:

779 vascular endothelial growth factor; NUCL: nucleotidase; PDE: phosphodiesterase;

780 HYA: hyaluronidase; KTC: prokineticin; PLA2: phospholipase A2; NGF: nerve growth

781 factor; NP: non-protein components. In the latter category, a conspicuous amount of

782 adenosine (2.4% of the total venom absorbance in the RP-HPLC separation; see Table

783 1, peak 2) was identified, as shown in Fig.3.

784

785 Figure 3: (A) Presence of selected nucleosides and FAD in D. polylepis venom shown

786 by spiking crude venom with 10 μg of nucleosides (adenosine, inosine, guanosine) or

787 FAD and separating the venom components by reverse-phase HPLC. If the peak of a

788 nucleoside coincides with the peak of a venom component, and if mass determination ® 789 (Q-Trap 3200 instrument, Applied Biosystems) yielded the same mass for the venom

790 component as calculated for the nucleoside, the venom component was judged to

791 consist of the corresponding nucleoside. (B) Collision-induced dissociation MS/MS

792 spectrum in positive mode showing the expected reporter ion transition for adenosine

793 268 → 136. D. polylepis venom contains a relatively large amount (2.4% of the whole

794 venom) of adenosine, traces of inosine and guanosine, but no FAD.

795

796 Figure 4: (A) Comparison of the phospholipase A2 activity between the venoms of D.

797 polylepis and M. nigrocinctus revealing negligible activity for D. polylepis venom. (B)

798 Comparison of the proteolytic activity between the venoms of D. polylepis, M.

799 nigrocinctus, and B. asper, evaluated on azocasein. D. polylepis venom shows

800 negligible proteinase activity.

801

802 Figure 5: ELISA-based immunoprofiling of antivenoms (SAVP: SAIMR Polyvalent

803 Snake Antivenom from South African Vaccine Producers’ SAIMR , VINS African:

804 Snake Venom Antiserum (African) from VINS Bioproducts Ltd., VINS Central Africa

805 Snake Venom Antiserum (Central African) from VINS Bioproducts Ltd., and a negative

806 control (Horse negative: normal serum from horses from Instituto Clodomiro Picado)

807 to the peptidic fractions of D. polylepis venom separated by RP-HPLC (see Materials

808 and Methods for details). Analyses were performed in triplicates. For identification of

809 venom fractions see Table 2.

810

811

812

813 Table 1: Assignment of the RP-HPLC isolated fractions of Dendroaspis polylepis venom to protein families by MALDI-TOF-TOF of selected peptide ions from in-gel trypsin-digested protein bands.

Peak % Mass▼ Mass Peptide ion MS/MS-derived sequence Conf Sc Protein family; related protein * (kDa) (Da) (%) m/z z 1 0.4 - - - 1 non-peptidic - - unknown

2 2.4 - 268.3 - 1 non-peptidic (Adenosine) - - non-peptidic (nucleosides)

3 0.1 - - - 1 non-peptidic - - unknown

4.i 3.7 10 6911.0, 1435.9 1 RXCYNHBSTTR 99 12 3FTx 6949.0, 2022.2 1 VBPGVGXHCCBSDBCNY 99 19 short neurotoxin-1, D. polylepis; P01416 6987.0 1585.0 1 VBPGVGXHCCBSDK 99 16 1279.8 1 XCYNHBSTTR 99 14

4.ii trace 10 6564.0 1045.7 1 BXPSFYYK 99 10 BPTI/Kunitz inhibitor 1816.0 1 BCXPFDYSGCGGNANR 99 15 dendrotoxin-delta, D. angusticeps; P00982 1799.0 1 ZCXPFDYSGCGGNANR 99 17

5a.i 1.1 12 6563.0 1045.7 1 BXPSFYYK 99 11 BPTI/Kunitz inhibitor 917.6 1 XPSFYYK 99 10 dendrotoxin-delta, D. angusticeps; P00982 1816.0 1 BCXPFDYSGCGGNANR 99 14 1798.9 1 ZCXPFDYSGCGGNANR 99 17

5a.ii 1.1 12 8020.0 2015.1 1 CCSTDNCNBFBFGBPR 99 11 3FTx 1190.7 1 VNXGCAATCPK 99 9 alpha-elapitoxin Dpp2c, D. polylepis; P01397

5b.i 4,0 10 6563.0 1045.7 1 BXPSFYYK 99 13 BPTI/Kunitz inhibitor 2015.1 1 ABBCXPFDYSGCGGNANR 99 19 dendrotoxin-delta, D. angusticeps; P00982 1815.9 1 BCXPFDYSGCGGNANR 99 19 1799.0 1 ZCXPFDYSGCGGNANR 99 20

5b.ii 4,0 10 8020.0 3548.0 1 TPSD(Qda)SBXCPPGE(Nda)XCYTBTWCDAWCSBR 99 15 3FTx alpha-elapitoxin Dpp2c, D. polylepis; P01397

6a 3,1 11 6563.0 1045.7 1 BXPSFYYK 99 12 BPTI/Kunitz inhibitor 1816.0 1 BCXPFDYSGCGGNANR 99 14 dendrotoxin-delta, D. angusticeps; P00982 1798.9 1 ZCXPFDYSGCGGNANR 99 16

6b.i 5.0 10 6563.0 2015.1 1 ABBCXPFDYSGCGGNANR 99 17 BPTI/Kunitz inhibitor 1045.7 1 BXPSFYYK 99 12 dendrotoxin-delta, D. angusticeps; P00982 1815.9 1 BCXPFDYSGCGGNANR 99 20 1798.9 1 ZCXPFDYSGCGGNANR 99 19

6b.ii 4.9 10 8080.0 2196.2 1 TPSD(Qda)SBXCPPGE(Nda)XCYTK 99 14 3FTx alpha-elapitoxin Dpp2c, D. polylepis; P01397

6b.iii trace 10 - 1330.7 1 TWCDAFCGD(Rca) 99 12 3FTx long neurotoxin A. labialis; ABX58167

7.i 5.3 10 6564.0 1045.7 1 BXPSFYYK 99 11 BPTI/Kunitz inhibitor 2015.1 1 ABBCXPFDYSGCGGNANR 99 15 dendrotoxin-delta, D. angusticeps; P00982 1816.0 1 BCXPFDYSGCGGNANR 99 18 1799.0 1 ZCXPFDYSGCGGNANR 99 17

7.ii 3.2 10 8010.0 1369.7 1 TWCDAWCSBR 99 15 3FTx 8080.0 2196.3 1 TPSD(Qda)SBXCPPGENXCYTK 99 16 alpha-elapitoxin Dpp2c, D. polylepis; P01397 2002.1 1 CCSTDNCNBFBFGBPR 99 12

7.iii trace 10 - 908.6 1 GBF(Mox)BPGK 96 7 Ribosomal protein L27e 60S ribosomal prot.L27 M. fulvius; U3FW60

8a 6.7 10 7137.0 1816.9 1 BCEGFTWSGCGGNSNR 99 21 BPTI/Kunitz inhibitor 1799.9 1 ZCEGFTWSGCGGNSNR 99 19 dendrotoxin-1, D. polylepis; P00979 1945.0 1 BBCEGFTWSGCGGNSNR 99 26 2073.1 1 BBBCEGFTWSGCGGNSNR 99 23 1236.8 1 XCXXHR(Nda)PGR 99 10 1143.7 1 XPAFYYNBK 99 15

8b.i 18.1 10 7137.0 1816.9 1 BCEGFTWSGCGGNSNR 99 21 BPTI/Kunitz inhibitor 1799.9 1 ZCEGFTWSGCGGNSNR 99 19 dendrotoxin-1, D. polylepis; P00979 1945.0 1 BBCEGFTWSGCGGNSNR 99 26 2073.1 1 BBBCEGFTWSGCGGNSNR 99 23 1236.8 1 XCXXHR(Nda)PGR 99 10 1143.7 1 XPAFYYNBK 99 15

8b.ii trace 10 - 2426.5 1 DTXFGXTTBNCPAGBNXCFXR 99 18 3FTx adrenergic toxin rho-EPTX-Da1b, D. angusticeps; P86419

9a.i 1.4 10 7179.0 1143.7 1 XPAFYYNBK 99 14 BPTI/Kunitz inhibitor 1798.9 1 ZCEGFTWSGCGGNSNR 99 15 dendrotoxin-1, D. polylepis; P00979

9a.ii trace 10 - 1451.8 1 XCPPGENXCYTK 99 12 3FTx 1369.7 1 TWCDAWCSBR 99 15 alpha-elapitoxin Dpp2c, D. polylepis; P01397

9a.iii trace 10 7549.0 2197.3 1 YSDXTWGCAATCPBPTNVR 91 7 3FTx muscarinic toxin-alpha, D. polylepis; P80494

9b.i 1.3 10 7549.0 2197.2 1 YSDXTWGCAATCPBPTNVR 99 25 3FTx 2329.3 1 SXFGXTTENCPDGBNXCFBK 99 19 muscarinic toxin-alpha, D. polylepis; P80494 1179.7 1 BWYYXNHR 99 12 1051.6 1 WYYXNHR 99 11

9b.ii 3.7 10 6560.0 1815.9 1 BCXPFDYSGCGGNANR 99 14 BPTI/Kunitz inhibitor 1798.9 1 ZCXPFDYSGCGGNANR 99 15 dendrotoxin-delta, D. angusticeps; P00982

9b.iii trace 10 - 1143.7 1 XPAFYYNBK 99 14 BPTI/Kunitz inhibitor dendrotoxin-1, D. polylepis; P00979

10.i 1.6 10 7456.0 2197.2 1 YSDXTWGCAATCPBPTNVR 99 14 3FTx muscarinic toxin-4, D. angusticeps; Q9PSN1

10.ii 1.0 10 6390.0 1815.9 1 BCXPFDYSGCGGNANR 99 14 BPTI/Kunitz inhibitor dendrotoxin-delta, D. angusticeps; P00982

10.iii trace 10 - 1623.9 1 GCTFTCPEXRPTGK 95.4 9 3FTx muscarinic toxin-like B. multicinctus; Q9W727

11a.i 0.6 11 6762.0 917.6 1 XPSFYYK 99 8 BPTI/Kunitz inhibitor dendrotoxin-delta, D. angusticeps; P00982

11a.ii trace 11 - 1143.7 1 XPAFYYNBK 99 14 BPTI/Kunitz inhibitor 1816.9 1 BCEGFTWSGCGGNSNR 95.1 10 dendrotoxin-1, D. polylepis; P00979

11a.iii trace 11 - 1369.7 1 TWCDAWCSBR 99 10 3FTx Hem-9, H. signata; R4FIK4

11b.i 2.9 10 6762.0 2015.1 1 ABBCXPFDYSGCGGNANR 99 16 BPTI/Kunitz inhibitor dendrotoxin-delta, D. angusticeps; P00982

11b.ii trace 10 - 1945.0 1 BBCEGFTWSGCGGNSNR 99 12 BPTI/Kunitz inhibitor 1798.9 1 ZCEGFTWSGCGGNSNR 99 15 dendrotoxin-1, D. polylepis; P00979

11b.iii trace 10 - 2197.3 1 YSDXTWGCAATCPBPTNVR 99 14 3FTx muscarinic toxin-4, D. angusticeps; Q9PSN1

12.i 0.7 10 7048.0 2450.2 1 GCGCPTA(Mox)WPYBTECCBGDR 99 12 3FTx 2122.0 1 GCGCPTA(Mox)WPYBTECCK 99 15 calciseptin, D. polylepis; P22947 989.6 1 RXCYXHK 99 11

12.ii trace 10 - 1143.7 1 XPAFYYNBK 99 14 BPTI/Kunitz inhibitor 1816.9 1 BCEGFTWSGCGGNSNR 99 14 dendrotoxin-1, D. polylepis; P00979 1799.9 1 ZCEGFTWSGCGGNSNR 99 15

12.iii trace 10 - 1369.7 1 TWCDAWCSBR 99 12 3FTx Hem-9, H. signata; R4FIK4

13a.i 0.5 13 7039.0 2122.0 1 GCGCPTA(Mox)WPYBTECCK 99 7 3FTx 2450.2 1 GCGCPTA(Mox)WPYBTECCBGDR 99 9 calciseptin, D. polylepis; P22947 1174.6 1 TCVENTCYK 99 9 989.7 1 RXCYXHK 99 11

13a.ii trace 13 - 1817.0 1 BCEGFTWSGCGGNSNR 99 10 BPTI/Kunitz inhibitor 1143.7 1 XPAFYYNBK 99 14 dendrotoxin-1, D. polylepis; P00979

13b.i 1.7 10 7039.0 2434.2 1 GCGCPTAMWPYBTECCBGDR 99 14 3FTx 2122.0 1 GCGCPTA(Mox)WPYBTECCK 99 14 calciseptin, D. polylepis; P22947 989.6 1 RXCYXHK 99 11 1779.0 1 TCVENTCY(Kca)MFXR 99 14

13b.ii trace 10 - 1143.7 1 XPAFYYNBK 99 13 BPTI/Kunitz inhibitor 1816.9 1 BCEGFTWSGCGGNSNR 99 17 dendrotoxin-1, D. polylepis; P00979

14.i 0.4 10 8608.0 2724.6 1 VCTPVGTSGEDCHPASHBXPFSGBR 99 11 Prokineticin 976.6 1 AVXTGACER 99 12 intestinal toxin MIT1, D. polylepis; P25687 1939.0 1 VCTPVGTSGEDCHPASHK 99 21 1294.7 1 GTCCAVSXWXK 99 18 2225.2 1 (Mox)HHTCPCAPNXACVBTSPK 99 25

14.ii 1.1 10 6558.0 1429.8 1 FCYHNTGMPFR 99 14 3FTx 1927.0 1 XXXBGCSSSCSETENNK 99 15 mambalgin-2, D. polylepis; P0DKS3

15.i 0.1 10 6984.0 1798.0 1 BCXPFXFSGCGGNANR 99 14 BPTI/Kunitz inhibitor 1669.9 1 CXPFXFSGCGGNANR 99 17 calcicludin, D. angusticeps; P81658 1164.6 1 WBPPWYCK 99 14 1010.6 1 FBTXGECR 99 11

15.ii 0.3 10 6569.0 1441.8 1 FCYHNXGMPFR 99 14 3FTx 1927.0 1 XXXBGCSSSCSETENNK 99 20 mambalgin-3, D.angusticeps; C0HJB0 1848.0 1 D(Mox)BFCYHNXG(Mox)PFR 99 8

16a.i 0.6 10 6983.0 1164.6 1 WBPPWYCK 99 9 BPTI/Kunitz inhibitor (?) 1798.0 1 BCXPFXFSGCGGNANR 99 14 calcicludin, D. angusticeps; P81658 1669.9 1 CXPFXFSGCGGNANR 99 18

16a.ii trace 10 - 1441.7 1 FCYHNXGMPFR 99 14 3FTx mambalgin-3, D.angusticeps; C0HJB0

16b.i 0.5 10 6983.0 1181.7 1 BBFSSFYFK 99 15 BPTI/Kunitz inhibitor 1053.6 1 BFSSFYFK 99 13 calcicludin, D. angusticeps; P81658 1164.6 1 WBPPWYCK 99 12 1798.0 1 BCXPFXFSGCGGNANR 99 19 1010.6 1 FBTXGECR 99 12 1669.9 1 CXPFXFSGCGGNANR 99 19

16b.ii trace 10 - 1369.7 1 TWCDAWCSBR 99 14 3FTx Hem-9, H. signata; R4FIK4

17.i 2.5 10 7384.0 970.6 1 WHYVXPR 99 11 3FTx muscarinic toxin-3, D. angusticeps; P81031

17.ii trace 10 1669.9 1 CXPFXFSGCGGNANR 99 16 BPTI/Kunitz inhibitor calcicludin, D. angusticeps; P81658

17.iii 1.3 10 7385.0 2296.2 1 GCVATC(Z)XPE(Nda)YDSXHCCK 99 22 3FTx muscarinic toxin-beta, D. polylepis; P80495

18a.i 0.1 25 - 1293.8 1 XDTACVCVXSR 99 13 Nerve growth factor 1798.0 1 H(Wox)NSYCTTTHTFVK 99 12 NGF-1, N. sputatrix; Q5YF90 1782.0 1 HWNSYCTTTHTFVK 99 21 1413.8 1 CRNPNPVPSGCR 99 9

18a.ii trace 25 - 962.5 1 BYFFETK 99 10 Nerve growth factor 975.6 1 ESHPVHNR 99 10 NGF-beta, N. natrix; B8QCJ9

18a.iii trace 25 - 970.6 1 WHYVXPR 99 9 3FTx muscarinic toxin-3, D. angusticeps; P81031

18b.i 0.1 15 - 1413.8 1 CRNPNPVPSGCR 99 10 Nerve growth factor 1782.0 1 HWNSYCTTTHTFVK 99 23 NGF-1, N. sputatrix; Q5YF90 1710.0 1 FXRXDTACVCVXSR 99 13 1097.6 1 NPNPVPSGCR 99 12 1293.7 1 XDTACVCVXSR 99 16

18b.ii 0.1 15 - 975.6 1 ESHPVHNR 99 11 Nerve growth factor 962.5 1 BYFFETK 99 11 NGF-beta, N. natrix; B8QCJ9

18c.i 0.1 10 7339.0 2281.2 1 GCVATCPXPENYDSXHCCK 99 11 3FTx 2202.2 1 SXFGXTTEDCPDGBNXCFK 99 14 muscarinic toxin-beta, D. polylepis; P80495

18c.ii trace 10 - 1669.9 1 CXPFXFSGCGGNANR 99 10 BPTI/Kunitz inhibitor 1010.6 1 FBTXGECR 91.7 8 calcicludin, D. angusticeps; P81658

18c.iii trace 10 - 1815.9 1 BCXPFDYSGCGGNANR 99 9 BPTI/Kunitz inhibitor dendrotoxin-delta, D. angusticeps; P00982

18c.iv trace 10 - 970.6 1 WHYVXPR 99 10 3FTx muscarinic toxin-3, D. angusticeps; P81031

18c.v trace 10 - 1143.7 1 XPAFYYNBK 99 12 BPTI/Kunitz inhibitor dendrotoxin-1, D. polylepis; P00979

19.i 1.4 10 7339.0 2358.3 1 SXFGXTTEDCPDGBNXCFBR 99 11 3FTx 2202.2 1 SXFGXTTEDCPDGBNXCFK 99 27 muscarinic toxin-beta, D. polylepis; P80495 2281.1 1 GCVATCPXPENYDSXHCCK 99 32

19.ii 1.4 10 7401.0 970.6 1 WHYVXPR 99 10 3FTx muscarinic toxin-3, D. angusticeps; P81031

19.iii trace 10 - 2491.4 1 S(Nda)SX(Wox)FXTSE(Nda)CPAGBNXCFK 98.5 11 3FTx muscarinic toxin-1, D. angusticeps; P60236

20a.i trace 10 - 1380.8 1 XPPXPXSXVEFR 94.6 9 Phospholipase A2 PLA2-Pse-8, P. modesta; R4FIQ4

20a.ii trace 10 - 1022.6 1 DFTBWXGR 99 11 - DNA/RNA-binding prot KIN17-like, C. horridus; T1DN91

20a.iii trace 10 - 1332.8 1 XFBPSCVVVBR man ma Vascular endothelial growth factor n VEGF, M. lebetina; P82475

20b 7.2 10 6472.0, 1310.6 1 CYPFTYSGCR 99 14 BPTI/Kunitz inhibitor 6510.0 dendrotoxin-B, D. polylepis; P00983

21a.i 0.2 67 - 1388.8 1 YXEFYVVVDNK 99 14 Metalloproteinase 3025.5 1 AA(Kdl)(Hox)DCDXSEXCTGBSAECPTDSFBR 99 11 SVMP-Aca-4, A. wellsi; R4G2D3

21a.ii - 67 - 1239.8 1 SVAVXBDHSBR 99 9 Metalloproteinase SVMP-Hop-14, H. bungaroides; R4FIM1

21a.iii - 67 - 1185.7 1 WRETDXXPR 99 8 Metalloproteinase SVMP-Hem-2, H. signata; R4G2W9

21b.i 0.2 60 - 1882.8 1 (Nda)GHPCBNNBGYCYNR 99 18 Metalloproteinase 1989.1 1 NDNABXXTGXDF(Nal)GTTVGR 99 23 SVMP-Hop-14, H. bungaroides; R4FIM1

21b.ii - 60 - 1853.0 1 TDXVSPPVCGNYFVEVG 99 15 Metalloproteinase pIII-SVMP, O. okinavensis; U3TBS9

21b.iii - 60 - 1185.7 1 WRETDXXPR 99 9 Metalloproteinase SVMP-Hem-2, H. signata; R4G2W9

21c 0.1 53 - 1255.9 1 FPXXSANXRPK 99 16 5'-nucleotidase 1389.9 1 XTXXHTNDVHAR 99 11 ecto-5'-nucleotidase, G. brevicaudus; B6EWW8 1344.6 1 CTG(Qda)DCYGGVAR 99 13 1448.8 1 VVSXNVXCTEC(Ram) 99 11

21d.i 0.2 49 - 2421.4 1 FHECNXGNXXCDAVVYNNXR 99 22 5'-nucleotidase 1448.8 1 VVSXNVXCTEC(Ram) 99 12 ecto-5'-nucleotidase-1; M. fulvius; U3FYP9 2689.6 1 ETPVXSNPGPYXEFRDEVEEXBK 99 19 1255.8 1 FPXXSANXRPK 99 15 1389.9 1 XTXXHTNDVHAR 99 13

21d.ii - 49 - 1344.6 1 CTGQdaDCYGGVAR 99 13 5'-nucleotidase ecto-5'-nucleotidase, G. brevicaudus; B6EWW8

22a 0.1 107 - 1371.8 1 AATYF(Wox)PGSEVK 99 8 Phosphodiesterase 1134.6 1 NPFYNPSPAK 99 11 phosphodiesterase-1, M. fulvius; U3FAB3 2305.3 1 ABRPDFSTXYXEEPDTTGHK 99 17

22b.i 0.5 62 - 1989.2 1 NDNABXXTGXDF(Nal)GTTVGR 99 23 Metalloproteinase SVMP-Hop-14, H. bungaroides; R4FIM1

22b.ii - 62 - 3025.5 1 AB(Hdl)DCD(Mox)PERCTGBSAECPTDSFBR 99 16 Metalloproteinase SVMP, A. engaddensis; Q9PT48

23.i 0.4 41 - 1256.8 1 VTX(Dna)XFGBWR 99 14 Metalloproteinase 941.5 1 GBGCGFCR 96.2 9 SVMP-1, M. fulvius; U3EPC7

23.ii - 41 - 1530.9 1 TBPAYBFSSCSVR 99 16 Metalloproteinase SVMP cobrin, N. kaouthia; Q9PVK7

23.iii - 41 - 1297.7 1 SAECPTDSFBR 99 10 Metalloproteinase SVMP-Hem-2, H. signata; R4G2W9

24 0.4 47 54892.0 2032.2 1 BHSDSNAFXHXFPESFR 99 23 Hyaluronidase 1904.1 1 HSDSNAFXHXFPESFR 99 23 hyaluronidase, E. pyramidum; A3QVN6 1700.0 1 AFHGXGVXDWENWR 99 17 1243.8 1 NDBXXWXWR 99 15

25a.i 0.3 73 - 1516.9 1 BYXEFYVVVDNK 99 17 Metalloproteinase carinatease-1, T. carinatus; B5KFV1

25a.ii - 73 - 1394.9 1 RPBCXXNBPXR 99 12 Metalloproteinase SVMP-Hop-14, H. bungaroides; R4FIM1

25a.iii - 73 - 3029.6 1 AABHDCD(Mox)(Pox)ERCTGBSAECPTDSFBR 99 14 Metalloproteinase SVMP, A. engaddensis; Q9PT48

25b 0.1 50 - 1904.1 1 HSDSNAFXHXFPESFR 99 9 Hyaluronidase 1243.8 1 NDBXXWXWR 99 11 hyaluronidase, E. ocellatus; A3QVN2

26.i 0.5 42 - 1297.7 1 SAECPTDSFBR 99 13 Metalloproteinase MTP-9, D. coronoides; F8RKV9

26.ii - 42 - 2203.2 1 RTBPAYBFSSCSVBEHBR 99 14 Metalloproteinase SVMP-1, M. fulvius; U3EPC7

26.iii - 42 - 1323.7 1 DPNYG(Mox)VEPGTK 99 14 Metalloproteinase atragin, N. atra; D3TTC2

26.iv - 42 - 1826.0 1 YXEFYVVVDNV(Mox)YR 99 10 Metalloproteinase mocarhagin, N. mossambica; Q10749

27 1.0 111 - 1685.0 1 TNDXVXPEEVEWXK 99 14 Metalloproteinase 1239.7 1 NPYGYBXAWK 99 14 endoplasmic reticulum aminopeptidase 1-like 1382.8 1 NVHXBBEHYSK 99 13 protein, C. horridus; T1E6L9 1462.9 1 TBEX(Pox)SXXFSVGR 97.8 10

* Cysteine residues are carbamidomethylated. Confidence (Conf) and Score (Sc) values are calculated by the Paragon algorithm of ProteinPilot® (ABsciex) ▼: estimated mass by SDS-PAGE under reducing conditions, in kDa. : observed isotope-averaged masses as determined by nESI-MS of selected RP-HPLC peaks, in Da. X: Leu/Ile; B: Lys/Gln; Z: pyroglutamate (2-oxo-pyrrolidone carboxylic acid). Possible, although unconfirmed/ambiguous amino acid modifications suggested by the automated identification software are shown in parentheses, with the following abbreviations: ox: oxidized; da: deamidated; ca: carbamyl; am: amide; na: Na cation; al: ammonia loss; dl: deltaH2C2; man: manually interpreted MS/MS spectrum.

Table 2: LD50s of Dendroaspis polylepis venom and the RP-HPLC isolated fractions

Toxicity Score1 LD Reported LD Peak % Protein family 50 50 % / LD (95% C.L.) (mg/kg) 50 (kg/mg)

Whole venom 0.68 0.25* 147.1 (0.57-1.09)

Whole venom 0.76 131.6 (PBS speedvac) (0.17-1.32)

Whole venom 0.69 (ACN/H O 144.9 2 (0.18-1.19) speedvac)

3FTx 0.08 4 3.6 0.09[54] 45.0 short neurotoxin-1, D. polylepis; P01416 (0.04-0.11)

3FTx (50%) 10.0 alpha-elapitoxin Dpp2c, D. polylepis; P01397 0.46 5 21.7 (1:1 mix2) BPTI/Kunitz inhibitor (50%) (0.16-0.76) dendrotoxin-delta, D. angusticeps; P00982

3FTx (40%) 12.6 alpha-elapitoxin Dpp2c, D. polylepis; P01397 0.53 6 23.8 (2:3 mix2) BPTI/Kunitz inhibitor (60%) (0.34-0.72) dendrotoxin-delta, D. angusticeps; P00982

3FTx (40%) 8.3 alpha-elapitoxin Dpp2c, D. polylepis; P01397 0.28 7 29.6 (2:3 mix2) BPTI/Kunitz inhibitor (60%) (0.00-0.56) dendrotoxin-delta, D. angusticeps; P00982

BPTI/Kunitz inhibitor 5.25 8 24.4 38[50] 4.65 dendrotoxin-1, D. polylepis; P00979 (2.48-8.31) BPTI/Kunitz inhibitor (20%) dendrotoxin-1, D. polylepis; P00979 6.3 3FTx (20%) 3.76 9 (1:1:3 1.7 muscarinic toxin-alpha, D. polylepis; P80494 (2.98-5.15) mix2) BPTI/Kunitz inhibitor (60%) dendrotoxin-delta, D. angusticeps; P00982

3FTx (60%) 2.5 muscarinic toxin-4, D. angusticeps; Q9PSN1 4.92 10 0.5 (3:2 mix2) BPTI/Kunitz inhibitor (40%) (3.69-13.56) dendrotoxin-delta, D. angusticeps; P00982

BPTI/Kunitz inhibitor 4.38 11 3.4 0.8 dendrotoxin-delta, D. angusticeps; P00982 (2.25-8.17)

3FTx (L-type Ca+2-channel blocker subfamily) 12 0.7 >1.5 <0.5 calciseptin, D. polylepis; P22947

3FTx (L-type Ca+2-channel blocker subfamily) 13 2.2 >5 <0.5 calciseptin, D. polylepis; P22947

Prokineticin (30%) 1.5 intestinal toxin MIT1, D. polylepis; P25687 14 >2.5 <1 (3:7 mix2) 3FTx (70%) mambalgin-2, D. polylepis; P0DKS3

BPTI/Kunitz inhibitor (20%) 1.4 calcicludin, D. angusticeps; P81658 15 >1 <1.5 (1:4 mix2) 3FTx (80%) mambalgin-3, D.angusticeps; C0HJB0

BPTI/Kunitz inhibitor 16 1.2 >2.5 <0.5 calcicludin, D. angusticeps; P81658

3.8 3FTx (30%) 17 >7.5 <0.5 (3:7 mix2) muscarinic toxin-3, D. angusticeps; P81031 3FTx (70%) muscarinic toxin-beta, D. polylepis; P80495

Nerve growth factor (33%) NGF-1, N. sputatrix; Q5YF90 0.3 Nerve growth factor (33%) 18 (1:1:1 >1 <0.5 NGF-beta, N. natrix; B8QCJ9 mix2) 3FTx (33%) muscarinic toxin-beta, D. polylepis; P80495

3FTx (50%) 2.8 muscarinic toxin-beta, D. polylepis; P80495 19 >5 <1 (1:1 mix2) 3FTx (50%) muscarinic toxin-3, D. angusticeps; P81031

BPTI/Kunitz inhibitor 20 7.2 N/A dendrotoxin-B, D. polylepis; P00983

Metalloproteinase (29%) SVMP-Aca-4, A. wellsi; R4G2D3 Metalloproteinase (29%) 0.7 SVMP-Hop-14, H. bungaroides; R4FIM1 21 (2:2:1:2 >1.15 <1 5'-nucleotidase (13%) mix2) ecto-5'-nucleotidase, G. brevicaudus; B6EWW8 5'-nucleotidase (29%) ecto-5'-nucleotidase-1; M. fulvius; U3FYP9

Phosphodiesterase (20%) 0.6 phosphodiesterase-1, M. fulvius; U3FAB3 22 >1 <1 (1:4 mix2) Metalloproteinase (80%) SVMP-Hop-14, H. bungaroides; R4FIM1

Metalloproteinase 23 0.4 >0.6 <1 SVMP-1, M. fulvius; U3EPC7

24 0.4 Hyaluronidase >0.3 <1.5 hyaluronidase, E. pyramidum; A3QVN6

Metalloproteinase (75%) 0.4 carinatease-1, T. carinatus; B5KFV1 25 >0.75 <1 (3:1 mix2) Hyaluronidase (25%) hyaluronidase, E. ocellatus; A3QVN2

Metalloproteinase 26 0.5 N/A MTP-9, D. coronoides; F8RKV9

Metalloproteinase N/A 27 1.0 endoplasmic reticulum aminopeptidase 1-like protein, C. horridus; T1E6L9

*http://snakedatabase.org/pages/LD50.php#legendAndDefinitions

1 Toxicity Score was defined as the ratio of protein fraction abundance (%) in the venom divided by its estimated (LD50) for

CD-1 mice by i.v. injection.

2Mix indicates that the fraction did not contain a pure, isolated toxin, but instead a mixture of 2-4 different toxins in variable ratios indicted in the table.

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