Article Isolation and Pharmacological Characterization of α-Elapitoxin-Ot1a, a Short-Chain Postsynaptic from the of the Western Desert Taipan, Oxyuranus temporalis

Carmel M. Barber 1, Muhamad Rusdi Ahmad Rusmili 1,2 and Wayne C. Hodgson 1,*

1 Monash Venom Group, Department of , Monash University, Clayton, VIC 3168, Australia; [email protected] 2 Department of Basic Medical Sciences, Kulliyyah of Pharmacy, International Islamic University Malaysia, Bandar Indera Mahkota 23800, Malaysia; [email protected] * Correspondence: [email protected]; Tel.: +61-3-9905-4861

Academic Editor: Bryan Grieg Fry Received: 22 December 2015; Accepted: 19 February 2016; Published: 29 February 2016

Abstract: Taipans (Oxyuranus spp.) are elapids with highly potent containing presynaptic (β) and postsynaptic (α) . O. temporalis (Western Desert taipan), a newly discovered member of this genus, has been shown to possess venom which displays marked in vitro neurotoxicity. No components have been isolated from this venom. We describe the characterization of α-elapitoxin-Ot1a (α-EPTX-Ot1a; 6712 Da), a short-chain postsynaptic neurotoxin, which accounts for approximately 30% of O. temporalis venom. α-Elapitoxin-Ot1a (0.1–1 µM) produced concentration-dependent inhibition of indirect-twitches, and abolished contractile responses to exogenous and carbachol, in the chick biventer cervicis nerve-muscle preparation. The inhibition of indirect twitches by α-elapitoxin-Ot1a (1 µM) was not reversed by washing the tissue. Prior addition of taipan antivenom (10 U/mL) delayed the neurotoxic effects of α-elapitoxin-Ot1a (1 µM) and markedly attenuated the neurotoxic effects of α-elapitoxin-Ot1a (0.1 µM). α-Elapitoxin-Ot1a displayed pseudo-irreversible antagonism of concentration-response curves to carbachol with a pA2 value of 8.02 ˘ 0.05. De novo sequencing revealed the main sequence of the short-chain postsynaptic neurotoxin (i.e., α-elapitoxin-Ot1a) as well as three other isoforms found in O. temporalis venom. α-Elapitoxin-Ot1a shows high sequence similarity (i.e., >87%) with other taipan short-chain postsynaptic neurotoxins.

Keywords: α-Elapitoxin-Ot1a; Oxyuranus temporalis; postsynaptic neurotoxin; antivenom; snake

1. Introduction The Oxyuranus genus consists of three species of highly venomous Australo-Papuan elapids; i.e., inland taipan (O. microlepidotus), coastal taipan (O. scutellatus; found in Australia and Papua New Guinea) and the more recently discovered O. temporalis (Western Desert taipan, [1]). Due to the remote location of O. temporalis, only a handful of specimens have been caught and, as such, limited information exists about this species [1–3]. We recently showed that O. temporalis venom displays marked post-synaptic neurotoxic activity in isolated skeletal muscle [4]. Typically taipan venoms contain presynaptic and postsynaptic neurotoxins and have also been shown to contain natriuretic-like peptides [5,6], prothrombin activators [6–9], reversible calcium channels blockers (i.e., taicatoxin, [6,10]), cysteine-rich secretory (CRISP) [6] and Kunitz-type plasma kallikrein inhibitors [6,11]. The presynaptic neurotoxins isolated from taipan venoms are

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Toxins 2016, 8, 58 2 of 12 paradoxin (O. microlepidotus,[12]), (O. scutellatus,[13]) and cannitoxin (O. s. canni,[14]), α β γ eacheach consistsconsists ofof threethree subunits ( α,, β andand γ)) with with molecular molecular masses masses between between 45 45 and and 47 47 kDa kDa [12–15]. [12–15 ]. AA number number of of postsynaptic postsynaptic neurotoxinsneurotoxins have been isolated from from taipan taipan venoms venoms including; including; oxylepitoxin oxylepitoxin-1‐1 α α ((O.O. microlepidotusmicrolepidotus,[, [16]),16]), α‐-scutoxinscutoxin 1 1 ( (O.O. scutellatus , ,[[17]),17]), α‐oxytoxin-oxytoxin 1 1(O. (O. s. s. canni canni, [17]),,[17]), taipan taipan toxin 1 1 ((O.O. scutellatusscutellatus,[, [18])18]) andand taipantaipan toxintoxin 22 ((O.O. scutellatusscutellatus,[, [18]).18]). These These short short-chain‐chain neurotoxins neurotoxins have have molecularmolecular massesmasses betweenbetween 6726–6789 Da. Many Many of of these these postsynaptic postsynaptic neurotoxins neurotoxins have have also also been been pharmacologicallypharmacologically characterized inin vitro vitro usingusing the the chick chick biventer biventer cervicis cervicis nerve nerve-muscle‐muscle preparation, preparation, withwith an an examination examination of of their their neurotoxicity neurotoxicity and and reversibility, reversibility, as as well well as as determination determination of potencyof (i.e. , pA(i.e.2, values)pA2 values) and theand effectiveness the effectiveness of antivenom of antivenom in preventing in preventing their their effects effects (as reviewed(as reviewed in [ 19in ]).[19]). TheThe aimaim ofof thethe presentpresent study was to isolate and pharmacologically pharmacologically characterize characterize the the major major short-chainshort‐chain postsynapticpostsynaptic neurotoxin from O. temporalis venom.venom.

2.2. Results Results 2.1. Fractionation of Venom via Reverse-Phase HPLC 2.1. Fractionation of Venom via Reverse‐Phase HPLC Fractionation of O. temporalis venom using a Jupiter semi preparative C18 column yielded Fractionation of O. temporalis venom using a Jupiter semi preparative C18 column yielded five five major peaks and a number of minor peaks (Figure1a). Peak two, eluting around 15 min, major peaks and a number of minor peaks (Figure 1a). Peak two, eluting around 15 min, showed showedmarked markedpostsynaptic postsynaptic neurotoxicity neurotoxicity in the chick in biventer the chick cervicis biventer nerve cervicis‐muscle nerve-muscle preparation. preparation. This peak α Thiswas peakchosen was for chosen further for analysis. further analysis. α‐Elapitoxin-Elapitoxin-Ot1a‐Ot1a (peak 2) (peak was 2)collected was collected and purified and purified using using an α ananalytical analytical C18 C18 column, column, where where α‐elapitoxin-elapitoxin-Ot1a‐Ot1a eluted eluted as a single as a peak single around peak aroundapproximately approximately 15 min 15(Figure min (Figure 1b). α‐1Elapitoxinb). α-Elapitoxin-Ot1a‐Ot1a (and its (andisoforms) its isoforms) was found was to found make toup make 30.1% up of 30.1%O. temporalis of O. temporalis venom venombased on based the area on the under area the under curve the of curve the HPLC of the profile. HPLC profile.

(a) (b) 2 3×106 5 3×106 3 2×106 2×106 4 1 1×106 1×106

Absorbance (214 nm) (214 Absorbance 0

Absorbance (214nm) 0

0 204060 0204060 Time (min) Time (min)

FigureFigure 1.1. RP-HPLCRP‐HPLC chromatograph of of ( (aa)) O.O. temporalis temporalis venomvenom run run on on a a Jupiter Jupiter semi semi preparative preparative C18 C18 columncolumn andand ((bb)) α‐α-elapitoxin-Ot1aelapitoxin‐Ot1a run on a Jupiter Jupiter analytical analytical C18 C18 column. column.

2.2. Intact Analysis with MALDI‐TOF Mass Spectrometry 2.2. Intact Protein Analysis with MALDI-TOF Mass Spectrometry Intact protein analysis of α‐elapitoxin‐Ot1a using MALDI‐TOF showed the molecular weight to Intact protein analysis of α-elapitoxin-Ot1a using MALDI-TOF showed the molecular weight to bebe 67126712 DaDa (Figure(Figure2 2).).

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Figure 2. MALDI‐TOF of α‐elapitoxin‐Ot1a indicating a molecular weight of 6712 Da. Proteins were Figure 2. MALDI-TOF of α-elapitoxin-Ot1a indicating a molecular weight of 6712 Da. Proteins were analysed in Linear mode with a mass range of 5kDa to 120kDa. analysed in Linear mode with a mass range of 5 kDa to 120 kDa.

2.3. Identification and de Novo Sequencing by LCMS/MS 2.3. IdentificationFigure 2. and MALDI de Novo‐TOF of Sequencing α‐elapitoxin by‐Ot1a LCMS/MS indicating a molecular weight of 6712 Da. Proteins were Proteinanalysed identification in Linear mode and with de a massnovo range sequencing of 5kDa towith 120kDa. PEAKS Studio 7 software (Version 7.0, BioinformaticsProtein identification Solution Inc., and Waterloo,de novo sequencingON, Canada, with2014) PEAKSgenerated Studio the following 7 software sequence (Version for 7.0, Bioinformaticsα‐2.3.elapitoxin Identification‐ SolutionOt1a: and de Inc., Novo Waterloo, Sequencing ON,by LCMS/MS Canada, 2014) generated the following sequence for α-elapitoxin-Ot1a:MTCYNQQSSQProtein identification AKTTTTCSGG and VSSCYRKTWSde novo sequencing DTRGTIIERG with PEAKS CGCPSVKKGI Studio 7 software ERICCGTDKC (Version NN 7.0, Bioinformatics Solution Inc., Waterloo, ON, Canada, 2014) generated the following sequence for This sequence was identified to have the highest signal by ESI‐LCMS/MS and de novo MTCYNQQSSQα‐elapitoxin‐Ot1a: AKTTTTCSGG VSSCYRKTWS DTRGTIIERG CGCPSVKKGI ERICCGTDKC NN sequencing. Three other isoforms of this short‐chain postsynaptic neurotoxin were also detected, howeverMTCYNQQSSQ only partial AKTTTTCSGG sequences could VSSCYRKTWS be detected DTRGTIIERG (data not shown). CGCPSVKKGI α‐Elapitoxin ERICCGTDKC‐Ot1a showed NN a high This sequence was identified to have the highest signal by ESI-LCMS/MS and de novo sequencing. degree of sequence similarity with short‐chain postsynaptic neurotoxins from other taipan species Three otherThis isoforms sequence of thiswas short-chainidentified to postsynaptic have the highest neurotoxin signal by were ESI also‐LCMS/MS detected, and however de novo only (>87%) (Figure 3 and Table 1). partialsequencing. sequences Three could other be detectedisoforms of (data this short not shown).‐chain postsynapticα-Elapitoxin-Ot1a neurotoxin were showed also adetected, high degree of sequencehowever similarity only partial with sequences short-chain could be postsynaptic detected (data neurotoxins not shown). α‐ fromElapitoxin other‐Ot1a taipan showed species a high (>87%) degree of sequence similarity with short‐chain postsynaptic neurotoxins from other taipan species (Figure3 and Table1). (>87%) (Figure 3 and Table 1).

Figure 3. Sequence alignment (from BLAST search) of α‐elapitoxin‐Ot1a with short‐chain postsynaptic neurotoxins from Oxyuranus spp. Shaded amino acids are similar to α‐elapitoxin‐Ot1a. Amino acids with (*) are fully conserved in all toxins, conserved amino acids with (.) are weakly similarFigure properties 3. Sequence group alignment and amino (from acids withBLAST (:) are search) strongly of α‐similarelapitoxin properties‐Ot1a group. with short‐chain Figure 3. Sequence alignment (from BLAST search) of α-elapitoxin-Ot1a with short-chain postsynaptic postsynaptic neurotoxins from Oxyuranus spp. Shaded amino acids are similar to α‐elapitoxin‐Ot1a. neurotoxins from Oxyuranus spp. Shaded amino acids are similar to α-elapitoxin-Ot1a. Amino acids Amino acids with (*) are fully conserved in all toxins, conserved amino acids with (.) are weakly with (*)similar are fully properties conserved group in and all amino toxins, acids conserved with (:) are amino strongly acids similar with properties (.) are weakly group. similar properties group and amino acids with (:) are strongly similar properties group.

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Table 1. Partial or Full N-terminal sequence and molecular mass (Da) of α-elapitoxin-Ot1a in Toxins comparison2016, 8, 58 to some other Australian elapid postsynaptic neurotoxins. 4 of 12

SpeciesTable 1. Partialα or-Neurotoxin Full N‐terminal MW sequence Partial/Full and molecularN-Terminal mass Sequence (Da) of α‐elapitoxin‐Ot1a in comparison to some other Australian elapid postsynapticMTCYNQQSSQ neurotoxins. AKTTTTCSGG VSSCYRKTWS DTRGTIIERG O. temporalis α-elapitoxin-Ot1a a 6712 CGCPSVKKGI ERICCGTDKC NN Species α‐Neurotoxin MW Partial/Full N‐Terminal Sequence b O. scutellatus α-scutoxin 1 6781 MTCYNQQSSQMTCYNQQSSE AKTTTTCSGG AKTTTTCSGG VSSCYRKTWS VSSCYKKTWY DTRGTIIERG DGRGTRIERG O. temporalis α‐elapitoxin‐Ot1a a 6712 O. scutellatus α-oxytoxin 1 b 6770CGCPSVKKGI MTCYNQQSSE ERICCGTDKC AKTTTTCSGG NN VSSC YKETWY DGRGTT O. scutellatus α‐scutoxin 1 b 6781 MTCYNQQSSE AKTTTTCSGG VSSCYKKTWY DGRGTRIERG O. microlepidotus oxylepitoxin-1 c 6789 MTCYNQQSSE AKTTTTCSGG VSSCYKETWY O. scutellatus α‐oxytoxin 1 b 6770 MTCYNQQSSE AKTTTTCSGG VSSCYKETWY DGRGTT c MTCYNQQSSE AKTTTTCSGG VSSCYKKTWS DGRGTIIERG O.O. scutellatus microlepidotus oxylepitoxinTaipan toxin‐1 1 d 67896726 MTCYNQQSSE AKTTTTCSGG VSSCYKETWY MTCYNQQSSECGCPSVKKGI AKTTTTCSGG ERICCRTDKC VSSCYKKTWS NN DGRGTIIERG O. scutellatus Taipan toxin 1 d 6726 CGCPSVKKGIMTCYNQQSSE ERICCRTDKC AKTTTTCSGG NN VSSCYKKTWS DIRGTIIERG O. scutellatus Taipan toxin 2 d 6781 MTCYNQQSSECGCPSVKKGI AKTTTTCSGG ERICCRTDKC VSSCYKKTWS NN DIRGTIIERG O. scutellatus Taipan toxin 2 d 6781 CGCPSVKKGIMTCCNQQSSQ ERICCRTDKC PKTTTTCAGG NN ETSCYKKTWS DHRGSRTERG α e P. colletti -elapitoxin-Pc1a 6759.6 MTCCNQQSSQ PKTTTTCAGG ETSCYKKTWS DHRGSRTERG P. colletti α‐elapitoxin‐Pc1a e 6759.6 CGCPHVKPGI KLTCCKTDEC NN CGCPHVKPGI KLTCCKTDEC NN MTCCNQQSSQ PKTTTTCAGG ESSCYKKTWS DHRGSRTERG P. porphyriacus α-elapitoxin-Ppr1 e 6746.5 MTCCNQQSSQ PKTTTTCAGG ESSCYKKTWS DHRGSRTERG P. porphyriacus α‐elapitoxin‐Ppr1 e 6746.5 CGCPHVKPGI KLTCCETDEC NN CGCPHVKPGI KLTCCETDEC NN f P.P. papuanus papuanus PapuantoxinPapuantoxin-1‐1 f 67386738 MTCCNQQSSQ MTCCNQQSSQ PKTTTT PKTTTT H.H. stephensi stephensi HostoxinHostoxin-1‐1 gg 66606660 MTPCNQQSSQ MTPCNQQSSQ PKTTK PKTTK Note:Note: Underlined Underlined aminoamino acid acid residues residues have have been deducedbeen deduced from the from sequences the ofsequences other short of chain otherα-neurotoxins; short chain α‐aneurotoxins;current study; a bcurrent[17]; c [ 16study,]; d [18 b ];[17],e [20 c]; [16],f [21 ];d g[18],[22]. e [20], f [21], g [22].

2.4.2.4. In In Vitro Vitro Neurotoxicity Neurotoxicity α‐α-Elapitoxin-Ot1aElapitoxin‐Ot1a (0.1 (0.1 μµM and 1 μµM) caused concentration concentration-dependent‐dependent inhibition of of twitches in thethe indirectly indirectly-stimulated‐stimulated chick chick biventer biventer preparation preparation (n ( =n 3,= Figure 3, Figure 4).4 At). 1 At μM, 1 α‐µM,elapitoxinα-elapitoxin-Ot1a‐Ot1a was stronglywas strongly neurotoxic neurotoxic with with a t90 a valuet90 value of of9.8 9.8 ± 0.4˘ 0.4 min min (Table (Table 2).2). α‐αElapitoxin-Elapitoxin-Ot1a‐Ot1a also also abolished contractilecontractile responsesresponses to to exogenous exogenous ACh ACh and CCh and while CCh only while reducing only KClreducing responses KCl by approximatelyresponses by approximately50% (Figure5). 50% (Figure 5).

(a) (b) 100 * 100 80 80

60 60

40 40 * (% ofinitial) (% ofinitial) Twitch height Twitch height 20 20

0 0 0204060 0 204060 Time (min) Time (min) toxin (0.1 M) toxin (1 M) toxin (0.1 M) + AV (10 U/mL) toxin (1 M) + AV (10 U/mL)

FigureFigure 4. EffectEffect ofof ( a(a))α α‐-elapitoxin-Ot1aelapitoxin‐Ot1a (0.1 (0.1µ μM)M) alone alone and and in thein the presence presence of taipan of taipan antivenom antivenom (AV; (AV;10 U/mL) 10 U/mL) or (b) αor-elapitoxin-Ot1a (b) α‐elapitoxin (1‐µOt1aM) alone (1 μM) and alone in the presenceand in the of taipanpresence antivenom of taipan (AV; antivenom 10 U/mL) (AV;on indirect 10 U/mL) twitches on ofindirect the chick twitches biventer of cervicis the chick nerve-muscle biventer preparation. cervicis nerve * p ‐

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Table 2. t90 values for elapid postsynaptic neurotoxins in chick biventer cervicis preparation.

Species Common Name Postsynaptic Neurotoxin t90 (min) at 1 µM O. temporalis Western Desert Taipan α-elapitoxin-Ot1a 9.83 ˘ 0.36 a O. scutellatus Coastal Taipan α-scutoxin 1 ~12 b O. scutellatus Papuan Taipan α-oxytoxin 1 ~25 b O. microlepidotus Inland Taipan oxylepitoxin-1 ~55 c H. stephensi Stephen’s Banded snake hostoxin-1 ~10 d A. sp. Seram Seram death adder Acantoxin IVa 9.7 ˘ 1.1 e a current study; b [17]; c [16]; d [22]; e [23]. Toxins 2016, 8, 58 5 of 12

150

100 Response

(% of original) 50

0 ACh CCh KCl toxin (0.1 µM) toxin (0.1 M) + AV (10 U/mL) toxin (1 M) toxin (1 M) + AV (10 U/mL) Figure 5. Effect of α‐elapitoxin‐Ot1a (0.1 μM or 1 μM) alone and in the presence of taipan antivenom Figure 5. Effect of α-elapitoxin-Ot1a (0.1 µM or 1 µM) alone and in the presence of taipan antivenom (AV; 1010 U/mL)U/mL) on contractile responses of the chick biventer cerviciscervicis nerve-musclenerve‐muscle preparation to acetylcholine (ACh; 1 mM), carbachol (CCh;(CCh; 2020 μµM)M) andand potassiumpotassium chloridechloride (KCl;(KCl; 4040 mM).mM).

Table 2. t90 values for elapid postsynaptic neurotoxins in chick biventer cervicis preparation. Washing the tissue, at 10 min intervals commencing at the t90 time point (i.e., time taken to produce 90%Species inhibition of nerve-mediatedCommon Name twitches)Postsynaptic after the addition Neurotoxin of α-elapitoxin-Ot1at90 (min) at 1 μ (1M µM), did a not produceO. any temporalis substantial Western recovery Desert of twitch Taipan height α‐ evenelapitoxin 3.5 h after‐Ot1a the addition9.83 of ±α 0.36-elapitoxin-Ot1a b (n = 3, dataO. not scutellatus shown). PreliminaryCoastal data, Taipan where α‐ the concentrationscutoxin 1 of α-elapitoxin-Ot1a~12 was ten-fold O. scutellatus Papuan Taipan α‐oxytoxin 1 ~25 b less (i.e., 0.1 µM) and a longer time of washing out was conducted did show that there was some O. microlepidotus Inland Taipan oxylepitoxin‐1 ~55 c degree of reversibilityH. stephensi in theStephen’s chick biventer Banded cervicissnake nerve-musclehostoxin preparation,‐1 i.e., recovery~10 d of around 33% after 280A. sp. min Seram (4 h and 40Seram min) afterdeath toxinadder addition toAcantoxin tissue (n IVa= 2, data not shown).9.7 ± 1.1 e These results suggest that the neurotoxic effectsa current of α-elapitoxin-Ot1a study, b [17], c [16], are d [22], poorly e [23]. reversible in vitro. The prior addition of taipan antivenom (10 U/mL) almost abolished the neurotoxic actions of α-elapitoxin-Ot1aWashing the tissue, (0.1 atµ M)10 min (Figure intervals4a) and commencing significantly at the delayed t90 time the point neurotoxic (i.e., time actions taken of to αproduce-elapitoxin-Ot1a 90% inhibition (1 µM) of (Figurenerve‐mediated4b). twitches) after the addition of α‐elapitoxin‐Ot1a (1 μM), did not produce any substantial recovery of twitch height even 3.5 h after the addition of α‐elapitoxin‐Ot1a 2.5.(n = CCh3, data Cumulative not shown). Concentration-Response Preliminary data, where Curves the concentration of α‐elapitoxin‐Ot1a was ten‐fold less (i.e., 0.1 μM) and a longer time of washing out was conducted did show that there was some degree In unstimulated chick biventer preparations, α-elapitoxin-Ot1a (10–30 nM) induced of reversibility in the chick biventer cervicis nerve‐muscle preparation, i.e., recovery of around 33% concentration-dependent non-parallel shifts with a marked depression of maximum response to after 280 min (4 h and 40 min) after toxin addition to tissue (n = 2, data not shown). These results CCh (Figure6). This indicates that α-elapitoxin-Ot1a is pseudo-irreversible in action. The modified suggest that the neurotoxic effects of α‐elapitoxin‐Ot1a are poorly reversible in vitro. Lew and Angus method was used to determine a pA value of 8.02 ˘ 0.05 for α-elapitoxin-Ot1a The prior addition of taipan antivenom (10 U/mL)2 almost abolished the neurotoxic actions of (Table3). α‐elapitoxin‐Ot1a (0.1 μM) (Figure 4a) and significantly delayed the neurotoxic actions of α‐elapitoxin‐Ot1a (1 μM) (Figure 4b).

2.5. CCh Cumulative Concentration‐Response Curves In unstimulated chick biventer preparations, α‐elapitoxin‐Ot1a (10–30 nM) induced concentration‐dependent non‐parallel shifts with a marked depression of maximum response to CCh (Figure 6). This indicates that α‐elapitoxin‐Ot1a is pseudo‐irreversible in action. The modified Lew and Angus method was used to determine a pA2 value of 8.02 ± 0.05 for α‐elapitoxin‐Ot1a (Table 3).

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4 control 3

2 * toxin 10 nM Response (g) Response 1 toxin 20 nM * toxin 30 nM 0 * -6 -5 -4 Log [CCh] (M) FigureFigure 6.6. EffectEffect ofof increasingincreasing concentrationsconcentrations ofof α‐α-elapitoxin-Ot1aelapitoxin‐Ot1a (10–30(10–30 nM;nM; nn == 3–4)3–4) onon cumulativecumulative concentration-responseconcentration‐response curves to carbacholcarbachol (CCh)(CCh) inin thethe chickchick biventerbiventer cerviciscervicis nerve-musclenerve‐muscle preparation.preparation. ** p < 0.05, significantly significantly different from control, one one-way‐way ANOVA followed by Bonferroni'ʹss multiplemultiple comparisonscomparisons test.test.

TableTable 3.3. pApA22 values for postsynaptic neurotoxins andand tubocurarine.tubocurarine.

Snake Species Common Name Toxin pA2 Value Snake Species Common Name Toxin pA Value Bungarus multicinctus Various kraits α‐ 8.712 ± 0.06 a a HoplocephalusBungarus multicinctus stephensi Stephen’sVarious Banded kraits snake α-bungarotoxinhostoxin‐1 8.718.45˘ ±0.06 0.32 b b OxyuranusHoplocephalus scutellatus stephensi Stephen’sCoastal Banded Taipan snake α‐ hostoxin-1scutoxin 1 8.458.38˘ 0.32± 0.59 c α c AcanthophisOxyuranus sp. scutellatus Seram SeramCoastal death Taipan adder acantoxin-scutoxin 1Iva 8.388.36˘ ±0.59 0.17 a Acanthophis sp. Seram Seram death adder acantoxin Iva 8.36 ˘ 0.17 a Oxyuranus temporalis Western Desert Taipan α‐elapitoxin‐Ot1a 8.02 ± 0.05 * Oxyuranus temporalis Western Desert Taipan α-elapitoxin-Ot1a 8.02 ˘ 0.05 * Oxyuranus scutellatus Papuan Taipan α‐oxytoxin 1 7.62 ± 0.04 c Oxyuranus scutellatus Papuan Taipan α-oxytoxin 1 7.62 ˘ 0.04 c d OxyuranusOxyuranus microlepidotus microlepidotus Inland Taipan oxylepitoxin-1oxylepitoxin‐1 7.167.16˘ 0.28± 0.28d e PseudechisPseudechis colletti colletti Collett’s snake α ‐α -elapitoxin-Pc1elapitoxin‐Pc1 7.047.04˘ 0.07± 0.07e PseudechisPseudechis prophyriacus prophyriacus Red bellied black snake α‐α-elapitoxin-Ppr1elapitoxin‐Ppr1 6.976.97˘ 0.03± 0.03e e PseudechisPseudechis papuanus papuanus Papuan black snake papuantoxin-1papuantoxin‐1 6.906.90˘ ±0.30 0.30f f NotNot applicable applicable ‐ - d-tubocurarined‐tubocurarine 6.296.29˘ 0.06± 0.06a a a [23],a [23 ];b b[22],[22]; c c[17],[17]; dd [16],[16]; e [20],[20]; ff [[21],21]; * * current current study. study.

3. Discussion 3. Discussion Snake venoms are comprised of numerous toxic and non‐toxic components. The toxic componentsSnake venoms often include are comprised neurotoxins of numerous which toxicassist and in non-toxicthe immobilization components. and The capture toxic componentsof prey but oftenalso may include cause neurotoxins clinical neurotoxicity which assist following in the immobilizationsystemic envenoming and capture in humans. of prey We buthave also recently may causestudied clinical O. temporalis neurotoxicity venom following and shown systemic that the envenomingreverse‐phase in HPLC humans. venom We profile have recently consists studied of only O.a few temporalis peaksvenom with the and major shown peak that theeluting reverse-phase around 15 HPLC min venom [4]. Previous profile consists studies of conducted only a few in peaks our withlaboratory the major have peak shown eluting that, around under 15 minthe [4same]. Previous reverse studies‐phase conducted HPLC conditions, in our laboratory short‐chain have shownpostsynaptic that, under neurotoxins the same elute reverse-phase from the reverse HPLC conditions,phase column short-chain between postsynaptic 15–17 min [16,17,21,22]. neurotoxins eluteTherefore, from O. the temporalis reverse phase venom column was investigated between 15–17 further min in [order16,17 ,to21 ,isolate22]. Therefore, and characterizeO. temporalis a shortvenom‐chain waspostsynaptic investigated neurotoxin further from in order the tovenom. isolate and characterize a short-chain postsynaptic neurotoxin fromα‐ theElapitoxin venom. ‐Ot1a, the first neurotoxin isolated from O. temporalis venom, was found to comprise 30.1%α -Elapitoxin-Ot1a,of the whole venom. the first Previous neurotoxin studies isolated have shown from O. that temporalis short‐chainvenom, postsynaptic was found neurotoxins to comprise 30.1%isolated of from the whole other venom. taipan Previousvenoms represent studies have between shown 1.1% that ( short-chaini.e., taipan toxin postsynaptic 2; [18]) to neurotoxins 9% of the isolatedvenom ( fromi.e., α‐ otheroxytoxin taipan 1; venoms [17]). Therefore, represent α‐ betweenelapitoxin 1.1%‐Ot1a (i.e., taipanrepresents toxin a 2; far [18 greater]) to 9% proportion of the venom of (O.i.e. ,temporalisα-oxytoxin venom 1; [17 ]).than Therefore, the shortα-elapitoxin-Ot1a‐chain neurotoxins represents isolated a from far greater other proportion taipan venoms. of O. temporalisTypically venomshort‐chain than post the‐synaptic short-chain neurotoxins neurotoxins have isolated molecular from masses other between taipan venoms. 6 and 7 kDa, Typically while short-chain long‐chain post-synapticpost‐synaptic neurotoxins have larger molecular molecular masses masses between of between 6 and 7 kDa, 7 and while 9 kDa long-chain [19]. α‐Elapitoxin post-synaptic‐Ot1a, neurotoxinswith a molecular have largermass of molecular 6712 Da, massesfalls within of between the range 7 and of the 9 kDa short [19‐chain]. α-Elapitoxin-Ot1a, neurotoxins. De withnovo asequencing molecular massof the of 15 6712 min Da, reverse falls within‐phase the HPLC range fraction of the short-chain revealed a neurotoxins. structural profileDe novo indicativesequencing of ofshort the‐chain 15 min postsynaptic reverse-phase neurotoxins HPLC fraction (i.e., revealedthe presence a structural of eight profile cysteine indicative residues of in short-chain identical positions to previously isolated short‐chain postsynaptic neurotoxins, Figure 7) (as previously reviewed [19]).

Toxins 2016, 8, 58 7 of 12 postsynaptic neurotoxins (i.e., the presence of eight cysteine residues in identical positions to previously isolated short-chain postsynaptic neurotoxins, Figure7) (as previously reviewed [19]). Toxins 2016, 8, 58 7 of 12

Figure 7. Structure of α‐elapitoxin Ot1a based on the structure of erabutoxin B [24]. Figure 7. Structure of α-elapitoxin Ot1a based on the structure of erabutoxin B [24]. α‐Elapitoxin‐Ot1a shares a high degree of sequence similarity with previously identified taipan α-Elapitoxin-Ot1ashort‐chain postsynaptic shares neurotoxins a high degree as well of sequenceas taipan short similarity‐chain postsynaptic with previously neurotoxins identified that taipan short-chainhave postsynapticonly been partially neurotoxins sequenced ( asi.e., α‐ wellscutoxin as taipan 1; [17]), short-chain α‐oxytoxin 1; postsynaptic [17]), oxylepitoxin neurotoxins 1; [16]). that However, compared to short‐chain postsynaptic neurotoxins from other seas snakes and terrestrial i.e. α α have onlyelapids, been α‐ partiallyelapitoxin sequenced‐Ot1a has much ( ,lower-scutoxin sequence 1; identity, [17]), -oxytoxini.e., 63% compared 1; [17]), to oxylepitoxin erabutoxin A 1; [16]). However,from compared Laticauda semifasciata to short-chain venom postsynaptic and 73% compared neurotoxins to α‐elapitoxin from other‐Pc1a from seas P. snakes colletti andvenom. terrestrial elapids,Deα-elapitoxin-Ot1a novo sequencing hasalso muchindicated lower the sequence presence identity,of three otheri.e., 63% isoforms compared of α‐elapitoxin to erabutoxin‐Ot1a. A from LaticaudaUnfortunately, semifasciata onlyvenom partial and sequences 73% compared of these isoforms to α-elapitoxin-Pc1a could be detected from andP. due colletti to theirvenom. similarDe novo sequencingamino also acid indicated sequences the separation presence of of these three isoforms other isoformswas not possible. of α-elapitoxin-Ot1a. Regardless, the combination Unfortunately, of only all four isoforms of α‐elapitoxin‐Ot1a represents a substantial proportion of O. temporalis venom partial sequences of these isoforms could be detected and due to their similar amino acid sequences (i.e., approximately 30%). separation ofThe these chick isoforms biventer wascervicis not nerve possible.‐muscle Regardless, preparation, the which combination contains both of all focally four‐ isoformsand of α-elapitoxin-Ot1amultiply‐innervated represents muscle a substantial fibres, represents proportion a simple of O. and temporalis effectivevenom method (i.e. of, approximatelyexamining the 30%). Theneurotoxic chick biventer activity of cervicisvenoms and nerve-muscle isolated components preparation, [25]. In this which preparation, contains α‐elapitoxin both focally-‐Ot1a and multiply-innervatedabolished nerve‐ musclemediated fibres, twitches represents much more a rapidly simple than and other effective elapid short method‐chain ofneurotoxins examining the neurotoxic(Table activity 2). In a ofprevious venoms study, and the isolated short‐chain components neurotoxins [ α‐25oxytoxin]. In this 1 (from preparation, the previouslyα-elapitoxin-Ot1a named O. s. canni venom) and α‐scutoxin 1 (from O. scutellatus venom) inhibited indirect twitches by 90% abolished nerve-mediated twitches much more rapidly than other elapid short-chain neurotoxins (t90 value) in approximately 25 and 12 min, respectively [17]. While oxylepitoxin‐1 (from α (Table2).O. In microlepidotus a previous venom) study, was the short-chainshown to have neurotoxins a t90 of approximately-oxytoxin 55 min 1 (from [16]. It the should previously be noted named O. s. cannithatvenom) these experiments and α-scutoxin were 1carried (from O.out scutellatus in an avianvenom) preparation inhibited (i.e., indirectchick biventer twitches cervicis by 90% (t90 value) innerve approximately‐muscle preparation) 25 and 12and min, the respectively potency of α‐ [17elapitoxin]. While‐ oxylepitoxin-1Ot1a may differ (from in mammalianO. microlepidotus preparations as has been previously observed for other α‐neurotoxins [20]. venom) was shown to have a t90 of approximately 55 min [16]. It should be noted that these experiments were carriedα‐ outElapitoxin in an avian‐Ot1a also preparation abolished (contractilei.e., chick responses biventer to cervicis nicotinic nerve-muscle receptor , preparation) as do other and the taipan short‐chain postsynaptic neurotoxins [16,17], indicating that α‐elapitoxin‐Ot1a has a α potencypostsynaptic of -elapitoxin-Ot1a mode of action. may In differaddition, in the mammalian inhibitory effects preparations of α‐elapitoxin as has‐Ot1a been on nerve previously‐mediated observed for othertwitchesα-neurotoxins were irreversible [20]. by washing. This is similar to hostoxin‐1 [22], but in contrast to other taipan α-Elapitoxin-Ot1apostsynaptic neurotoxins, also abolished i.e., α‐oxytoxin contractile 1 [17] and responses oxylepitoxin to‐1 nicotinic [16], which receptor were reversible agonists, by as do other taipanwashing short-chain of the tissue. postsynaptic neurotoxins [16,17], indicating that α-elapitoxin-Ot1a has a postsynapticCumulative mode ofconcentration action. In‐response addition, curves the to inhibitory CCh, in the effects absence of andα -elapitoxin-Ot1apresence of increasing on nerve- concentrations of α‐elapitoxin‐Ot1a, resulted in a concentration‐dependent decrease in the maximal mediated twitches were irreversible by washing. This is similar to hostoxin-1 [22], but in contrast response to CCh with no parallel shift in the cumulative concentration response curve to CCh, to otherindicating taipan postsynaptic a pseudo‐irreversible neurotoxins, interactioni.e., α of-oxytoxin α‐elapitoxin 1 [17‐Ot1a] and with oxylepitoxin-1 skeletal muscle [16 nicotinic], which were reversible by washing of the tissue.

Cumulative concentration-response curves to CCh, in the absence and presence of increasing concentrations of α-elapitoxin-Ot1a, resulted in a concentration-dependent decrease in the maximal Toxins 2016, 8, 58 8 of 12 response to CCh with no parallel shift in the cumulative concentration response curve to CCh, indicating a pseudo-irreversible interaction of α-elapitoxin-Ot1a with skeletal muscle nicotinic acetylcholine receptors (nAChRs). That is, the toxin dissociates from the nAChR at such a slow rate that re equilibration is not achieved within the time frame of the experiment. Similarly, α-scutoxin 1 showed pseudo-irreversible activity in the avian preparation, while α-oxytoxin 1 was found to be reversible, as evidenced by a rightward parallel shift of the cumulative concentration-response curves to CCh with no depression of the maximal response [17]. Using the modified Lew Angus method of analysis, the pA2 value for α-elapitoxin-Ot1a was calculated to be 8.02 ˘ 0.05, which is approximately 5ˆ less potent that α-bungarotoxin but 186ˆ more potent than d-tubocurarine [23] (Table3). Although the clinical effects of envenoming by O. temporalis are unknown, it was important to elucidate whether the commercially available CSL taipan antivenom, which is raised against the venom of O. scutellatus, offers protection against the neurotoxic effects of α-elapitoxin-Ot1a. The in vitro inhibitory effects of α-elapitoxin-Ot1a were abolished or markedly delayed, depending on the concentration of toxin tested, indicating that the antivenom was effective under these conditions. Interestingly, in a previous study, taipan antivenom had little effect on the in vitro neurotoxic effects of oxylepitoxin-1 [16] while, unfortunately, the effect of antivenom against the taipan short-chain neurotoxins α-scutoxin 1 and α-oxytoxin 1 was not investigated [17]. However, this type of in vitro data should be interpreted with caution given the added complexities of treating envenomed humans with antivenom, and the often considerable delay between envenoming and the administration of antivenom. Although it is clear that O. temporalis venom contains a high proportion of the short-chain postsynaptic neurotoxin α-elapitoxin-Ot1a (plus three other isoforms), whether or not this neurotoxin is able to bind at human nAChRs is unknown. Hart et al. [26] showed that Pseudechis colletti venom, which was later shown to contain mostly/only short-chain postsynaptic neurotoxins [20], was far less effective at blocking the actions of α-bungarotoxin at the human nAChR than nAChRs at the chick biventer neuromuscular junction. Similar results have been observed with other short-chain postsynaptic neurotoxins, including the sea snake short-chain postsynaptic neurotoxin erabutoxin [27,28]. In conclusion, this study reports the isolation and characterization of α-elaptitoxin-Ot1a, the first component isolated from O. temporalis venom. α-Elaptitoxin-Ot1a is a potent, pseudo-irreversible short-chain postsynaptic neurotoxin, whose inhibitory actions, in in vitro skeletal muscle preparations, can be prevented or delayed with prior administration of CSL Taipan antivenom. α-Elaptitoxin-Ot1a represents 30.1% of O. temporalis venom and also shares high amino acid sequence similarity with many other short-chain postsynaptic neurotoxins from Australian elapids.

4. Materials and Methods

4.1. Venom Supply O. temporalis venom was obtained as previously described [4] from two live specimens of O. temporalis held at the Adelaide Zoo (Adelaide, South Australia, Australia). After extraction, the venom was frozen with dry ice, transferred into a ´20 ˝C freezer and then lyophilized. After lyophilization, the venom was stored at 4–8 ˝C until required. The venom samples were then pooled, reconstituted with 0.15 M NaCl, aliquoted, re-frozen and lyophilized.

4.2. Purification α-Elapitoxin-Ot1a All chromatography separations were performed using a high-performance liquid chromatography (HPLC) system (Shimadzu, Kyoto, Japan). Freeze-dried O. temporalis venom was dissolved in Milli-Q water (Millipore Corporation, Billerica, MA, USA) to give a stock solution of 10 mg/mL. Samples were briefly vortexed, centrifuged at 14,000 rpm for 2 min and then applied to a Phenomenex (Phenomenex, Torrance, CA, USA) Jupiter semi-preparative (5 µm C18 300Å, 250 mm ˆ 10 mm) C18 column that was equilibrated with 95% Toxins 2016, 8, 58 9 of 12 solvent A (0.1% trifluoroacetic acid, TFA) and 5% solvent B (90% acetonitrile (ACN) in 0.09% TFA). The samples were then eluted with the following gradient conditions of solvent B at a flow rate of 2 mL/min; 0%–20% B over 5 min, 20%–60% B between 5–40 min, 60%–80% B between 40–45 min, and finally 80%–0% B between 45–50 min. The eluent was monitored at 214 nm. Individual peaks were collected manually, frozen at ´80 ˝C and then subsequently freeze-dried. α-Elapitoxin-Ot1a (i.e., the peak at 15 min from the semi preparative reverse-phase HPLC) was further purified on a Phenomenex Jupiter analytical (150 mm ˆ 2 mm, 5 µm, 300 Å) C18 after equilibrating with solvent A (0.1% TFA). The sample was then eluted using the following gradient conditions of solvent B (90% ACN in 0.09% TFA) at a flow rate 0.2 mL/min: 0%–20% over 0–5 min, 20%–60% in between 5 and 40 min and then 60%–80% over 40–45 min. The eluent was monitored at 214 nm. Purified α-elapitoxin-Ot1a was collected, frozen at ´80 ˝C and then subsequently freeze-dried ready for further analysis.

4.3. Mass Spectrometry and Amino Acid Sequencing

4.3.1. Intact Protein Analysis Using Matrix Associated Laser Desorption Time of Flight (MALDI-TOF) Mass Spectrometry Intact protein analysis of α-elapitoxin-Ot1a was made using a MALDI TOF/TOF 4700 Proteomics Analyzer (Applied Biosystems; Foster City, CA, USA) with results analyzed using 4000 Series Explorer version 3.0 software (Applied Biosystems; Foster City, CA, USA) with 15 point smoothing applied. Samples were mixed 1:1 with 10 mg/mL α-cyano-4-hydroxycinnamic acid matrix (Laser BioLabs, Sophia-Antipolis, Valbonne, France) in 50% Acetonitrile 0.1% TFA and spotted onto the MALDI target plate. Proteins were analyzed in linear mode with a mass range of 5 kDa to 120 kDa.

4.3.2. In-Solution Digestion of Sample in Preparation for Electrospray-Ionization Coupled with Mass-Spectrometry/Mass Spectrometry (ESI-LCMS/MS) Protein (200 ng of the reverse-phase HPLC fraction eluting at 15 min) was mixed with 25 µL of 100 mM ammonium bicarbonate, 25 µL of trifluroethanol and 1 µL of 200 mM dithiothretiol (DTT) before being briefly vortexed, centrifuged and incubated at 60 ˝C for 1 h. Iodoacetamide (4 µL of 200 mM) was added and left in the dark at room temperature for 1 h. DTT (1 µL) was added and left to incubate at room temperature for another 1 h. Samples were diluted with Milli-Q grade water and ammonium bicarbonate to achieve pH 7–9. Trypsin was added based on a ratio of 1:20 (enzyme:protein) and incubated overnight at 37 ˝C. Formic acid (1 µL) was added at the end of the incubation to stop enzyme activity. The samples were dried in a vacuum concentrator and stored at ´20 ˝C prior to analysis. Finally, 10 µL of 0.1% formic acid was added to the dried sample before it was loaded into an ESI-LCMS/MS system.

4.3.3. Nanoflow Liquid Chromatography ESI-LCMS/MS The digested sample was loaded into a Agilent C18 300 Å Large Capacity Chip (Agilent Technologies, Santa Clara, CA, USA). The column was equilibrated by 0.1% formic acid in MilliQ water (solution A) and peptides were eluted with an increasing gradient of 90% ACN in 0.1% formic acid (solution B) by the following gradient; 3%–50% solution B from 0–30 min, 50%–95% solution B from 30–32 min, 95% solution B from 32–39 min and 95%–3% solution B from 39–47 min. The polarity of the Q-TOF was set at positive, capillary voltage at 2150 V, fragmentor voltage at 300 V drying gas flow of 5 L/min and gas temperature of 300 ˝C. The spectrum was scanned in auto MS/MS mode over a range of 110–3000 m/z for MS scan and 50–3000 m/z range for MS/MS scan. The spectrum was analyzed by using Agilent MassHunter data acquisition software (Agilent Technologies, Santa Clara, CA, USA).

4.3.4. Protein Identification by Automated de Novo Sequencing De novo sequencing was conducted by using PEAKS Studio (Version 7.0, Bioinformatics Solution, Waterloo, ON, Canada, 2014). The identity search and de novo sequencing were performed by Toxins 2016, 8, 58 10 of 12 comparing de novo sequence tags with NCBInr Serpentes database (version April 2014, National Center for Biotechnology Information, Rockville Pike, MD, USA, 2014) in PEAKS DB and modes. Carbamidomethylation was set as fixed modification and maximum missed cleavage was set at three. Parent mass and precursor mass tolerance was set at 0.1 Da. By using false detection rate (FDR) < 0.1% and ´10log protein (´10logP) > 60, identified proteins were accepted if they have at least two peptides detected and one unique peptide. Maximum variable post-translational modification was set at four.

4.4. Neurotoxicity Studies To examine the reversibility of the toxin, α-Elapitoxin-Ot1a (1 µM) was added to the organ baths and left in contact with the preparation until the t90 time point was reached (i.e., the time at which 90% inhibition of the pre-venom/toxin twitch height was achieved). The tissue was then washed at 10 min intervals. For studies examining the effectiveness of antivenom, tissue preparations were set up as above. Taipan antivenom (3 or 10 U/mL) was added to the organ bath 10 min prior to the addition of α-elapitoxin-Ot1a (0.1 µM or 1 µM).

4.5. CCh Cumulative Concentration-Response Curves

In order to determine the potency (i.e., pA2 value) of α-elapitoxin-Ot1a, cumulative concentration–response curves to CCh were obtained in unstimulated chick preparations. Tissues were set up at 1 g tension on metal ring holders, allowed to equilibrate for 20 min, then α-elapitoxin-Ot1a (10–30 nM) or vehicle (Milli-Q water) was added to separate organ baths and left in contact with the tissue for 1 h. Following this incubation period, cumulative concentration-response curves to CCh were obtained in the presence of toxin or vehicle.

4.6. Data Analysis The percentage of α-elapitoxin-Ot1a was determined from area under the curve analysis of the whole venom semi-preparative reverse-phase HPLC chromatograms. The area under curve for the peak representing α-elapitoxin-Ot1a (and its isoforms) was expressed as a percentage of the area under the curve for all peaks in the whole venom chromatogram. For neurotoxicity experiments, twitch height was measured at regular time intervals and was expressed as a percentage of the pre-toxin twitch height. Contractile responses to ACh, CCh and KCl were expressed as a percentage of original responses. The time taken for 90% inhibition of the twitch response (t90 values) was used to compare the potency of α-elapitoxin-Ot1a with other elapid postsynaptic neurotoxins. In order to determine antagonist potency (i.e., pA2), cumulative concentration-response curves to CCh, obtained in the chick biventer, in the absence or presence of α-elapitoxin-Ot1a, were analyzed using the modified Lew Angus method (PRISM 6.0 GraphPad Software, San Diego, CA, USA, 2014). For antivenom studies, an unpaired t test was used to analyze whether there was statistical significant difference (* p < 0.05) between α-elapitoxin-Ot1a alone and α-elapitoxin-Ot1a in the presence of taipan antivenom at the end of a 60 min time period.

4.7. Chemicals and Drugs The following chemicals and drugs were used: acetylcholine chloride (Sigma-Aldrich, St. Louis, MO, USA), acetonitrile (ACN, Merck, Darmstadt, Germany), ammonium bicarbonate (Sigma-Aldrich), carbamylcholine chloride (carbachol; Sigma-Aldrich), dithiothretiol (Merck), formic acid (Sigma-Aldrich), iodoacetamide (GE Healthcare, Uppsala, Sweden), LCMS grade acetonitrile (Fisher Scientific, Loughborough, UK), potassium chloride (KCl, Ajax Finechem Pty. Ltd., Taren Point, Australia), proteomics grade bovine trypsin (Sigma-Aldrich), taipan antivenom (CSL Ltd., Melbourne, Australia), trifluoroacetic acid (TFA, Auspep, Melbourne, Australia), d-tubocurarine (Sigma-Aldrich) Toxins 2016, 8, 58 11 of 12 and trifluroethanol (Sigma-Aldrich). All chemicals were dissolved or diluted in MilliQ water for unless otherwise stated.

Acknowledgments: We are most grateful to Terry Morley (Adelaide Zoo, South Australia) and Nathan Dunstan (Venom Supplies, Tanunda, South Australia) for the supply of O. temporalis venom. Author Contributions: C.M.B. and W.C.H. conceived and designed the experiments. C.M.B. and M.R.A.R. performed the experiments and analyzed the data. C.M.B., M.R.A.R. and W.C.H. wrote the paper. Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations The following abbreviations are used in this manuscript:

ACh acetylcholine CCh carbachol HPLC high performance liquid chromatography ESI-LCMS/MS electrospray-ionization coupled with mass-spectrometry/mass spectrometry

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