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Sequences, geographic variations and molecular phylogeny of venom phospholipases and threefinger toxins of eastern India Bungarus fasciatus and kinetic analyses of its Pro31 phospholipases A2

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phylogeny of venom phospholipases and threefinger toxins

of eastern India Bungarus fasciatus and kinetic analyses

of its Pro31 phospholipases A

2

Inn-Ho Tsai1, Hsin-Yu Tsai1, Archita Saha2and Antony Gomes2

1 Institute of Biological Chemistry, Academia Sinica, Taiwan, Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan 2 Department of Physiology, University of Calcutta, Kolkata, India

Snakes of the genus Bungarus are commonly known as kraits, which are characterized by their banded skin pattern. They are distributed from Pakistan through southern Asia to Indonesia and central China [1,2]. In the past, more than 20 proteins were purified and sequenced from pooled venom of Bungarus fasciatus

(Bf), which was obtained from either the Miami Serpentarium Laboratory or south-eastern Asia. The proteins include eight variants of phospholipases A2 (EC3.1.1.4, PLAs) [3–6], four isoforms of threefinger toxins (3FTx) [7–10], at least one Kunitz protease inhibitors [10–12], a factor-X activator [13], an

Keywords

Bungarus fasciatus; cDNA cloning; phospholipase A2; phylogenetic analysis;

threefinger toxins; venom geographic variation

Correspondence

I.-H. Tsai, Institute of Biological Chemistry, Academia Sinica, Taiwan; Institute of Biochemical Sciences, National Taiwan University; POB 23–106, Taipei, Taiwan Fax: +886 223635038

E-mail: bc201@gate.sinica.edu.tw Database

The sequence data were deposited in the GenBank database with the accession num-bers: DQ508406, DQ508411-14 for KBf-VI, KBf-grIB, KBf-II, KBf-Va, and KBf-X, DQ768745 for KBf-III, DQ835584 for Vb-2, respectively; DQ508407-10 for 3FTx-LI, -LK, -RK and -RI, and DQ835582-3 for VIIIa and 3FTx-LT, respectively

(Received 30 June 2006, revised 16 October 2006, accepted 17 November 2006) doi:10.1111/j.1742-4658.2006.05598.x

Eight phospholipases A2 (PLAs) and four three-finger-toxins (3FTx) from the pooled venom of Bungarus fasciatus (Bf) were previously studied and sequenced, but their expression pattern in individual Bf venom and possible geographic variations remained to be investigated. We herein analyze the individual venom of two Bf specimens from Kolkata (designated as KBf) to address this question. Seven PLAs and five 3FTx were purified from the KBf venoms, and respective cDNAs were cloned from venom glands of one of the snakes. Comparison of their mass and N-terminal sequence revealed that all the PLAs were conserved in both KBf venoms, but that two of their 3FTx isoforms were variable. When comparing the sequences of these KBf-PLAs with those published, only one was found to be identi-cal to that of Bf Vb-2, and the other five were 94–98% identiidenti-cal to those of Bf II, III, Va, VI and XI-2, respectively. Notably, the most abundant PLA isoforms of Bf and KBf venoms contain Pro31 substitution. They were found to have abnormally low kcatvalues but high affinity for Ca2+. Phylogenetic analysis based on the sequences of venom group IA PLAs showed a close relationship between Bungarus and Australian and marine Elapidae. As the five deduced sequences of KBf-3FTx are only 62–82% identical to the corresponding Bf-3FTx from the pooled venom, the 3FTx apparently have higher degree of individual and geographic variations than the PLAs. None of the KBf-3FTx was found to be neurotoxic or very lethal; phylogenetic analyses of the 3FTx also revealed the unique evolution of Bf as compared with other kraits.

Abbreviations

Bf, Bungarus fasciatus; diC16PC,L-dipalmitoyl phosphatidylcholine; diC6PC,L-dicaproyl phosphatidylcholine; 3FTx, threefinger toxin; KBf, Kolkata B. fasciatus; PLA, phospholipase A2.

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acetylcholine esterase [14] and other enzymes [15]. The numbers of isoforms for PLA and 3FTx from the pooled Bf venom were high, but the intraspecies or the geographic variations of this venom species have not been explored.

Intra-species variations of venom proteins [16] such as PLAs have been well documented for several viperid species [17,18], but are less well explored for elapid venom. In order to better understand the proteomics and variations of Bf venom, we studied individual venom of two specimens of Bf from Kolkata, India (designated as KBf) by a comparative proteomic and genomic approach. The venom PLA and 3FTx iso-forms were purified and characterized. After the mRNA was prepared from KBf venom glands, cDNAs corresponding to the two toxin families were amplified and cloned using specifically designed primers. The amino acid sequence and mass of the PLA and 3FTx were predicted from the cDNA sequences, matched with those of the purified KBf venom proteins as well as PLA and 3FTx isoforms reported for pooled Bf venom.

The three most abundant PLAs in Bf venom are Va, Vb-2 and VI (comprising60% of the proteins in pooled venom); similar PLA isoforms are also abun-dant in the KBf venoms. These enzymes bear a Pro31 substitution near the highly conserved Ca2+ binding loop [19] and are characterized with low enzymatic activities [3], but show membrane-interfering activities and moderate lethality to mice [20,21]. By kinetic study, we further determined their abnormally low kcat values toward phospholipids substrates, but high Ca2+ binding affinity. Finally, phylogenetic analyses of the elapid PLAs and the krait 3FTx were carried out to

better understand the intrageneric and intergeneric variations of kraits and their position in the Elapidae biosystematics.

Results and Discussion

Purification and characterization of venom proteins

To assure that the observed proteins sequence varia-tions between the individual and pooled Bf venom could be attributed to geographic variations, venom samples were collected from two KBf near Kolkata in different seasons for this study. Crude venom was dissolved in buffer and fractionated by Superdex G75 gel filtration on a Pharmacia FPLC system (Fig. 1). Eluted fractions were collected and lyophilized sepa-rately. Pooled fractions B and C (Fig. 1) were then purified by reversed phase HPLC on a C18-column. The chromatographic profiles of the two KBf venoms were not identical (Fig. 2). Homogeneities of each protein peak were examined by SDS⁄ PAGE. Abun-dance of a protein in the crude venom was estimated based on the relative peak area of its UV absorbance at 280 nm and expressed as percentage content (w⁄ w), assuming equal extinction coefficient for all the proteins (Table 1).

A total of seven PLAs and five 3FTx were purified from each KBf venom, and were analyzed by auto-matic sequencing and mass spectrophotometry. The results were listed in Table 1. All these venom pro-teins showed a single mass peak by ESI-MS spectro-metry, except that the PLA KBf-II contained a substantial amount of the oxidized form (13 019 Da)

Fig. 1. Gel filtration of crude venoms of two KBf (samples 1 and 2). Venom powder (15–20 mg) of KBf was dissolved in 200 lL of deionized water and loaded onto a Superdex G75 (HR10⁄ 30) column. The elution step was carried out on a FPLC system with an equilibration buffer containing 0.1Mammonium acetate (pH 6.24) at a flow rate of 0.5 mL min)1. Fractions (B), (B¢), (C) and (C¢) were pooled separately.

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besides the native form (13 003 Da). The KBf-PLAs were also matched with previously reported PLA var-iants of the pooled Bf venom [3–6]; only one of them was found to be identical to Vb-2, with the others being 94% similar to the other five Bf PLA isoforms (Fig. 3). Two inactive PLA homologs, with N-ter-minal sequence either identical to Bf Ala49-PLA [5] or with a single substitution Val3ILe, were purified from both KBf venoms. The differences in their molecular masses (Table 1) and HPLC elution time (Fig. 2) may be attributed to this single mutation at position 3. The novel PLAs were thus named after their orthologous or closest Bf-PLA isoforms as: KBf-Va, KBf-VI, KBf-Vb-1, KBf-II, KBf-III, and KBf-A49, respectively (Table 1). Like the pooled venom, Vb-2, KBf-Va, and KBf-VI together

com-prised about 55–60% of the individual venom mass. Notably, two Bf-PLAs, X-1 (13 025 Da) and XI-2 (13 342 Da) [4,10], were absent in both KBf venom, although a highly similar PLA (designated as KBf-X) was cloned (see next session).

Various 3FTx subtypes were purified from the two KBf venoms and annotated as 3FTx-LI, -LK, LF, -LT, -RK and -RI, respectively, according to their first and second amino acid residues (Table 1). The individ-ual KBf venoms have identical sets of PLAs and several conserved 3FTx (3FTx-LT and 3FTx-RK), but two of their 3FTx show sequence and mass variations (Table 1). In particular, the major 3FTx-LI (-LK) in sample 1 KBf and 3FTx-LF in sample 2 KBf were very different. PLAs and 3FTx are common elapid venom families and are known to undergo accelerated

Fig. 2. Purification of venom proteins by RP-HPLC. Protein fractions from gel filtration were re-solubilized separately and injected into a Vydac RP-C18 column. For (B) and (B¢), elution started with 20% buffer B for 5 min followed by a linear gradient of buffer B for 25 min; for (C) and (C¢), the elution started with 15% buffer B for 5 min followed by a linear gradient of buffer B for 25 min, flow rate was 1.0 mLÆmin)1. Venom protein PLAs and 3FTx were purified and confirmed by ESI-MS and pH-stat enzyme assays. Their annotations are the same as in Table 1.

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Table 1. Inventory of PLAs and 3FTx purified from KBf venom. Masses were determined by ESI-MS spectrometry. PLA annotations follow those previously published or cloned (PL-II, accession number AF387594).

PLA or 3FTx

% content

(w⁄ w) Mass (Da) N-Terminal sequence determined

Both KBf

KBf Va 11 13079 ± 1 NLLQFKNMIQ CAGSRLWVAY

Vb-2 15 13093 ± 1 NLLQFKNMIQ CAGSRLWVAY

KBf VI 23 13051 ± 1 NLYQFKNMIE CAGTRTWLAY

KBf II 7 13003 ± 1 NLLQFKNMIE CAGTRTWMAY

KBf-III 0.7 13412 ± 1 NLFQFKNMIQ CAGTRSWTDY

KBf-A49 0.6 13170 ± 1 NMIQFKSMVQ CTSTRPWLDY

KBf-A49¢ 0.4 13156 ± 1 NMVQFKSMVQCTSTRPWLDY kBf, number 1 3FTx-LI 5.5 6455 ± 1 LICYSSSMNKDSKT 3FTx-LK 1.9 6401 ± 1 LKCHTTQFRNIET 3FTx-LT 0.4 7421 ± 1 LTCLICPEKYCQKVHTXR VIIIa 0.4 7420 ± 1 LTCLICPERYCQKVHTXR 3FTx-RK 0.5 7305 ± 1 RKCLTKYSQDNESSKT kBf, number 2 3FTx-LI 0.1 6374 ± 1 LICYSSPMSKETKTCQKWET 3FTx-LF 2.4 6882 ± 1 LFCYKTPSTKGYQICEKWQT 3FTx-LTa 0.5 7421 ± 1 LTCLICPEKYCQKVHT VIIIaa 0.5 7420 ± 1 LTCLICPERYCQKVHT 3FTx-RK 1.2 7305 ± 1 RKCLTKYSQDNESSKT

aKBf3F-LT and VIIIa were co-purifed as revealed by N-terminal sequencing and mass analysis.

Fig. 3. Alignment of amino acid sequences of KBf PLAs and related venom PLAs. Single-letter codes of amino acids are used, conserved residues are reversed out, and gaps are marked with hyphens. The numbering system of Renetseder et al. [58] has been adopted. Acces-sion numbers for B. fasciatus PLAs are as follows: Vb-2, P00609; Va, P00628; VI, P00627; II, Q90WA8; III, P14615; for B. candidus group IB, GenBank AAO84769.

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evolution [22]. Intra-species venom variations usually result from quantitatively differential expression or minor structural changes of the venom proteins [18]. It is rather surprising that the venom 3FTx showed such a high degree of individual variation in the KBf speci-mens. Our results thus suggested that mutational rates of the exon of the 3FTx genes are much faster than those of the PLA genes, leading to high variation of KBf-3FTx.

Cloning and cDNA sequencing

Venom glands of only one of the KBf specimens were used for total RNA extraction. We have used facile methods to clone many toxin cDNAs from the Bf venom glands after cDNAs corresponding to the major toxin families had been amplified by PCR. This is a relatively economical and efficient approach to clone and determine protein sequences of the toxin families. It is also a powerful tool to study tissue-specific mRNAs expressed in low levels. Distinct clones were selected and sequenced at least twice, and then transla-ted into amino acid sequences. Seven PLA clones were identified from about 50 sequenced cDNA clones and their full amino acid sequences were thus deduced (Fig. 3). The venom PLA precursors contain a con-served 27-residue signal peptide which is similar to those of other elapid venom PLAs (Table 2). The pre-dicted enzyme regions also closely matched masses and partial sequences of the purified PLAs (Table 1). Using the same approach, a total of six 3FTx were cloned, sequenced and matched with the protein purified.

Their 21-residue signal peptides were also very con-served (Table 2).

Although two KBf-A49 as well as KBf-Vb-1 venoms were purified (Table 1), we failed to clone their cDNA. There are probably some distinct mutations in 5¢-UTR of the cDNA templates, leading to insufficient priming during the PCR reactions. The Ala49 mutants are rather unique among the elapid venom PLAs, and mutations of Asp49Ala, Tyr28Asn and Gly30Asp at their catalytic Ca2+ binding sites [5] presumably abol-ish the enzymatic activity of KBf-A49 (Table 3). Nev-ertheless, we have cloned a group IB PLA (with pancreatic loop) and designated it as KBf-grIB. Its protein sequence is 84% identical to the group IB PLA cloned from the Malayan krait Bungarus candidus [23] (Fig. 3). The group IB PLAs were never been purified from Bungarus venoms, possibly because of degeneration.

Table 2. cDNA deduced venom PLAs and 3FTx of KBf. The isoelectric point (pI) and molecular mass were predicted from each protein sequence. ND, not determined.

Encoded protein Calculated mass (Da) Predicted pI Number

of clones Signal peptide sequence

PLA KBf -Va 13077 8.0 4 MYPAHLLVLLAVCVSLLGAANIPPQPL Vb-2 13091 8.0 5 MYPAHLLVLLAVCVSLLGAANIPPQPL KBf-VI 13051 8.0 7 MYPAHLLVLLAVCVSLLGAANIPPQSL KBf-II 13003 8.0 2 MYPAHLLVLLAVCVSLLGAANIPPQSL KBf-III 13411 5.3 5 ND KBf-Xa 13177 8.9 2 MYPAHLLVLLAVCVSLLGAANIPPQPL

KBf-grIBa 14141 4.8 3 MYPAHLLVLLAVCVSLLGAS I IPPQPL

3FTx 3FTx-LI 6455 8.2 5 MKTLLLTLVVVTIVCLDLGYT 3FTx-LK 6401 8.7 4 MKTLLLTLVVVTIVCLDLGYT 3FTx-LT 7421 8.7 2 MKTLLLTLVVVTIVCLDLGYT VIIIa 7420 8.7 9 MKTLLLTLVVVTIVCLDLGYT 3FTx-RK 7305 9.5 1 MKTLLLTLVVVTIVCLELGYT 3FTx-RI* 6968 8.7 3 MKTLLLTLVVLTIVCLDLGHT

aCould not be isolated from both KBf venoms.

Table 3. Enzymatic activities of purified venom PLAs toward zwit-terionic micellar substrates. Initial hydrolysis rate of 3 mMdiC16PC

in the presence of 6 mMTriton X-100, 10 mMCaCl2and 0.1MNaCl was measured with a pH-stat apparatus. Data of Vb-2 and VI were taken from [3].

PLA Specific activity (lmolÆmg)1Æmin)1)

KBf-Va 23 Bf-Vb-2 27 Bf-VI 9.8 KBf-II 25 KBf-A49 < 0.5 KBf-III 45

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Alignment and comparison of amino acid sequences

Complete amino acid sequences of KBf-PLA paralogs deduced from cDNA sequences were aligned pairwise with those of Bf-PLAs obtained by protein sequencing (Fig. 3). Apparently, only one of the KBf PLAs is identical to the previously reported Vb-2, while the other five are 94–98% identical to Bf Va, Vb-1, VI, XI-2 (or X-1) [3,5,10] and PL-II (from Chinese Bf, accession number AF387594), respectively. Although its cDNA has been cloned, KBf-X is not expressed in both KBf venoms. The previously reported X-1 and XI-2 [10] are structurally very similar to KBf-X and they possibly represent the allelic variants of KBf-X in different individual snakes.

We also deduced the full protein sequences of five 3FTx from cDNA sequences (Table 2). The KBf-3FTx are all basic proteins with 57–62 amino acid resi-dues and four disulfide bonds, except 3FTx-LT and VIIIa, which contain 65 residues and a fifth disulfide bond in the loop I region. The venom 3FTx of Bf and KBf may be putatively classified into five types with distinct N-terminal sequences (i.e. LI, LK, LT, RK or RI). They were aligned and compared with those of the 3FTx purified from the pooled Bf venom [7,8,10], or the most related sequences identified by a blast search (Fig. 4). Notably, only VIIIa is conserved in both KBf and Bf venom samples; the amino acid sequences of the other four KBf-3FTx appeared to be 62–82% identical to the published sequences of Bf-IV, fasiatoxin, VIIIa, and VII, respectively. Besides many amino acid substitutions, KBf 3FTx-LI and 3FTx-LK are shorter than their apparent Bf-3FTx orthologs (IV and fasciatoxin, respectively) by five or six residues at the C-terminus (Fig. 4). Thus, geographic variations of 3FTx are greater than those of PLAs in this venom species. Notably, all the four-disulfide-containing 3FTx of this species include a Trp residue at their loop II (Fig. 4), which is rather uncommon among elapid

venom 3FTx [24]. We also found that 3FTx-LT is identical to a weak neurotoxin NTX4 (AY611643) pre-sent in B. candidus venom, while 3FTx-RK is 84% identical to bucain [25] of B. candidus.

Calcium binding and kinetic parameter of the P31-PLAs

Four PLAs (Va, Vb-2, VI and II) of KBf and Bf con-tain a Pro at position 31 and are hereafter referred to as P31-PLAs. Their functions appear to resemble cobra ‘direct lytic factors’ or cytotoxins, which cause membrane depolarization, muscle necrosis and moder-ate lethality [20,21,26]. These enzymes showed very low hydrolytic activities toward various kinds of micelles and mono-dispersed substrates in vitro (Table 3) [8,27]. Other P31-PLAs were also found in Australian and marine elapid venoms, including Pa-13, Pa-15 from Pseudechis australis, pseudexin B from Pseudechis porphyriacus[28–30], and LcPLH from Lat-icauda colubrina[31]. They are usually abundant in the venom and show low catalytic activities. Thus, the evo-lution of P31-PLAs in elapid venom bears a similarity to the Lys49-PLAs [32] in pitviper venom in the sense that they are all basic PLAs present in relatively high content and retain interfacial or membrane binding properties in spite of the low catalytic activities.

In fact, many of the inactive Lys49 PLAs from crota-lid venoms also contain Pro31 [32], while other viperid venom PLAs usually contain Trp31 [17,32]. Group IA or elapid venom PLAs with higher catalytic activities usually contain Lys, Arg or Leu at position 31 [33,34]. Previous studies of pancreatic PLA mutants revealed that replacements of Leu31 or Arg31 by other amino acids reduced the enzymatic activities considerably [34,35]. Position 31 is at the entrance of the substrate cleft and is one of the major interface-recognition sites of PLAs [19,32,36]. It is thus reasonable to speculate that Pro31 substitution may affect either Ca2+binding and⁄ or configuration of the oxyanion-hole at the amide

Fig. 4. Alignment of amino acid sequences of 3FTx of Bf and other related species. Single-letter codes of amino acids are used, conserved residues are reversed out, and gaps are marked with hyphens. Asterisks denote the eight conserved Cys residues. SwissProt accession numbers or references are as follows: fasicatoxin, P14534; VII-1, P10808; VI and VIIIa [10], bucain (from B. candidus venom), P83346.

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backbone of Gly30 and thus the kinetic properties of the PLA reactions.

To better understand whether the Ca2+ binding was affected by Pro31 substitution, we carried out kinetic analyses of the P31-PLAs at different concentrations of CaCl2 (Fig. 5). Our results showed that the P31-PLAs can bind Ca2+ with a dissociation constant of 13–49 lm, suggesting a stronger binding than many other catalytically active venom PLAs, which have a Ca2+dissociation constant of 100 lm (Fig. 5). We also compared the kinetic properties of Bf VI (a P31-PLA) with those of Bf X-1 (containing K31) using l-dipalmi-toyl phosphatidylcholine (diC16PC) in Triton X-100 (1 : 2, molar ratio) and monodispersed l-dicaproyl

phosphatidylcholine (diC6PC; Fig. 6B). The turnover rate (kcat) of Bf VI calculated from double reciprocal plots was about 10-fold lower than that of Bf X-1, while their apparent Km values were rather similar (Fig. 6). Thus, it is very likely that the P31 substitution prevents the backbone amide of Gly30 from forming an essential oxyanion hole in the transition state, thus reducing kcatby10-fold.

The Ca2+-dependent hydrolysis of 2-acyl ester of lecithin substrate by P31-PLAs has been confirmed [37]. The enzymes have a preference to interact with the zwitterionic micelles (diC16PC and Triton X-100) rather than the anionic micelles (diC16PC and deoxycholate) [3]. However, substrate binding to group I PLAs was

Fig. 5. Ca2+-binding affinity of two

Pro31-PLAs (Bf Va and VI) and a K31-PLA (Bf-X-1). The initial rate of hydrolysis of 3 mM

diC16PC in the presence of 6 mMTriton

X-100 was measured by pH-stat at pH 7.3 and 37C with 0.1MNaCl at different CaCl2

concentrations. The 1⁄ Vmaxvalues

deter-mined from double reciprocal plots were further plotted against reciprocals of CaCl2

concentrations to determine the Ca2+ affinity of the PLA.

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found to be independent of the Ca2+ binding. This is in contrast with group II PLAs, whose substrate bind-ing was facilitated >10-fold upon enzyme bindbind-ing to Ca2+[38]. It has been shown in other esterases that the contribution of the oxyanion hole to the transition-state stabilization reaches 20 kJÆmol)1, and accounts for a 100-fold increase of catalytic rates [39]. Because the P31-PLAs could effectively hydrolyze a chromo-genic pseudo-substrate, 4-nitro-3-octanoyloxybenzoate [3], the transition state or mechanism of hydrolysis of this ester is probably different from that of the phos-pholipid micelles.

Functions or toxicity of the 3FTx

The elapid venom 3FTx are a large multigene family and recent phylogenetic analyses of all the 3FTx revealed that kraits’ venom may contain type I and II (short or long chain) a-neurotoxins and many ‘orphan groups’ whose functional roles are not clear [40]. The major 3FTx in KBf (sample 1) are 3FTx-LI and -LK (i.e. ‘orphan group XVIII’), which were either not at all or only weakly neurotoxic, as tested in pharmacological studies using the chick biventer cervicis [41] or rat phrenic nerve diaphragm [42]. Sur-prisingly, 3FTx-LI and -LK found in KBf sample 1 venom (Table 1) are not conserved in KBf sample 2 venom. The lethal dose (LD)50 (2.1 mgÆkg)1) for venom of number 1 KBf used in this particular study was slightly higher than previously reported (1.3– 1.5 mgÆkg)1) for the pooled venom from several sup-pliers [1]. Mice administered with a lethal dose of KBf venom did not show typical neurotoxic symp-toms. The only postsynaptic neurotoxin previously isolated, albeit with low yield, from the pooled Bf venom was VII-1 [8] (belonging to type I a-neurotox-in [40]), but we failed to isolate a similar protea-neurotox-in from these two KBf venoms (Table 1). This can prob-ably explain why the KBf venom has weaker lethality than the pooled Bf venom.

Notably, VIIIa and 3FTx-LT appears to be con-served in the venoms of both KBf and Bf; they are similar to B. candidus NTX4 and Naja melanoleuca s4c11 (SwissProt P01400), which belong to the ‘orphan group II’ [40] or unconventional 3FTx [43]. Another protein 3FTx-RK (belonging to ‘orphan group III’) is conserved in both KBf venoms, and is very similar to bucain from B. candidus venom [22] and a 3FTx cloned from Bungarus multicinctus (AJ006137 [44]). These 3FTx are present in moderate quantities and their targets remain to be identified. The fact that all isolated Bf venom proteins are less toxic (LD50> 4 lgÆg)1 in mice) [10] than the crude venom (LD50 of 1.3–2.1 lgÆg)1 in mice) suggests that synergisms between venom components are import-ant.

Phylogenetic analyses of krait PLAs and 3FTx The results in the present study suggested that previ-ously reported Bf-PLA isoforms, including III, Va, Vb-2, VI, A49, and II (which was cloned from the Chinese Bf), are probably paralogous to each other, as they coexist in a single KBf venom. A cladogram (Fig. 7) was built based on the amino acid sequences of 34 representative group IA PLAs with the king

Fig. 6. Lineweaver)Burk plots of the hydrolysis of lecithins by Bf VI and X-1. Initial reaction rates were measured by pH-stat at pH 7.3 and 37C with 0.1MNaCl and 6 mMCaCl2. The value of

kcatwas calculated by dividing the Vmaxwith the enzyme

concentra-tion. (A) Hydrolysis of mixed micelles of diC16PC and Triton X-100

(1 : 2); the PLA used was 0.14 lM Bf VI (d) or 0.057 lMBf X-1 (s). (B) Hydrolysis of diC6PC; the PLA used was 1.4 lMBf VI (d)

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cobra venom group IB PLA as an out-group; all the KBf-PLAs except KBf A49 were included. The genus Bungarus appears to be monophyletic, as all the krait PLAs except KBf-III are allied together in this robust tree. Topology of this PLA tree is also in accord with a species tree based on the mtDNA sequences, showing that Bungarus contains three lineages represented by Bf, Bungarus flaviceps and other Bungarus species, respectively [1,40,45]. Notably, venom PLAs of differ-ent genera of elapids are clearly resolved with high bootstrap supports in the phylogenetic tree (Fig. 7). The sea snakes have been shown [45,46] to be diphylet-ic within the Australian and marine elapid clade (with the laticaudines and hydrophiines having separate ori-gins). Notably, the PLA tree (Fig. 7) revealed that Bungarus is closer to Australian and marine elapid snakes than to the Asian cobra or king cobra; the rela-tionship has not been shown in previous phylogenetic trees of elapid venom PLAs [45–47]. Our data thus support a novel phylogenetic relationship for reinter-pretation of the systematics of these elapid genera.

In addition, a cladogram of kraits’ 3FTx was built based on the amino acid sequences (Fig. 8). It has been pointed out that type I and type II a-neurotoxins are ubiquitous among elapid venoms, but that orphan groups III, IV, V, IX, XVII, XVIII and XIX of 3FTx are restricted to kraits’ venom [40]. The tree in Fig. 8 shows that Bf venom contains only four paralogous 3FTx, i.e. type I a-neurotoxin and orphan groups II, III, and XVIII. In contrast, venoms of B. multicinctus, B. candidus and B. flaviceps have special type II a- and j-neurotoxins [40,48] and orphan groups IV, V, IX, XVII, or XIX, while sharing the orphan groups II and III with Bf venom. Notably, the neurotoxic PLAs (b-bungarotoxins) are present in venom of all kraits except Bf [48,49]. It is thus likely that Bf is an unique and primitive krait lineage. Speciation of Bf possibly took place before the other kraits evolved distinct 3FTx-orphan groups and strong type II neurotoxins and b-bungarotoxins, and before B. flaviceps lineage split from other neurotoxic kraits including B. caelu-rus, B. multicinctus and B. candidus [1,48].

Fig. 7. Phylogenetic analysis of group IA venom PLAs. The dataset includes amino acid sequences of selected group IA elapid venom PLAs. Amino acid substitutions at position 31 were shown in parentheses. A group IB PLA purified from king cobra Ophiopagus hannah was used as the out-group. Values above the branches indicate the percentage of 1000 bootstrap replicates. Species names and accession numbers are as follows: Acanthophis antarcticus: acanthin I and II, P81236 and P81237; Bungarus caeruleus: PL, PL-1, -2 and -3, AF297663, AAS20530, AAR19228-9; Bungarus flavicpes: PL-I and -II, Ab112359–60; B. multicinctus: 0702209 A; Haemachatus haemachatus: P00595; Laticauda colubrina: K31 and P31, P10116 and P10117; Laticauda laticuadata: PC17 and PL, BAB72251 and CAA68449; Laticauda semifasci-ata: PL I, BAB72247; Naja atra: CAA51694; Naja kaouthia: P00596; Naja m. mossambica: P00602; Naja naja: acidic PLA, CAA45372; Notechis scutellatus: notexin, P00608; Oxyuranus scutellatus: OS2 AAB33760; P. australis: PA11 and PA13, P04056 and P04057; P. porphy-riacus: pseudexin A and B, P20258 and P20259; and O. hannah: acidic I and II, P80966 and Q9DF33.

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Summary and conclusions

Intrageneric and intraspecies variations of kraits’ venom have been investigated by proteomic and tran-scriptomic analyses herein and in other recent studies [40,48,49]. We have cloned and sequenced from a KBf specimen a total of seven PLAs and six 3FTx KBf; among them 11 were novel sequences (Table 2). Major findings or conclusions from this study are: (a) Individ-ual Bf venom contains almost as many paralogous PLAs and 3FTx variants as the pooled venom. (b) The small and nonenzymatic 3FTx show much greater geo-graphic and individual variations than the PLAs in this venom species. (c) Pro31 substitution in ‘cardiotoxin-like PLAs’ is an evolutionary strategy to reduce the enzyme turnover rates but retain high affinity for bind-ing to Ca2+ and the membrane interface. (d) Kraits are possibly genetically related to Australian and mar-ine elapids. (e) Bf venom has evolved distinct PLA and 3FTx subtypes which are not found in other kraits’ venoms, and their functions remain to be elucidated. Apparently, Bf split from other krait species in very ancient times and evolved with non-neurotoxic venom strategy. It is also worth noting that the prey of Bf and king cobra consists mainly of snakes and reptiles,

which are distinct from those of other kraits (e.g. B. candidus and B. multicinctus, which prey on small catfishes, eels and rodents [50]).

Experimental procedures

Materials

Crude venom was milked from two individual specimen of Bf (Calcutta Snake Park, Kolkata, India). Venom glands were dissected after killing one of the snakes. The tissue was preserved for several weeks in the RNAlater solution (Ambion, Austin, TX, USA) before extraction of mRNA for preparation of the cDNA. Modification and restriction enzymes and the pGEM-T vector were purchased from Promega (Madison, WI, USA). Phospholipid substrate was from Avanti-Biochemical (Alabaster, AL, USA). Triton X-100 and sodium deoxycholate were from Sigma Chemical Co. (St Louis, MO, USA). All buffers and chemi-cals were reagent grade.

Venom protein purification

Lyophilized venom (15–20 mg) was dissolved in a small volume of 100 mm ammonium acetate (pH 6.24) followed Fig. 8. Phylogenetic analysis of kraits’ venom 3FTx. The dataset used includes full amino acid sequences so far available for 3FTx of krait venoms, except a possibly erroneous Q9W727 [40]. H. haemachatus cytotoxin P24776 was used as the out-group. Values above branches indicate the percentage of 1000 bootstrap replicates. In addition to those directly shown in the tree, the accession numbers and references are as follows: B. candidus (Bc): a-bgtx CAD92407, bucain P83346, bucaindin P81782, candiduxin 1 and 2 AAL30057 and 8, candoxin AAN16112, ntx4 AAT38875, wtx 1–3 AAL30059-61; B. flaviceps (Bfl): j-flavitoxin P15815; B. multicinctus (Bm): a-bgtx CAB51843, c-bgtx CAD01082, j, j1a, j1b, j2, j3, j5, j6-bgtx CAA69971, AAL30054-5, P15816, CAA72434, O12962, Q9W729; and B. fasciatus (Bf): Bf-IV [59], BfVII-1 P10808, VIIIa [10], fasciatoxin P14534. ntx, neurotoxin.

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by centrifugation at 9000 g for 5 min on a Kubota (Tokyo, Japan) KM-15200 centrifuge equipped with angle rotor RA2724. The supernatant was applied to a Super-dex-G75 gel filtration column and eluted with the same buffer on a FPLC system. Fractions containing PLAs and 3FTx were further purified by reverse-phase HPLC on a Vydac C18 column (Vydac; 4.6· 250 mm). Elution was carried out in a gradient containing buffers A and B, which were made of 0.07% (v⁄ v) trifluoroacetic acid in distilled water and acetonitrile, respectively. Proteins col-lected from the elution peaks were dried in a vacuum-cen-trifuge device (Labconco, Kansas City, MO, USA). Protein concentrations in stock solutions were determined with a dye-based protein determination kit from Bio-Rad (Hercules, CA, USA) [51].

Determination of protein sequences and masses

The N-terminal sequences of purified proteins were deter-mined by a gas-phase amino acid sequencer coupled with a phenylthiohydantoin amino acid analyzer (model 477 A; Perkin Elmer, Foster City, CA, USA). The molecular weight of each purified protein (dissolved in 0.1% acetic acid with 50% acetonitrile by volume) was analyzed under positive mode by ESI-MS on a mass spectrometer (Sciex API100, Perkin Elmer). Purity of the venom pro-tein was assessed by SDS⁄ PAGE and N-terminal sequen-cing.

Cloning of venom toxins

The mRNA from Bf venom glands was extracted using the mRNA extraction kit. Their complementary DNA (cDNA) was prepared using the cDNA synthesis kit according to the manufacturer’s instructions (Stratagene, La Jolla, CA, USA). PCR primers were synthesized based on the con-served regions of the cDNA sequences encoding homologs of elapid venom PLAs [23] and 3FTx [24] , respectively. For amplification of 3FTx, primer 1 was 5¢-ATGAAAAC TCTGCTGCTGACCTTG-3¢ and primer 2 was 5¢-CTCAA GGAAWTTAGSCAC TCRKAGAG-3¢. For amplification of PLAs, the primers 3 and 4 used were 5¢-GCAGTTTGT

GTCTCCCTCTTAGGA-3¢ and 5¢-CACAGTCCTTGA

GCTGAAGCTTCTC-3¢. In addition, primer 5

(5¢-CAG(C,T)(C,A)TCTCAATCTCTT(T,C)-3¢) was designed based on the N-terminal sequence of KBf-III to replace pri-mer 3 for PCR. Pripri-mers 1, 3 and 5 were in the sense orien-tation of the 5¢-end sequence, whereas primers 2 and 4 were in the antisense direction of a conserved region at the 3¢-end untranslated region.

PCR was conducted using cDNA of Bf venom glands as templates in the presence of SuperTaq DNA polymerase (HT Biotech, Cambridge, UK) [52]. The conditions of each of the 30 cycles were set to 92C for 1.0 min during denaturation, 52C for 1.0 min during annealing, and 72 C for 1.0 min

during extension. As examined by 1% agarose gel electro-phoresis, DNA fragments at the expected size for PLA, 3FTx and KuI were specifically amplified. After treating with poly-nucleotide kinase, the product was inserted into the pGEM-T vector (Promega Biotech) that was then used to transform Escherichia coli strain JM109 [53]. The plasmid DNA was extracted from white transformants and was further exam-ined for its restriction pattern by agarose gel electrophoresis. The cloned cDNA was sequenced by the DNA-Sequencing-System (model 373 A; PE-Applied Biosystems, Foster City, CA, USA).

PLA assay and kinetic analysis

Micelles of 3 mm diC16PC with 3 mm sodium deoxycholate

or 6 mm Triton X-100, or diC6PC and 100 mm NaCl were

prepared in a glass-Teflon tissue homogenizer, and 2.5 mL of the solution was transfer to a reaction cup with a ther-mostat of the pH-stat apparatus (Radiometer, Copenhagen, Denmark). With constant stirring, 10 mm CaCl2was added

directly before addition of the enzyme. Release of acid dur-ing substrate hydrolysis was followed by pH-stat titration at pH 7.4 and 37C with 8 mm NaOH. The initial hydro-lysis rate was corrected to the nonenzymatic rate in each experiment. The affinity of Ca2+was determined kinetically at different concentrations of CaCl2following the published

methods [3,38].

Neuromuscular effects

Neurotoxicity of purified Bf-venom proteins was assessed on chick biventer cervicis [42] and rat phrenic nerve dia-phragm neuromuscular preparation [43]. Leghorn chicks (10 days old) were anaesthetized with chloroform and the biventer cervicis muscle was dissected out. One end of the muscle was tied with a oxygenator tube and the other end was tied with Brodie’s lever. The muscle was passed through the platinum electrode. The preparation was sus-pended in 4 mL oxygenated (95% O2+ 5% CO2) Tyrode’s

solution (137 mm NaCl, 2.7 mm KCl, 1.8 mm CaCl2,

1.2 mm MgCl2, 11.9 mm NaHCO3, 0.4 mm NaH2PO4 and

5.5 mm glucose) at room temperature (29 ± 1C).

Male albino rats (150 ± 10 g) were killed by stunning and the hemidiaphragm with attached phrenic nerve was dissected out with a small portion of the anterior chest wall to serve as an anchor for the platinum electrode. The pointed end of the diaphragm segment was attached to Brodie’s lever with a thread and the nerve was threaded through the platinum electrode. The chick or rat prepar-ation was suspended in 6 mL oxygenated Tyrode solution at room temperature (29 ± 1C). The preparation was stimulated with a square wave electronic stimulator at 8–12 V of 0.5 ms duration and 10-s pulse. Muscle contrac-tions were recorded by Brodie’s lever on a rotating smoked drum.

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Lethal effects

Lethal potency of purified PLA was determined in ICR adult mice of 30 g body weight. The PLA was injected intraperitoneally with 0.1 mL protein prepared in sterile phosphate-buffered saline. Six mice were used to obtain the median LD50of each dosage. LD50and its confidence limit

at 95% probability were calculated [54].

Phylogenetic analysis of Bungarus venom PLA2

The alignment of amino acid sequences was prepared using the cllustal w program [55]. Cladograms were construc-ted based on the aligned sequences by a neighbor-joining algorithm using the phylip program [56], and the degree of confidence for the internal linage was determined by boot-strap methods [57].

Animals

Animals (mice, rats and chicks) were treated according to institutional guidelines for the care and use of experimental animals under the approval of the University of Calcutta, India.

Acknowledgements

This work was supported by grants from Academia Sinica and National Science Council, Taiwan, ROC. The authors thank C. S. Liu for his encouragement and advice, and for his generosity in providing B. fas-ciatusvenom PLAs for kinetic studies.

References

1 Slowinski JB (1994) A phylogenetic analysis of Bungarus (Elapidae) based on morphological characters. J Herpe-tol 28, 440–4462.

2 Tan NH & Ponnudurai G (1990) A comparative study of the biological properties of krait (genus Bungarus) venoms. Comp Biochem Physiol 95C, 105–109. 3 Liu CS, Chen JM, Chang CH, Chen SW, Tsai IH, Lu

HS & Lo TB (1990) Revised amino acid sequences of the three major phospholipases A2from Bungarus

fasciatus(banded krait) venom. Toxicon 28, 1457–1468. 4 Liu CS, Leu HL, Chang CS, Chen SW & Lo TB (1989)

Amino acid sequence of a neutral phospholipase A2

(III) in the venom of Bungarus fasciatus. Int J Pept Protein Res 34, 257–261.

5 Liu CS, Kuo PY, Chen JM, Chen SW, Chang CH, Tseng CC, Tzeng MC & Lo TB (1992) Primary struc-ture of an inactive mutant of phospholipase A2in the

venom of Bungarus fasciatus (banded krait). J Biochem (Tokyo) 112, 707–713.

6 Liu CS, Chang CS, Leu HL, Chen SW & Lo TB (1988) The complete amino-acid sequence of a basic phospholi-pase A2in the venom of Bungarus fasciatus. Biol Chem

Hoppe Seyler 369, 1227–1233.

7 Liu CS, Hsiao PW, Chang CS, Tzeng MC & Lo TB (1989) Unusual amino acid sequence of fasciatoxin, a weak reversibly acting neurotoxin in the venom of the banded krait, Bungarus fasciatus. Biochem J 259, 153–158. 8 Liu CS, Chen JP, Chang CS & Lo TB (1989) Amino

acid sequence of a short chain neurotoxin from the venom of banded krait (Bungarus fasciatus). J Biochem (Tokyo) 105, 93–97.

9 Jiang MS, Haggblad J & Heilbronn E (1986) Interaction with chick myotube cholinergic receptors of an alpha-neurotoxin isolated from venom of the banded krait (Bungarus fasciatus). Toxicon 24, 713–719.

10 Liu CS & Lo TB (1994) Chemical studies of Bungarus fasciatusvenom. J Chinese Biochem Soc 23, 69–75. 11 Liu CS, Wu TC & Lo TB (1983) Complete amino

acid sequences of two protease inhibitors in the venom of Bungarus fasciatus. Int J Pept Protein Res 21, 209– 215.

12 Chen C, Hsu CH, Su NY, Lin YC, Chiou SH & Wu SH (2001) Solution structure of a Kunitz-type chymo-trypsin inhibitor isolated from the elapid snake Bun-garus fasciatus. J Biol Chem 276, 45079–45087. 13 Zhang Y, Xiong YL & Bon C (1995) An activator of

blood coagulation factor X from the venom of Bungarus fasciatus. Toxicon 33, 1277–1288.

14 Cousin X, Creminon C, Grassi J, Meflah K, Cornu G, Saliou B, Bon S, Massoulie J, Bon C, Cousin X et al. (1996) Acetylcholinesterase from Bungarus venom: a monomeric species. FEBS Lett 387, 196–200. 15 Yost DA & Anderson BM (1983) Adenosine

dipho-sphoribose transfer reactions catalyzed by Bungarus fas-ciatusvenom NAD glycohydrolase. J Biol Chem 258, 3075–3080.

16 Warrell DA (1997) Geographic and intraspecies vari-ation in the clinical manifestvari-ations of envenoming by snakes. In Venomous Snakes (Thorpe RS, Wuster W, Malhotra A, eds). pp. 189–204. Clarendon Press, Oxford.

17 Tsai IH, Wang YM, Chen YH, Tsai TS & Tu MC (2004) Venom phospholipases A2of bamboo viper

(Tri-meresurus stejnegeri): molecular characterization, geo-graphic variations and evidence of multiple ancestries. Biochem J 377, 215–223.

18 Tsai IH, Wang YM & Chen YH (2003) Variations of phospholipases A2in the geographic venom samples of

pit vipers. J Toxicol -Toxin Rev 22, 651–662.

19 Scott DL (1997) Phospholipase A2: structure and

cata-lytic properties. In Kini, RM, ed. Venom Phospholipase A2Enzymes: Structure, Function and Mechanism.Wiley,

(13)

20 Qi YH, Gong H, Wieland SJ, Fletcher JE, Conner GE & Jiang MS (1989) Effect of a phospholipase A2with

cardiotoxin-like properties, from Bungarus fasciatus snake venom, on calcium-modulated potassium cur-rents. Toxicon 27, 1339–1349.

21 ShiauLin SY, Huang MC & Lee CY (1975) A study of cardiotoxic principles from the venom of Bungarus fas-ciatus(Schneider). Toxicon 13, 189–192.

22 Chang LS, Chung C, Liou JC, Chang CW & Yang CC (2003) Novel neurotoxins from Taiwan banded krait (Bungarus multicinctus) venom: purification, characterization and gene organization. Toxicon 42, 323–330.

23 Tsai IH, Hsu HY & Wang YM (2002) A novel phos-pholipase A2from the venom gland of Bungarus

candi-dus: cloning and sequence-comparison. Toxicon 40, 1363–1367.

24 Utkin YN, Kukhtina VV, Maslennikov IV, Eletsky AV, Starkov VG, Weise C, Franke P, Hucho F & Tsetlin VI (2001) First tryptophan-containing weak neurotoxin from cobra venom. Toxicon 39, 921–927.

25 Watanabe L, Nirthanan S, Rajaseger G, Polikarpov I, Kini RM & Arni RK (2002) Crystallization and preli-minary X-ray analysis of bucain, a novel toxin from the Malayan krait Bungarus candidus. Acta Crystal D Biol Crystal 58, 1879–1881.

26 Xu K (1986) Membrane active polypeptides from venom of Bungarus fasciatus. Biomed Res 7 (Suppl.), 89–93. 27 Chang WC, Lee ML & Lo TB (1983) Phospholipase A2

activity of long-chain cardiotoxins in the venom of the banded krait (Bungarus fasciatus). Toxicon 21, 163–165. 28 Schmidt JJ & Middlebrook JL (1989) Purification,

sequencing and characterization of pseudexin phospho-lipases A2from Pseudechis porphyriacus (Australian

red-bellied black snake). Toxicon 27, 805–818. 29 Nishida M, Terashima M & Tamiya N (1985) Amino

acid sequences of phospholipases A2, from the venom

of an Australian elapid snake (king brown snake, Pseu-dechis australis. Toxicon 23, 87–104.

30 Takasaki C, Yutani F & Kajiyashiki T (1990) Amino acid sequences of eight phospholipases A2from the

venom of Australian king brown snake, Pseudechis australis. Toxicon 28, 329–339.

31 Takasaki C, Kimura S, Kokubun Y & Tamiya N (1988) Isolation, properties and amino acid sequences of a phospholipase A2and its homologue without activity

from the venom of a sea snake, Laticauda colubrina, from the Solomon Islands. Biochem J 253, 869–875. 32 Wang YM, Pong HF & Tsai IH (2005) Unusual

phos-pholipases A2in the venom of two primitive tree viper

Trimeresurus puniceusand Trimeresurus boneenesis. FEBS J 272, 3015–3025.

33 Kuipers OP, Kerver J, van Meersbergen J, Vis R, Dijk-man R, Verheij HM & de Haas GH (1990) Influence of size and polarity of residue 31 in porcine pancreatic

phospholipase A2on catalytic properties. Protein Eng 3,

599–603.

34 Chang LS, Lin SR & Chang CC (1998) Identification of Arg-30 as the essential residue for the enzymatic activity of Taiwan cobra phospholipase A2. J Biochem (Tokyo) 124, 764–768.

35 Yu BZ, Janssen MJW, Verheij HM & Jain MK (2000) Control of the chemical step by leucine-31 of pancreatic phospholipase A2. Biochemistry 39, 5702–5711.

36 Qin S, Pande AH, Nemec KN, He X & Tatulian, SA (2005) Evidence for the regulatory role of the N-term-inal helix of secretory phospholipase A2from studies on

native and chimeric proteins. J Biol Chem 280, 36773– 36783.

37 Chang WC, Lee ML & Lo TB (1983) Phospholipase A2

activity of long-chain cardiotoxins in the venom of the banded krait (Bungarus fasciatus). Toxicon 21, 163–165. 38 Teshima K, Kitagawa Y, Samejima Y, Kawauchi S,

Fujii S, Ikeda K, Hayashi K & Omori-Satoh T (1989) Role of Ca2+in the substrate binding and catalytic

functions of snake venom phospholipases A2. J Biochem

(Tokyo) 106, 518–527.

39 Magnusson AO, Rotticci-Mulder JC, Santagostino A & Hult K (2005) Creating space for large secondary alco-hols by rational redesign of Candida antarctica lipase B. Chembiochem 6, 1051–1056.

40 Fry BG, Wuster W, Kini RM, Brusic V, Khan A, Venkataraman D & Rooney AP (2003) Molecular evo-lution and phylogeny of elapid snake venom three-finger toxins. J Mol Evol 57, 110–129.

41 Ginsborg BL & Warriner J (1960) The isolated chick biventer cervicis nerve-muscle preparation. Br J Phar-macol 15, 410–411.

42 Bulbring E (1946) Observation on the isolated phrenic nerve diaphragm preparation of the rat. Br J Pharmac Chemother 1, 38–42.

43 Nirthanan S, Gopalakrishnakone P, Gwee MC, Khoo HE & Kini RM (2003) Non-conventional toxins from elapid venoms. Toxicon 41, 397–407.

44 Qian YC, Fan CY, Gong Y & Yang SL (1998) cDNA cloning and sequence analysis of six neurotoxin-like proteins from Chinese continental banded krait. Bio-chem Mol Biol Int 46, 821–828.

45 Slowinski JB & Lawson R (2002) Snake phylogeny: evidence from nuclear and mitochondrial genes. Mol Phylogenet Evol 24, 194–202.

46 Tsai IH (1997) Phospholipases A2from Asian snake

venom. J Toxicol -Toxin Rev 16, 79–113.

47 Slowinski JB, Knight A & Rooney AP (1997) Inferring species trees from gene trees: a phylogenetic analysis of the Elapidae (Serpentes) based on the amino acid sequences of venom proteins. Mol Phylogenet Evol 8, 349–362.

48 Yanoshita R, Ogawa Y, Murayama N, Omori-Satoh T, Saguchi K, Higuchi S, Khow O, Chanhome L,

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Samej-ima Y & Sitprija V (2006) Molecular cloning of the major lethal toxins from two kraits (Bungarus flaviceps and Bungarus candidus). Toxicon 47, 416–424.

49 Singh G, Gourinath S, Sarvanan K, Sharma S, Bha-numathi S, Betzel Ch, Yadav S, Srinivasan A & Singh TP (2005) Crystal structure of a carbohydrate induced homodimer of phospholipase A2 from Bungarus caeruleusat 2.1A˚ resolution. J Struct Biol 149, 264– 272.

50 Coborn J (1991) Atlas of Snakes of the World, p. 430, TFH Publication Co., Neptune City, CA.

51 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein util-izing the principle of protein-dye binding. Anal Biochem 72, 248–254.

52 Mullis KB & Faloona FA (1987) Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol 155, 335–350.

53 Maniatis T, Fritsch EF & Sambrook J (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

54 Litchfield JT & Wilcoxon FJ (1949) A simplified method of evaluating does-effect experiments. J Phar-macol Exp Therap 96, 99–113.

55 Thompson JD, Higgins DG & Gibson TJ (1994) improving the sensitivity of progressive multiple sequence alignment through sequence weighting, posi-tion-specific gap penalties and weight matrix choice. Nucleicl Acids Res 22, 4673–4680.

56 Felsenstein J (1992) Phylip: The Phylogeny Interfence Package, Version 3.573. University of Washington. Department of Genetics, Seattle, WA.

57 Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791. 58 Renetseder R, Brunie S, Dijkstra BW, Drenth J & Sigler

PB (1985) A comparison of the crystal structures of phospholipase A2from bovine pancreas and Crotalus

atroxvenom. J Biol Chem 260, 11627–11636. 59 Liu CS, Chen JP, Chang CM, Chen SW & Lo TB

(1991) Amino acid sequence of a fasciatoxin-homologue, fasciatoxin-II in the venom of Bungarus fasciatus (banded karit) J Chinese Biochem Soc 20, 33–39.

數據

Fig. 1. Gel filtration of crude venoms of two KBf (samples 1 and 2). Venom powder (15–20 mg) of KBf was dissolved in 200 lL of deionized water and loaded onto a Superdex G75 (HR10 ⁄ 30) column
Fig. 2. Purification of venom proteins by RP-HPLC. Protein fractions from gel filtration were re-solubilized separately and injected into a Vydac RP-C18 column
Table 1. Inventory of PLAs and 3FTx purified from KBf venom. Masses were determined by ESI-MS spectrometry
Table 3. Enzymatic activities of purified venom PLAs toward zwit- zwit-terionic micellar substrates
+5

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