Phospholipases A2 from Callosellasma rhodostoma venom gland cloning and sequencing of 10 of the cDNAs, three-dimensional modelling and chemical modification of the major isozyme.

Callosellasma rhodostoma (Malayan pitviper) is a monotypic Asian pitviper of medical importance. Three acidic phospholipases A2 (PLA2s) and one basic PLA2-homolog were purified from its venom while 10 cDNAs encoding distinct PLA2s were cloned from venom glands of a Thailand specimen of this species. Complete amino-acid sequences of the purified PLA2s were successfully deduced from their cDNA sequences. Among the six un-translated PLA2 cDNAs, two apparently result from recombination of its Lys49-PLA2 gene with its Asp49-PLA2 genes. The acidic PLA2s inhibit platelet-aggregation, while the noncatalytic PLA2-homolog induces local edema. This basic PLA2-homolog contains both Asp49 and other, unusual substitutions unique for the venom Lys49-PLA2 subtype (e.g. Leu5, Trp6, Asn28 and Arg34). Three-dimensional modelling of the basic protein revealed a heparin-binding region, and an abnormal calcium-binding pocket, which may explain its low catalytic activity. Oxidation of up to six of its Met residues or coinjection with heparin reduced its edema-inducing activity but methylation of its active site His48 did not. The distinct Arg/Lys-rich and Met-rich region at positions 10-36 of the PLA2 homolog presumably are involved in its heparin-binding and the cell membrane-interference leading to edema and myotoxicity.     

Unusual venom phospholipases A2 of two primitive tree vipers Trimeresurus puniceus and Trimeresurus borneensis

To explore the venom diversity of Asian pit vipers, we investigated the structure and function of venom phospholipase A2 (PLA2) derived from two primitive tree vipers Trimeresurus puniceus and Trimeresurus borneensis. We purified six novel PLA2s from T. puniceus venom and another three from T. borneensis venom. All cDNAs encoding these PLA2s except one were cloned, and the molecular masses and N-terminal sequences of the purified enzymes closely matched those predicted from the cDNA. Three contain K49 and lack a disulfide bond at C61-C91, in contrast with the D49-containing PLA2s in both venom species. They are less thermally stable than other K49-PLA2s which contain seven disulfide bonds, as indicated by a decrease of 8.8 degrees C in the melting temperature measured by CD spectroscopy. The M110D mutation in one of the K49-PLA2s apparently reduced its edematous potency. A phylogenetic tree based on the amino-acid sequences of 17 K49-PLA2s from Asian pit viper venoms illustrates close relationships among the Trimeresurus species and intergeneric segregations. Basic D49-PLA2s with a unique Gly6 substitution were also purified from both venoms. They showed edema-inducing and anticoagulating activities. It is notable that acidic PLA2s from both venoms inhibited blood coagulation rather than platelet aggregation, and this inhibition was only partially dependent on enzyme activity. These results contribute to our understanding of the evolution of Trimeresurus pit vipers and the structure-function relationships between various subtypes of crotalid venom PLA2.     

莽山烙铁头蛇毒液蛋白质组学研究(英文)

目的收集一条中国特有品种莽山烙铁头蛇的新鲜毒液,对其蛇毒蛋白作生化分析。方法先以电泳胶片法分析比较莽山烙铁头蛇毒与中国大陆及台湾、日本琉球各地其它品种的烙铁头蛇和各地其他品种的烙铁头蛇的粗蛇毒,在凝胶电泳中展现之异同。然后利用高效液相色谱法(HPLC)将莽山烙铁头蛇毒中的磷脂酶分离,再以质谱仪等鉴定,并与以往相同方法分析莽山烙铁头蛇所得到的磷脂酶分析结果比较。结果我们的研究证实了莽山烙铁头蛇毒含唯一主要的磷脂酶,其含量大约占蛇毒总重量的58%,与中国其他烙铁头蛇品种及日本琉球烙铁头蛇的蛇毒某些碱性磷脂酶之氨基酸序列及蛋白结构特别相似。此碱性磷脂酶主要毒性是水肿、局部炎症及肌肉坏死。结论莽山烙铁头蛇咬伤的临床表现为抗凝与出血,局部炎症坏死等特点,亦可由其蛇毒成分得到印证与解释。     

Snake venomics and antivenomics of Protobothrops mucrosquamatus and Viridovipera stejnegeri from Taiwan: Keys to understand the variable immune response in horses

The proteomes of the venoms of the snakes Viridovipera stejnegeri and Protobothrops mucrosquamatus from Taiwan were characterized by N-terminal sequencing, MALDI-TOF mass fingerprinting, and collision-induced dissociation tandem mass spectrometry of in-gel generated tryptic peptides. Proteins belonging to the following toxin classes were identified: metalloproteinase, phospholipase A(2) (PLA(2)), serine proteinase, C-type lectin-like, CRISP, l-amino acid oxidase, disintegrin, and peptides (vasoactive and inhibitors of SVMPs). Nine horses were immunized with a mixture of these venoms. All horses developed a satisfactory immune response against lethality of the venom of V. stejnegeri, whereas only three horses reached the accepted neutralizing potency against the venom of P. mucrosquamatus. Antivenoms were prepared from pools of 'good responder' (GR) and 'poor responder' (PR) horses and compared by antivenomics and neutralization tests. A similar neutralizing response was observed between the GR and PR antivenoms against the venom of V. stejnegeri, whereas antivenom from PR had a lower neutralizing activity against effects of P. mucrosquamatus venom than antivenom from GR. The low potency of the plasma of some horses against this venom is a consequence of the low immunogenicity of the neurotoxic PLA(2) trimucrotoxin. Our results provide clues for innovating the immunization scheme to generate improved antivenoms.     

Geographic variations,cloning, and functional analyses of the venom acidic phospholipases A2 of Crotalus viridis viridis

Geographic venom samples of Crotalus viridis viridis were obtained from South Dakota, Wyoming, Colorado, Oklahoma, Texas, New Mexico, and Arizona. From these samples, the phospholipases A(2) (PLA(2)s) were purified and their N-terminal sequences, precise masses, and in vitro enzymatic activities were determined. We purified two to four distinct acidic PLA(2)s from each sample; some of them displayed different inhibition specificities toward mammalian platelets. One of the acidic PLA(2)s induced edema, but had no anti-platelet activity. There was also a common basic PLA(2) myotoxin in all the samples. We have cloned five acidic PLA(2)s and several hybrid-like nonexpressing PLA(2)s. Molecular masses and N-terminal sequences of the purified PLA(2)s were matched with those deduced from the cDNA sequences, and the complete amino acid sequences of five novel acidic PLA(2)s were thus solved. They share 78% or greater sequence identity, and a cladogram based on the sequences of many venom acidic PLA(2)s of New World pit vipers revealed at least two subtypes. The results contribute to a better understanding of the ecogenetic adaptation of rattlesnakes and the structure-activity relationships and evolution of the acidic PLA(2)s in pit viper venom.     

Cloning and Identification of a Complete cDNA Coding for a Bactericidal and Antitumoral Acidic Phospholipase A2 from Bothrops jararacussu Venom

In order to better understand the function of acidic phospholipases A2 (PLA2s) from snake venoms, expressed sequence tags (ESTs) that code for acidic PLA2s were isolated from a cDNA library prepared from the poly(A)+ RNA of venomous glands of Bothrops jararacussu. The complete nucleotide sequence (366 bp), named BOJU-III, encodes the BthA-I-PLA2 precursor, which includes a signal peptide and the mature protein with 16 and 122 amino acid residues, respectively. Multiple comparison of both the nucleotide and respective deduced amino acid sequence with EST and protein sequences from databases revealed that the full-length cDNA identified (BOJU III--AY145836) is related to an acidic PLA2 sharing similarity, within the range 55-81%, with acidic phospholipases from snake venoms. Moreover, phylogenetic analysis of amino acid sequences of acidic PLA2s from several pit viper genera showed close evolutionary relationships among acidic PLA2s from Bothrops, Crotalus, and Trimeresurus. The molecular modeling showed structural similarity with other dimeric class II PLA2s from snake venoms. The native protein BthA-I-PLA2, a nontoxic acidic PLA2 directly isolated from Bothrops jararacussu snake venom, was purified and submitted to various bioassays. BthA-I-PLA2 displayed high catalytic activity and induced Ca2+-dependent liposome disruption. Edema induced by this PLA2 was inhibited by indomethacin and dexamethasone, thus suggesting involvement of the cyclo-oxygenase pathway. BthA-I-PLA2 showed anticoagulant activity upon human plasma and inhibited phospholipid-dependent platelet aggregation induced by collagen or ADP. In addition, it displayed bactericidal activity against Escherichia coli and Staphylococcus aureus and antitumoral effect upon breast adrenocarcinoma as well as upon human leukemia T and Erlich ascitic tumor. Following chemical modification with p-bromophenacyl bromide, total loss of the enzymatic and pharmacological activities were observed. This is the first report on the isolation and identification of a cDNA encoding a complete acidic PLA2 from Bothrops venom, exhibiting bactericidal and antitumoral effects.     

Purification, characterization and cytokine release function of a novel Arg-49 phospholipase A(2) from the venom of Protobothrops mucrosquamatus

Group IIA phospholipase A(2) (PLA(2)) are major components in Viperidae/Crotalidae venom. In the present study, a novel PLA(2) named promutoxin with Arg at the site 49 has been purified from the venom of Protobothrops mucrosquamatus by chromatography. It consists of 122 amino acid residues with a molecular mass of 13,656 Da assessed by MALDI-TOF. It has the structural features of snake venom group IIA PLA(2)s, but has no PLA(2) enzymatic activity. Promutoxin shows higher amino acid sequence identity to the K49 PLA(2)s (72-95%) than to D49 PLA(2)s (52-58%). Promutoxin exhibits potent myotoxicity in the animal model with as little as 1 microg of promutoxin causing myonecrosis and myoedema in the gastrocnemius muscle of mice. Promutoxin is also able to stimulate the release of IL-12, TNFalpha, IL-6 and IL-1beta from human monocytes, and induce IL-2, TNFalpha and IL-6 release from T cells, indicating that this snake venom group IIA PLA(2) is actively involved in the inflammatory process in man caused by snake venom poisoning.     

Effect of crotapotin on the biological activity of Asp49 and Lys49 phospholipases A(2) from Bothrops snake venoms

Myonecrosis, in addition to edema and other biological manifestations, are conspicuous effects of Bothrops snake venoms, some of them caused by phospholipases A(2) (PLA(2)s). Asp49-PLA(2)s are catalytically active, whereas Lys49-PLA(2)s, although highly toxic, have little or no enzymatic activity upon artificial substrates, due to a substitution of lysine for aspartic acid at position 49. Crotapotin (CA), the acidic counterpart of crotoxin PLA(2) (CB), is a PLA(2)-like protein from Crotalus durissus terrificus snake venom, and is considered a chaperone protein for CB, able to increase its lethality about ten fold, but to inhibit the formation of the rat paw edema induced by carrageenin and by snake venoms. In this study, we demonstrate that CA significantly inhibits the edema induced by BthTX-I (23% inhibition), BthTX-II (27%), PrTX-I (25%), PrTX-III (35%) and MjTX-II (10%) on the mouse paw. CK levels evoked by isolated Asp49 or Lys49-PLA(2)s were reduced by 40% to 54% in the presence of CA and, in all cases, the membrane damaging activity of the toxins was also reduced. Circular dichroism spectra of the PLA(2)s in the presence and absence of CA showed that there was not any detectable secondary structural modification due to association between CA and the myotoxins. However, Fourier Transformed Infrared (FT-IR) analysis indicated that ionic and hydrophobic contacts contributed to stabilize this interaction.     

Venomic analysis and evaluation of antivenom cross-reactivity of South American Micrurus species

Coral snakes from Micrurus genus are the main representatives of the Elapidae family in South America. However, biochemical and pharmacological features regarding their venom constituents remain poorly investigated. Here, venomic analyses were carried out aiming at a deeper understanding on the composition of M. frontalis, M. ibiboboca, and M. lemniscatus venoms. In the three venoms investigated, proteins ranging from 6 to 8 kDa (3FTx) and 12 to 14 kDa (PLA(2)) were found to be the most abundant. Also, the N-terminal sequences of four new proteins, purified from the M. lemniscatus venom, similar to 3FTx, PLA(2) and Kunitz-type protease inhibitor from other Micrurus and elapid venoms are reported. Cross-reactivity among different Micrurus venoms and homologous or heterologous antivenoms was carried out by means of 2D-electrophoresis and immunoblotting. As, expected, the heterologous anti-Elapid venom displayed the highest degree of cross-reactivity. Conversely, anti-M. corallinus reacted weakly against the tested venoms. In gel digestions, followed by mass spectrometry sequencing and similarity searching, revealed the most immunogenic protein families as similar to short and long neurotoxins, weak neurotoxins, PLA(2), β-bungarotoxin, venom protein E2, frontoxin III, LAO and C-type lectin. The implications of our results for the production of Micrurus antivenoms are discussed.     

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

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 identical to that of Bf Vb-2, and the other five were 94-98% identical 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 k(cat) values 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.     

Snake venomics of the Brazilian pitvipers Bothrops cotiara and Bothrops fonsecai. Identification of taxonomy markers

We report the proteomic characterization of venom of the pitvipers Bothrops cotiara and Bothrops fonsecai. Crude venoms were fractionated by reverse-phase HPLC, followed by SDS-PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and CID-MS/MS. Each venom contained around 30 proteins in the range of 7-110 kDa belonging to only 8 (B. cotiara) and 9 (B. fonsecai) families which may target the hemostatic system, albeit distinctly distributed among the two species. B. cotiara and B. fonsecai share medium-sized disintegrins, disintegrin-like/cysteine-rich (DC) fragments, snake venom vascular endothelial growth factor, cysteine-rich secretory proteins, serine proteinases, C-type lectins, l-amino acid oxidase, and Zn(2+)-dependent metalloproteinases. In addition, B. fonsecai expresses a high abundance PLA(2) molecule (13,890 Da), whereas PLA(2) molecules were not detected in B. cotiara's venom. This striking finding is in line with previous biochemical analyses showing the absence of phospholipasic activity in the venom of B. cotiara. The potential adaptive significance of the lack of PLA(2) molecules is enigmatic, and alternative explanations are discussed. B. fonsecai is morphologically extremely similar to B. cotiara. Our comparative proteomic analysis shows that compositional differences between their venoms can be employed as a taxonomy signature for unambiguous species identification independently of geographic origin and morphological characteristics.     

Snake venomics and antivenomics of the arboreal neotropical pitvipers Bothriechis lateralis and Bothriechis schlegelii

We report the comparative proteomic characterization of the venoms of two related neotropical arboreal pitvipers from Costa Rica of the genus Bothriechis, B. lateralis (side-striped palm pit viper) and B. schlegelii (eyelash pit viper). The crude venoms were fractionated by reverse-phase HPLC, followed by analysis of each chromatographic fraction by SDS-PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and collision-induced dissociation tandem mass spectrometry of tryptic peptides. The venom proteomes of B. lateralis and B. schlegelii comprise similar number of distinct proteins belonging, respectively, to 8 and 7 protein families. The two Bothriechis venoms contain bradykinin-potentiating peptides (BPPs), and proteins from the phospholipase A 2 (PLA 2), serine proteinase, l-amino acid oxidase (LAO), cysteine-rich secretory protein (CRISP), and Zn (2+)-dependent metalloproteinase (SVMP) families, albeit each species exhibit different relative abundances. Each venom also contains unique components, for example, snake venom vascular endothelial growth factor (svVEGF) and C-type lectin-like molecules in B. lateralis, and Kazal-type serine proteinase inhibitor-like proteins in B. schlegelii. Using a similarity coefficient, we estimate that the similarity of the venom proteins between the two Bothriechis taxa may be <10%, indicating a high divergence in their venom compositions, in spite of the fact that both species have evolved to adapt to arboreal habits. The major toxin families of B. lateralis and B. schlegelii are SVMP (55% of the total venom proteins) and PLA 2 (44%), respectively. Their different venom toxin compositions provide clues for rationalizing the distinct signs of envenomation caused by B. schlegelii and B. lateralis. An antivenomic study of the immunoreactivity of the Instituto Clodomiro Picado (ICP) polyvalent antivenom toward Bothriechis venoms revealed that l-amino acid oxidase and SVMPs represent the major antigenic protein species in both venoms. Our results provide a ground for rationalizing the reported protection of the ICP polyvalent antivenom against the hemorrhagic, coagulant, defibrinating, caseinolytic and fibrin(ogen)olytic activities of Bothriechis ( schlegelii, lateralis) venoms. However, these analyses also evidenced the limited recognition capability of the polyvalent antivenom toward a number of Bothriechis venom components, predominantly BPPs, svVEGF, Kazal-type inhibitors, some PLA 2 proteins, some serine proteinases, and CRISP molecules.     

Inhibition of enzymatic and pharmacological activities of some snake venoms and toxins by Mandevilla velutina (Apocynaceae) aqueous extract

Phospholipases A(2) (PLA(2)) are multifunctional proteins which exhibit varied biological activities correlated to the structural diversities of the sub-classes. The crude aqueous extract from subterranean system of Mandevilla velutina, a plant found in Brazilian savanna, was assayed for its ability to inhibit biological activities of several snake venoms and isolated PLA(2)s. The extract induced total inhibition of the phospholipase activity of Crotalus durissus terrificus venom and only partial inhibition of Bothrops venoms. When assayed against purified toxins, the highest efficacy was detected against CB and crotoxin, while almost ineffective against PLA(2)s from the genus Bothrops. Although M. velutina crude extract significantly inhibited the myotoxic activity of C. d. terrificus venom and CB, it produced only partial inhibition of either Bothrops jararacussu venom or its main myotoxins BthTX-I (basic Lys49), BthTX-II (basic Asp49) and BthA-I-PLA(2) (acidic Asp49). The extract exhibited also full inhibition of hemorrhage caused by Bothrops alternatus, Bothrops moojeni and Bothrops pirajai snake venoms, but partial inhibition (90%) of that induced by B. jararacussu venom. The extract was ineffective to inhibit the fibrinogenolytic activity of B. moojeni, B. alternatus and B. pirajai crude venoms, while their caseinolytic activity was only partially inhibited. No inhibition of the anticoagulant activity, although partial reduction of the edema-inducing activity of C. d. terrificus and B. alternatus crude venoms, CB, PrTX-I, BthTX-I and crotoxin was observed. Besides extending survival of mice injected with lethal doses of C. d. terrificus and B. jararacussu venoms, M. velutina extract decreased to 50% the lethality of mice. Extracts of 18 month old micropropagated plants were able to partially neutralize the effect of the crude venoms and toxins.     

Glycan structures and intrageneric variations of venom acidic phospholipases A(2) from Tropidolaemus pitvipers

Most of the phospholipases A(2) (PLA(2) ; EC3.1.1.4) variants isolated so far from snake venoms are nonglycosylated enzymes. In the present study, we purified an active glycosylated PLA(2) and an inactive nonglycosylated Lys49-like PLA(2) from two geographical venom samples of Tropidolaemus. The PLA(2) variants from the two samples have rather different N-terminal sequences, implying that the samples were probably derived from two species (Tropidolaemus?subannulatus and Tropidolaemus?wagleri). The active PLA(2) s from Sulawesi and Sumatra venoms were designated as Tsu-E6 and Twa-E6, respectively, as a result of the presence of their conserved Glu6 residue. Tsu-E6 inhibited ADP-induced aggregation of mouse and human platelets. Twa-E6 stimulated the aggregation of mouse platelets but inhibited the aggregation of human platelets. Both PLA(2) s were found to be glycosylated at Asn14. Using MALDI-TOF analysis, the released glycans were shown to comprise complex type oligosaccharides without sialylation. This is the first glycan structure of the snake venom PLA(2) to be solved. Furthermore, the enzymatic removal of glycans from both PLA(2) s did not significantly alter their effects on lipid hydrolysis and platelet aggregation. The thermostability of glycosylated Twa-E6 was also found to be as good as that of other homologous PLA(2) s. The presence of these oligosaccharides in PLA(2) s warrants further analyses, which may provide useful insights into the functional regulation of these biomolecules.     

Biochemical and pharmacological characterization of PhTX-I a new myotoxic phospholipase A2 isolated from Porthidium hyoprora snake venom

This paper reports the biochemical and pharmacological characterization of a new myotoxic PLA(2) (EC 3.1.1.4) called PhTX-I, purified from Porthidium hyoprora venom by one step analytical chromatography reverse phase HPLC. The homogeneity of the PhTX-I fraction and its molecular mass were initially evaluated by SDS-PAGE and confirmed by MALDI-TOF spectrometry, indicating a molecular mass of 14.249Da and constituted of a single polipeptidic chain. Amino acid sequence was determined by "de novo sequencing," in tandem mass spectrometry, belonging to D49-PLA(2) enzyme class and exhibiting high identity (44-90%) with other myotoxics PLA(2) from snake venoms. The enzymatic investigation showed maximal activity at pH 8 and 35-45°C. This activity was dependent on Ca(2+), other cations (Mg(2+), Mn(2+), Cd(2+) and Zn(2+)) reduced notably the enzymatic activity, suggesting that the arrangement of the catalytic site presents an exclusive structure for Ca(2+). Ex vivo, whole venom and PhTX-I PLA(2) caused blockade of the neuromuscular transmission in young chick biventer cervicis preparations similar to other isolated snake venom toxins from the Bothrops genus. In vivo, both induced local myotoxicity and systemic interleukin-6 response upon intramuscular injection, additionally, induced moderate footpad edema. In vitro, both induced low cytotoxicity in skeletal muscle myoblasts, however PhTX-I PLA(2) was able to lyse myotubes.     

N49 phospholipase A2, a unique subgroup of snake venom group II phospholipase A2

A novel phospholipase A2 (PLA2) with Asn at its site 49 was purified from the snake venom of Protobothrops mucrosquamatus by using SP-Sephadex C25, Superdex 75, Heparin-Sepharose (FF) and HPLC reverse-phage C18 chromatography and designated as TM-N49. It showed a molecular mass of 13.875 kDa on MALDI-TOF. TM-N49 does not possess enzymatic, hemolytic and hemorrhagic activities. It fails to induce platelet aggregation by itself, and does not inhibit the platelet aggregation induced by ADP. However, it exhibits potent myotoxic activity causing inflammatory cell infiltration, severe myoedema, myonecrosis and myolysis in the gastrocnemius muscles of BALB/c mice. Phylogenetic analysis found that that TM-N49 combined with two phospholipase A2s from Trimeresurus stejnegeri, TsR6 and CTs-R6 cluster into one group. Structural and functional analysis indicated that these phospholipase A2s are distinct from the other subgroups (D49 PLA2, S49 PLA2 and K49 PLA2) and represent a unique subgroup of snake venom group II PLA2, named N49 PLA2 subgroup.     

一种新的烙铁头蛇毒肌肉毒素

用分子筛和快速蛋白质液相色谱从烙铁头（ＴＲｒｉｍｅｒｅｓｕｒｕｓ ｍｕｃｒｏｓｑｕａｍａｔｕｓ）蛇毒中分离了一个新的碱性肌肉毒素,命名为ＴＭＰＢ。它的分子量为１６０００,等电点为９．２．用蛋白质序列仪测定了其Ｎ端２４个氨基酸残基,ＴＭＰＢ与其他两个从同种蛇毒中分离到的碱性磷酯酶Ａ２的同源性分别为４１．７％和５４．２％《