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Mini-Reviews in Organic Chemistry, 2014, 11, 15-27 15 Purification and Characterization of Venom Components as Source for Antibiotics

Leidy Johana Vargas Muñoza* and Sebastián Estrada-Gómeza,b aPrograma de Ofidismo/Escorpionismo, Facultad de Química Farmacéutica, Universidad de Antioquia UdeA, Medellín, Colombia., Calle 70 # 52-2, A.A. 1226, Medellín, Colombia; bFacultad de Química Farmacéutica, Universidad de Antioquia UdeA, Medellín, Colombia

Abstract: The extensive use of antibiotics in medicine, food industry, and agriculture has led to a frequent emergence of multidrug-resistant bacteria, which creates an urgent need for new antibiotics. It is now widely recognized that venom proteins could play a promising role against multidrug-resistant bacteria. Different proteins with antibacterial activity have been characterized from the venoms of , spiders and scorpions in the last decade. This review summarizes the proteins and peptides that have been purified and characterized from different venoms with antibacterial activity.

Keywords: Antimicrobial activity, , spider, scorpion, venom, peptides.

1. INTRODUCTION During the last decade, diverse proteins with antibacterial activity have been characterized from different sources like Bacterial infections are among the 10 leading causes of plants, , mammals, microorganisms and venoms death worldwide according to the World Health Organization (snake, scorpion and spider venoms) being the last but the [1]. The presence and current emergence of multiple- main focus in this review. resistant strains make the risk of these infections more threatening since a treatment becomes unreachable. In fact, bacterial resistance has been the principal aspect responsible 2. SNAKE VENOMS for increasing morbidity, mortality and health care costs of Snake venoms are cocktails of enzymatic and non- bacterial infections [2]. Therefore, research for new antimi- enzymatic proteins used for both immobilization and pre- crobials or antibacterial prototypes is continuously necessary digestion of prey. The most common enzymes present in for drug design and development seeking the treatment of these venoms include acetylcholinesterases, L-amino acid infections involving multidrug-resistant microorganisms [1, oxidases, desintegrins, serine proteinases, metalloprotein- 3] It is also of considerable interest to explore and develop ases, lectins and phospholipases A2 [6]. These cocktails have antimicrobials with a new mechanism(s) of action which can been tested against different microorganism; from the Vi- potentially avoid the appearance of drug resistance. Nature is peridae, the experimental data revealed that the venoms from a wide source of antimicrobial peptides since they are ubiq- Agkistrodon rhodostoma, atrox and B. jararaca uitous as part of the innate immune system and host defense exhibited a promising antibacterial activity against some of mechanisms. They have been increasingly recognized as a the Gram-positive and Gram-negative bacteria like Entero- critical first line of defense against many pathogens isolated coccus faecalis, Staphylococcus epidermidis and Staphylo- from various sources. coccus aureus [7]; while venoms from Crotalus viridus Antimicrobial resistance is a natural phenomenon which helleri, C. atrox and C. horridus horridus inhibited the entails an inherent risk, associated to antimicrobial drugs growth only from aerobic like S. epidermidis, Pseu- use, comprising the intrinsic and acquired resistance. The domona aeruginosa, and Enterobacter cloacae [8]. Ciscotto intrinsic is referred to a non-genetic related resistance [4], et al. [9], tested B. jararacussu crude venom against differ- while the acquired resistance, is achieved by antibiotics use, ent bacteria. It was active against Gram-positive bacteria and occurs by mutations in the bacterial genome or by hori- such as Eubacterium lentum, Peptostreptococcus anaero- zontal transference of genetic information [4]. The most im- bius, Propionibacterium acnes, S. aureus and S. epidermidis, portant antibiotics resistance mechanisms are related with the and Gram-negative bacteria such as Porphyromonas gin- antibiotic drawing diminution, increase of antibiotic efflux, givalis, Prevotella intermedia, P. aeruginosa, and Salmo- inactivation or modification of antibiotic targets, hydrolysis nella typhimurium. Antimicrobial activity of this venom, was or any chemical modification of the antibiotic structure and associated with the presence of proteins like LAAO and/or any new resistance acquired by horizontal genetic transmis- phospholipase A2 due to variation in content and quality of sion [4, 5]. these proteins in snake venom. Moreover, the Australian elapid Pseudechis australis inhibited S. aureus and Es- cherichia coli [10]. *Address correspondence to this author at the Calle 70 # 52-2, Medellín Colombia, zip code: 050010. Medellin, Colombia; Tel: (0574) 219 6649; Several toxins from these venoms have been isolated and Fax: (0574) 2631914; E-mail: [email protected] characterized, especially L- amino acid oxidases (LAAOs)

1875-6298/14 $58.00+.00 © 2014 Bentham Science Publishers 16 Mini-Reviews in Organic Chemistry, 2014, Vol. 11, No. 1 Muñoz and Estrada-Gómez and phospholipase A2 (PLA2), enhancing antimicrobial activ- and physiochemical properties, inactivation by pH changes ity as seen below in this review. or freezing and others [12]. Now, LAAOs, have emerged as an interesting object of study for their enzymology, struc- 2.1. LAAOs tural biology, pharmacology and because of their relatively L-amino acid oxidases (LAAOs, E.C.1.4.3.2) are enanti- easy purification. Recently, more and more SV-LAAOs have been characterized with distinct molecular mass, substrate oselective flavoenzymes catalyzing the stereospecific oxida- preference, interactions with platelets [11c, 13], induction of tive deamination of a wide range of L-amino acids to form - hemorrhage [11d] and apoptosis [14], equally antileishma- keto acids, ammonia and hydrogen peroxide (H2O2). LAAOs have been reported in , Crotalidae and Elapidae nial [15] and antibacterial activities [10]. snakes, usually as FAD-(flavin adenine dinucleotide) or First reports about LAAO antimicrobial activity came in FMN- (flavin mononucleotide) homodimeric binding glyco- the early 70s when Skarnes et al. [16] reported the antimi- proteins. Flavins in LAAOs are responsible for the yellow crobial activities elicited by C. adamanteus LAAOs. However, color of snake venom and contribute to toxicity via the oxi- since then few LAAOs were isolated till 1990 and later in dative stress arising from the production of H2O2 [9, 11]. year 2000 when the interest increased in these proteins lead- Before 1990s, the studies in the field of snake venom ing to a rise in their isolation and characterization. (Table 1) LAAOs (SV-LAAOs) used to be focused on their enzymatic summarizes SV-LAAOs reported with antimicrobial activity.

Table 1. LAAOs Isolated with Antibacterial Activity. Gram (G) Negative (-) and Positive (+)

LAAO Isolated from Snake Venom Antimicrobial Activity Properties References

Crotalus adamanteus Active against G() P. aeruginosa y E. coli Optimum pH 6.5-7.0 Skarnes [16]

More active against G(+) Staphylococcus mutans, than C. durissus cascavella 120 kDa, dimer, pI 5.4 Toyama et al. [22] G() Xanthomonas axonopodis pv passiflorae

Agkistrodon halys Active against G() E. coli and G(+) Bacillus subtilis 60.7 kDa, optimum pH: 8.8 Zhang et al. [53]

Bothrops alternatus Active against G() E. coli, and G(+) B. subtilis 123 kDa, homodimer, pI 5.37 Stabeli et al. [11d]

Active against G(+) S. aureus, G() P. aeruginosa and B. marajoensis 67 kDa, monomer, acidic Torres et al. [54] Candida albicans.

Active against G() E. coli, Salmonella typhimurium, P. B. moojeni 140 kDa, homodimer; pI 4.8 Stabeli et al. [21] aeruginosa and G(+) S. aureus

B. pauloensis More active against G() E. coli than G(+) S. aureus. 65 kDa, homodimeric, pI 6.3 Rodrigues et al. [55]

B. pirajai Active against G() E. coli and P. aeruginosa 130 kDa, homodimer; pI 4.9 Izidoro et al. [56]

Active against K. pneumoniae, P. aeruginosa and P. B. mattogrosensis 60 kDa, homodimer. Okubo et al., [25] mirabilis,

Protobothrops Active against G(+) B. megaterilum and S. aureus, G() 110 kDa, homodimer Lu et al. [11c] (Trimeresurus) jerdonii E. coli and P. aeruginosa

More active against G(+) S. aureus than G() E. coli Trimeresurus mucrosquamatus 110 kDa, homodimer Wei et al. [11e] and B. dysenteriae.

Vipera lebetina More active against G() E. coli than G(+) B. subtilis. 140 kDa, homodimer, pI 5.3 Tonismagi et al. [23]

Daboia russellii Active against G(+) S. aureus; G() P. aeruginosa and 100 kDa, homodimer; pI 8.8 Zhong et al. [24] siamensis E. coli.

Naja naja oxiana More active against G(+) B. subtilis than G() E. coli 110 kDa, homodimer Zhong et al. [24] (Naja oxiana)

Active against Aeromonas sp and G(+) B. subtilis and S. Pseudechis australis 142 kDa, dimer, 56 kDa Stiles et al. [10] aureus. A. hydrophila

Active against gram-positive and gram-negative three isoforms: 80, 60.8 and Bothrops jararaca Ciscotto et al. [9] bacteria. 48.1 kDa, monomer

124.4 kDa, homodimer, pI Agkistrodon blomhoffii ussurensis Active against S. aureus. Sun et al. [57] 4.7

Active against P. aeruginosa, K. pneumoniae and Ophiophagus hannah 135 kDa, homodimer, pI 4.5 Lee et al. [58] E. coli.

Agkistrodon halys pallas Active against E. coli. 60.7 kDa, pI 8.8 Liu et al., [59] Purification and Characterization of Venom Components Mini-Reviews in Organic Chemistry, 2014, Vol. 11, No. 1 17

The antimicrobial mechanism is not fully well under- different physicochemical properties could also be attractive stood, but some researches indicate that the production of for interaction with bacterial cell membranes. In silico struc- H2O2 may be involved in this process since the effects of tural analyses produced the first report of LAAO fragments SV-LAAOs could be suppressed or abolished in presence of with antimicrobial activity. catalase enzymes. The production of limited but adequate amount of H2O2 selectively attacks microorganism since 2.2. PLA2 human host cells, tissues or normal flora are less sensitive to this amount of reactive oxygen species (ROS) [17]. Al- Phospholipase A2 (PLA2) enzymes hydrolyze the sn-2 bond of phospholipids resulting in the release of a fatty acid though the amount of H2O2 produced is insufficient to gener- ate such effect, SV-LAAOs bind to the bacterial cell surface and lysophospholipid [26]. In nature five types of PLA2 have been described: secretory (sPLA2), cytosolic (cPLA2), resulting in relatively high local concentrations of H2O2, so 2+ that tiny amounts of SV-LAAOs could inhibit bacterial Ca independients (iPLA2), platelet active acetylhidrolase growth significantly [9]. On the other hand, the glycosylation (PAF-AH) and lisosomal PLA2. sPLA2 enzymes of II group of SV-LAAOs might also play a functional role in antimi- are principal constituents of Viperidae snake venom [27], are crobial activity, since deglycosylation affects the activity of small molecular size proteins (14 kDa) expressed in a num- SV-LAAOs, indicating that carbohydrates may play a role ber of cell types and present in various body fluids [28]. mediating the enzymatic and biological activities of LAAOs First report about PLAs with antibacterial activity was [18]. given by Peter Elsbach, et al. [29]. They reported a PLA2 The antimicrobial activities of SV-LAAOs showed a with bactericidal activity present in permeability-increasing non-selective specificity (between Gram-positive and Gram- protein, against Gram-negative bacteria such as Escherichia negative) due to their different binding affinities toward di- coli and Salmonella typhimurium. Subsequently a lot of verse bacteria, which means that SV-LAAOs can be used works have been done, (Table 2) summarize snake venom as new pharmacological agents against specific microbial PLA2 reported with antimicrobial activity. infections. The bactericidal mechanism of action of sPLA2 enzymes, Regarding purification, techniques are relatively easy to depends on whether the bacteria are Gram-positive or Gram- obtain homogenous LAAO from snake venom. Crude venom negative. As a rule, hydrolysis of the phospholipid compo- has been fractionated using Sephadex G-200 gel filtration nent of the bacterial cell membrane, by sPLA2s, is involved chromatography and Mono-Q HR 5/5 high performance an- in the antibacterial activity of Gram-positive [30] and Gram- ion exchange chromatography to yield the purified enzyme negative [31]. Foreman-Wykert et al., [32] reported sPLA2s as well as size exclusion chromatography, DEAE [19] and able to penetrate the peptidoglycan envelope of Gram- CM-Sepharose ion exchange chromatography. For example, positive bacteria and to gain access to the cell membrane the LAAO from C. rhodostoma venom can be obtained using phospholipids. These bacteria are more vulnerable to sPLA2 a simple two-steps procedure: Sephadex gel filtration chro- during the logarithmic growth phase (when the bacterial cells matography followed by Mono-Q high-performance ion ex- are dividing) than at the stationary phase. The sites of the change chromatography [20]. BmooLAAO-I, from Bothrops bacterial envelope engaged in cell growth seem to be the moojeni snake venom, was purified using sequential CM preferential sites for the action of sPLA2. On the other hand, Sepharose, ion-exchange and phenyl-Sepharose chromatog- the activity against Gram-negative bacteria is different be- raphy allowing a high degree of purity [21] whereas, LAAO cause sPLA2s cannot penetrate the lipopolysaccharide enve- from Crotalus durissus cascavella venom was purified, to a lope and reach the phospholipid layer. Therefore, sPLA2s are high degree of molecular homogeneity, using a combination capable to hydrolyze the phospholipid of the bacterial cell of molecular exclusion and ion-exchange chromatography membrane, only after the lipopolysaccharide-rich layer is system [22] in the same way, the LAAO from Vipera lebet- disrupted as a result of the action of other host defense ina venom was purified to homogeneity using same chroma- agents such as the bactericidal/permeability-increasing pro- tography procedure used in the C. durissus cascavella tein or the membrane attack complex of complement [33]. venom but needed a third step of hydrophobic chromatogra- The most abundant cell membrane phospholipid of the phy [23]. Zhong et al., [24] reported the isolation of DRS- Gram-positive bacterium S. aureus is phosphatidylglycerol LAAO purified from Daboia russellii siamensis venom by [34] and that of the Gram-negative bacterium E. coli is ion-exchange, gel filtration and affinity chromatographies. phosphatidylethanolamine [35]. Although sPLA2s are not Techniques like gel filtration chromatography and reversed- substrate specific, they prefer phosphatidylethanolamine as phase HPLC chromatography were used to isolate the LAAO substrate over phosphatidylcholine, which is the dominant from B. mattogrosensis snake venom [25]. phospholipid component of mammalian cell membranes SV-LAAOs have been researched as a possible source of [36]. antimicrobial tools for biotechnological and pharmaceutical On the other hand, purification of PLA2 needs a combina- purposes. Complete sequences have been fragmented in or- tion of different steps, such as gel filtration followed by ion- der to reduce the size and maintain the antimicrobial activity. exchange and reverse-phase HPLC, because PLA2 shares the Okubo et al., [25] fragmented complete Bothrops mat- same molecular mass with its isoforms or other proteins togrosensis LAAO (BmLAO) according to exposed charges from snake venom like lectins [28]. For example, BnpTX-I and hydrophobic moment, since most antimicrobial peptides and II, were isolated from Bothrops neuwiedi pauloensis with activity against Gram-positive and Gram-negative bac- snake venom through three chromatographic steps: ion- teria are cationic and show at least one hydrophobic face. exchange chromatography on CM-Sepharose, gel filtration BmLAO fragments yielded were synthesized and were found on Sephadex G-50 and reverse phase HPLC on a C18 col- to be functional. Data demonstrate that other regions with umn [37]. Barbosa et al., [2b] purified, by ion-exchange 18 Mini-Reviews in Organic Chemistry, 2014, Vol. 11, No. 1 Muñoz and Estrada-Gómez

Table 2. Snake Venom PLA2 with Antibacterial Activity

PLA2 Isolated from Snake Venom Antibacterial Activity Characteristic References

Daboia russelli siamensis 15.0 kDa, monomeric Daboiatoxin

Crotalus durissus terrificus Actives against 23.5 kDa, basic Samy et al. [60] Crotoxin B Burkholderia pseudomallei.

Pseudechis australis 13.2 kDa Mulgatoxin

Bothrops asper Bactericidal against gram-positives and 30 kDa as dimers Páramo et al. [4] Miotoxina II gram-negatives

Bothrops neuwiedi pauloensis Active against Escherichia coli and S. 14 kDa, monomeric, 28 kDa as dimers, Rodrígues et al. [37] BnpTX-I y II aureus pI 7.8

Bungarus fasciatus Active against E. coli and S. aureus. 14 kDa, homodimeric, pI 7.54 Xu et al. [61] BFPA

Bothrops moojeni Active against E. coli and Candida Stábeli et al. [62] 14 kDa, basic MjTX-II, albicans. Lomonte et al. [63]

Bothrops jararacussu Xanthomonas axonopodis. pv. passiflorae 15 kDa monomeric Barbosa et al. [2b] BthTx-I y BthTx-II

Bothrops atrox Active against E. coli 15 kDa homodimer, pI 8.9 Nuñez et al. [64] Myotoxin I

Active against Xanthomonas axonopodis Crotalus durissus collilineatus 14 kDa, pI 8.3 Toyama et al. [65] passiflorae

Porthidium nasutum Active against S. aureus 15 kDa, acidic, pI 4.6 Vargas et al. [66] PnPLA2

Bothrops brazili 14 kDa monomer and 28 kDa dimer, pI Inhibiting E. coli and C. albicans growth. Costa et al. [67] MTX-I and II 8.0 and 8.2.

Bothrops neuwiedi pauloensis Bactericidal to a G(-) bacteria, E. coli. 14 kDa, pI 8.9 Soares et al. [68] BnSP-7 chromatography and reverse phase HPLC, myotoxin I candidate for further evaluation of its antimicrobial potential (BthTx-I; Lys 49) and II (BthTX-II; Asp 49) from Bothrops in vivo. jararacussu venom. Paramo et al. evidenced the bactericidal activity of the 2.3. Others synthetic peptide myotoxin II-(115-129) derived from the Samy et al., [39] reported a snake venom metalloprotein- myotoxin II sequence from Bothrops asper snake venom. ases (SVMPs) from zinc-dependent enzymes family, and The bactericidal effect was observed on a variety of Gram- isolated from Agkistrodon halys (pallas, Chinese viper) negative and Gram-positive organisms, and differences in snake venom as antibacterial. The toxin is a single chain individual susceptibilities were evidenced. The bactericidal polypeptide with a molecular weight of 23146.61 and N- potency of the peptide is, in general, comparable to those of terminal sequence (MIQVLLVTICLAVFPYQGSSIILES) other well characterized cationic peptides. Equally, San- which displays antibacterial effect against Proteus vulgaris, tamaría et al. [38] synthesized 10 peptide variants, based on Proteus mirabilis (Gram-negative bacteria), S. aureus the original C-terminal sequence 115–129 of myotoxin II (Gram-positive bacteria, which exhibited more antibacterial and its triple Tyr/Trp substituted peptide p115-W3. In vitro properties) and the multi-drug resistant Burkholderia pseu- assays for bactericidal activity of these peptides suggest a domallei with an MIC value ranging from 1.875–60 mM. general correlation between the number of tryptophan substi- Authors suggest that this metalloproteinase exerts its antimi- tutions introduced and microbicidal potency, both against crobial effect by altering membrane packing and inhibiting Gram-negative (Salmonella typhimurium) and Gram-positive mechanosensitive targets. (S. aureus) bacteria. The peptide variant pEM-2 (KKWRWWLKALAKK) showed a reduced toxicity to- On the other hand, from the venom of inland taipan wards muscle cells, while retaining high bactericidal po- Oxyuranus microlepidotus, an acidic protein with 50-amino- tency. The authors of this paper concluded that the bacteri- acid (omwaprin) was isolated and characterized. It showed a cidal property of pEM-2, combined with its relatively low concentration-dependent antibacterial activity against se- toxicity towards eukaryotic cells validates it as a promising lected Gram-positive strains such as Bacillus megaterium Purification and Characterization of Venom Components Mini-Reviews in Organic Chemistry, 2014, Vol. 11, No. 1 19 and Staphylococcus warneri. This selective antibacterial methods of these peptides, where the most commonly used is activity was attributed to several factors, including charge the reverse phase HPLC using a combination of different density and structure of lipopolysacharides in the case of columns (C4, C5, C8, C18) solvents and gradients. (Table 4) Gram-negative bacteria, or lipid composition of the cyto- summarizes some characterization parameters of different plasmic membrane and the electrostatic potential across this AMPs. Peptides identification and structural characterization membrane in Gram-positive bacteria [40]. are commonly carried out using matrix-assisted laser desorp- tion ionization time of flight spectrometry (MALDI-TOF Rádis-Baptista et al. [41] reported the purification and MS) and amino acid analysis is normally carried out using cloning of C-type lectin named crotacetin. It was active Edman degradation microsequencer. against the Gram-negative bacteria Xanthomonas axonopo- dis pv. Passiflorae which was able to induct membrane rup- ture and cytoplasmic vacuolation. 3.2. Cytolytic Peptides (CP) Cytolytic peptides are the main source of antimicrobial 3. ARACHNID VENOMS compounds from arachnid venoms. These peptides are linear (lacking disulphide bridges), short (<50 amino acids), cati- 3.1. Spider and Scorpions onic and amphiphilic molecules, establishing the biggest Spiders and scorpions venoms are a complex mixture of group of antimicrobial peptides (AMP). These peptides re- neurotoxins, enzymes, proteins, antimicrobial, neurotoxic quire to assume an amphipathic structure with a positive and cytolytic peptides, nucleotides, salts, amino acids and charge (-helix structure) which allows their interaction neurotransmitters which affects both, vertebrates and inver- (permeabilization/incorporation) with the anionic eukaryotic tebrates [42]. The production of this arsenal is “manufac- and prokaryotic (Gram negative and Gram positive) cells tured” on venom glands which are located, in spiders, in the membrane surface due to its hydrophobic potential causing cephalothorax (Araneomorphae) or in the chelicerae (Myga- membrane disruption and tissue necrosis. The activity does lomorphae) [42, 43], while in scorpions, the venom gland is not depend on a strict organization of the amino acid se- located in last tail segment (telson) where the sting is lo- quence [42e, 47], just the presence of polar charged residues cated. Spiders venom components can be divided in three is required. These kinds of peptides are quite abundant in groups: low (<1 kDa), medium (<10 kDa) and high (>10 nature (around 150 reported worldwide, 17 from arachnids, kDa) molecular weight compounds. The low molecular antimicrobial sequence database) as part of different innate weight compounds are commonly known as acylpolyamines, immune systems. which possess an aromatic moiety linked to a hydrophilic lateral chain affecting mainly glutamic receptors, although 3.3. Spider Cytolytic Peptides (CP) they also express some affinity to some sodium and calcium voltage-gated ionic channels [44]. Medium and high molecu- Spider cytolytic peptides are rather common in both, lar weight compounds correspond to peptides and proteins venom and hemolymph, although most isolations and char- expressing neurotoxic and enzymatic activities (catalytic) acterizations of these active peptides come from the hemo- like the ones expressed by different cationic antimicrobial lymph. Only few peptides had been characterized from the molecules. Molecular mass oscillates between 5 kDa and venom and were found mainly in the araneomorphae in- 120 kDa (only in latrodotoxins), and the main targets are fraorder in the spiders genus Cupiennius, Lachesana, Lycosa striated muscle cells (in the case of sphingomyelinases), and Oxyopes [46e, 48]. These peptides consist of short voltage-dependent sodium (Na+v), potassium (K2+v), and chains (bellow 37 amino acid) showing a hydrophobic N- calcium (Ca2+v) channels, as well as calcium and potassium terminal and a C-terminus, composed of polar charged resi- ligand-dependent channels (pre and post-synaptic) and cho- dues, and with activity against Gram-negative and Gram- linergic receptors [42, 45]. In a very similar way, scorpion positive bacteria. Kuhn-Nentwig et al. isolated and charac- venoms mixture can be divided into disulfide-rich peptides terized 4 AMPs’ from the spider Cupiennius salei (Ctenidae) and non disulfide-rich peptides. Disulfide-rich peptides are named cupiennin 1a, 1b, 1c and 1d, active against E. coli the major components in this venom containing around 30 or (ATCC 25922), P. aeruginosa (ATCC 27853), S. aureus 70 amino acids residues and three or four disulfide bridges (ATCC 29213) and E. fecalis (ATCC 29212). All were sepa- [46]. The main targets of these toxins are the ionic channels rated using a five step protocol including gel filtration (Su- like sodium (Na+v), potassium (K2+v), chlorine (Cl-) or cal- perdex 75 HR 10/30), cationic exchange chromatography cium (Ca2+v) located in the nervous system, blocking or gat- (Mono S HR 10/10 column) eluted with a salt gradient (0- ing them and exhibiting a neurotoxic activity. The non- 100% NaCl 2M pH 5.5, 55 minutes) and successive reverse disulfide rich compounds were discovered recently. The phase HPLC (RP-HPLC) steps using a C4 (nucleosil 300-5 main characteristic of these molecules is the lacking of disul- column), C18 (nucleosil 120-5 column) and a C8 (nucleosil fide bridges, the diverse sequence, hemolytic, immune 100-5 column). Different gradients were used in RP-HPLC modulating, antibacterial and insecticidal activities, and also (Table 3). Peptides molecular mass was examined by electro these molecules have relatively low molecular mass (1-4 spray ionization mass spectrometry on a single stage quad- kDa). Most of these peptides possess an amphipathic - rupole (ESI-MS). N-terminal sequence and amino acid com- helical structure like that reported for different cationic an- position were analyzed using Edman degradation technique timicrobial molecules [46]. in sequencer equipment (Procise cLC 492). Peptides charac- The presence of these short, linear peptides exhibiting an terization included molecular mass, number of amino acids, amphipathic -helical structure, lacking disulfide bridges in theoretical isoelectric point (Table 4) and structural features arachnid venoms, corresponds with the presence of diverse (not shown) [48d]. Kozlov et al. reported the isolation and cytolytic peptides. (Table 3) describes different isolation characterization of 7 short linear cystein free AMPs from the 20 Mini-Reviews in Organic Chemistry, 2014, Vol. 11, No. 1 Muñoz and Estrada-Gómez

Table 3. Different Gradients used to Isolation and Purification of AMP’s from Spider Venoms

AMP Method Gradient References

SPIDERS Flow Rate: 0.5 ml/min 0-15 min 100% Solvent A 10 min 1% Solvent B RP-HPLC C4 column 120 min 0.4% Solvent B Solvent A: Trifluoroacetic acid (TFA) 0.1% in H2O Solvent B: TFA 0.1% in acetonitrile (ACN) Flow Rate: 0.5 ml/min Cupiennin 1a-d [48d] 0-10 min 22% of Solvent B in A

RP-HPLC C18 column 115 min of 22%-40% Solvent B 10 min of 40%-100% Solvent B Same solvent as above Flow Rate: 0.5 ml/min

RP-HPLC C8 column Constant 36% Solvent B Same solvent as above Flow Rate: 0.3 ml/min 0-35 min linear gradient (LG) 0%-70% Solvent B RP-HPLC C5 column Solvent A: TFA 0.1% in H2O Solvent B: TFA 0.1% in ACN

Latarcins (Ltc) 1, 2a, 3a, Flow Rate: 0.3 ml/min [48b] 3b, 4a, 4b, 5 RP-HPLC Amide column 0-40 min 20%-60% Solvent B Same solvent as above Flow Rate: 50 l/min

RP-HPLC C18 column 0-40 min 20%-60% Solvent B Same solvent as above Flow Rate: 2.0 ml/min 0-60 min LG 0%-60% Solvent B RP-HPLC C18 column Solvent A: TFA 0.1% in H2O Solvent B: TFA 0.1% in ACN Flow Rate: 1.0 ml/min Oxyopinins Cation exchange HPLC 0-60 min LG 10mM-1M Buffer A [46e] Buffer A: Sodium Phosphate Buffer pH: 6.5, 30% ACN Flow Rate: 1.0 ml/min

RP-HPLC C4 column 0-60 min LG 0%-60% Solvent B (Purification) Solvent A: Hepta-fluorobutyric acid (HFBA) 0.1% in H2O Solvent B: HFBA 0.1% in ACN Flow Rate: 1.0 ml/min 0-60 min LG 0.5%/min Solvent B RP-HPLC C8 column Solvent A: TFA 0.1% in H2O Solvent B: ACN 99.9% in H2O Flow Rate: 1.0 ml/min 0-60 min LG 0.33%/min Solvent B Lycotoxins RP-HPLC C8 column [48e] Solvent A: TFA 0.5% in H2O Solvent B: n-propyl alcohol 99.5% in H2O Flow Rate: 1.0 ml/min

RP-HPLC C4 column 0-60 min LG 0.25%/min Solvent B (Purification) Solvent A: HFBA 0.1% in H2O Solvent B: ACN 99.9% in H2O Purification and Characterization of Venom Components Mini-Reviews in Organic Chemistry, 2014, Vol. 11, No. 1 21

Table 3. Contd…..

AMP Method Gradient References

SCORPIONS

Flow Rate: 1.0 ml/min 0-240 min LG 0%-60% Solvent B AamAP1 - 2 RP-HPLC C5 column [52a] Solvent A: TFA 0.1% in H2O Solvent B: ACN 99.9% in TFA

Flow Rate: 2.0 ml/min 0-60 min LG 5%-65% Solvent B RP-HPLC C4 column Solvent A: TFA 0.1% in H2O Solvent B: ACN 99.9% in TFA

Flow Rate: 0.8 ml/min 0-60 min LG 5%-35% Solvent B Im-1 RP-HPLC C18 column [46g] Solvent A: Formic acid (FA) 0.1% in H2O Solvent B: ACN 99.9% in FA

Flow Rate: 0.05 ml/min 0-80 min LG 10%-50% Solvent B RP-HPLC C18 column (Purification) Solvent A: HFBA 0.1% in H2O Solvent B: ACN 99.9% in HFBA

Flow Rate: 2.0 ml/min 0-60 min LG 0%-60% Solvent B RP-HPLC C18 column Solvent A: TFA 0.1% in H2O Solvent B: ACN 99.9% in TFA

Flow Rate: 1.0 ml/min 0-75 min LG 0-100% Buffer A Pandinins 1-2 Cation exchange HPLC [46d] Buffer A: 0.5 Acetic acid in 1M ammonium acetate pH: 5.9

Flow Rate: 2.0 ml/min 0-60 min LG 0%-60% Solvent B RP-HPLC C4 column (Purification) Solvent A: TFA 0.1% in H2O Solvent B: ACN 99.9% in TFA

Sephadex G-50 Not described

Flow Rate: 1.0 ml/min 0-45 min LG 0%-45% Solvent B RP-HPLC C18 column Solvent A: TFA 0.1% in H2O Bactridine 1-6 Solvent B: ACN 99.9% in TFA [46f]

Flow Rate: 1.0 ml/min 0-60 min LG 25%-35% Solvent B RP-HPLC C18 column Solvent A: TFA 0.1% in H2O Solvent B: ACN 99.9% in TFA 22 Mini-Reviews in Organic Chemistry, 2014, Vol. 11, No. 1 Muñoz and Estrada-Gómez

Table 4. Characterization of Spider Venom AMP’s

AMP Molecular Mass Amino Acids Charge pH=7 pI References

SPIDERS

ESI-MS: 3814.50 Da Cupiennin 1a 35 +8 11.30 Theory: 3814.59 Da

ESI-MS: 3800.25 Da Cupiennin 1b 35 +8 11.30 Theory: 3800.57 Da [48d] ESI-MS: 3769.75 Da Cupiennin 1c 35 +8 11.30 Theory: 3770.48 Da

ESI-MS: 3795.13 Da Cupiennin 1d 35 +8 11.30 Theory: 3795.55 Da

MALDI: 3071.5 Da Ltc 1 25 Between +5 and +10 10 Theory: 3071.8 Da

MALDI: 2901.0 Da Ltc 2a 26 Between +5 and +10 10 Theory: 2900.8 Da

MALDI: 2481.7 Da Ltc 3a 20 Between +5 and +10 10 Theory: 2481.4 Da

MALDI: 2424.6 Da Ltc 3b 20 +5 10 [48b] Theory: 2424.3 Da

MALDI: 2900.9 Da Ltc 4a 24 Between +5 and +10 10 Theory: 2900.6 Da

MALDI: 2882.3 Da Ltc 4b 24 Between +5 and +10 10 Theory: 2882.6 Da

MALDI: 3428.1 Da Ltc 5 28 +10 10 Theory: 3427.9 Da

MALDI: 5221.2 Da Oxyopinin 1 48 Not reported Not reported Theory: 5221.3 Da

MALDI: 4126.9 Da Oxyopinin 2a 37 Not reported Not reported Theory: 4127.1 Da

MALDI: 4146.9 Da Oxyopinin 2b 37 Not reported Not reported [46e] Theory: 4146.9 Da

MALDI: 4064.8 Da Oxyopinin 2c 37 Not reported Not reported Theory: 4064.7 Da

MALDI: 4156.9 Da Oxyopinin 2d 37 Not reported Not reported Theory: 4156.8 Da

MALDI: 2843.4 Da Lycotoxin I 25 Not reported Not reported Theory: 2843.5 Da [48e] MALDI: 3206.0 Da Lycotoxin II 27 Not reported Not reported Theory: 3205.8 Da Purification and Characterization of Venom Components Mini-Reviews in Organic Chemistry, 2014, Vol. 11, No. 1 23

Table 4. Contd…..

AMP Molecular Mass Amino Acids Charge pH=7 pI References

SCORPIONS

MALDI: 1931.2 Da AamAP1 18 Not reported Not reported Theory: 1932.3 Da [52a] MALDI: 1881.1 Da AamAP2 18 Not reported Not reported Theory: 1982.0 Da

Im-1 MALDI: 3000-4500 56 Not reported Not reported [46g]

MALDI: 4799.2 Da Pandinin 1 44 Not reported 10.28 Theory: 4799.5 Da [46d] MALDI: 2612.6 Da Pandinin 2 24 Not reported 10.52 Theory: 2612.1 Da

Bactridine 1 MALDI: 6921 Da 61 Not reported Not reported

Bactridine 2 MALDI: 7363 Da 64 Not reported Not reported

Bactridine 3 MALDI: 7226 Da 60 Not reported Not reported [46f] Bactridine 4 MALDI: 7011 Da 60 Not reported Not reported

Bactridine 5 MALDI: 7101 Da 60 Not reported Not reported

Bactridine 6 MALDI: 7173 Da 60 Not reported Not reported venom of the spider Lachesana tarabaevi showing an am- Crude venom was fractioned using RP-HPLC with a C8 col- phipathic  helix structure in membrane-mimicking envi- umn. Active fractions were purified using either a C8 column ronment active against some Gram-negative E. coli and P. or a C4 column. Different gradients were used in RP-HPLC aeruginosa, and some Gram-positive B. subtilis and A. globi- (Table 3). Peptides molecular mass and amino acid analysis formis. They fractionated the pure venom using RP-HPLC were examined using MALDI-TOF MS [48e]. In a similar on a C5 Jupiter column, collected fractions were separated by way as described above, Bneli and Yigit, reported the anti- RP-HPLC in an amide column and a final purification of the bacterial activity of the web spider venom Agelena labyrin- compounds was performed using a C18 column. Different thica against B. subtilis, Shigella sp., S. aureus, P. aerugi- gradients were used in RP-HPLC (Table 3). Peptides mo- nosa and two E. coli different strains, based on the presence lecular mass was examined using MALDI-TOF MS. As of AMP [50]. mentioned before, amino acids sequences analysis (N- Terminal) was carried out using Edman degradation [48b]. 3.4. Scorpion Cytolytic Peptides (CP) With the same spider venom, but most recently, Kuzmeknov Scorpion venom is the main source for different CP be- et al. isolated five new peptides featuring insect toxicity, longing to the AMP family. A wide collection of these pep- disulfide-rich N-terminal resembling ICK motif and a C- tides have been identified, in different scorpion species, terminal tail sharing structural and functional properties with showing a variable amino acid length between 13 and 47 AMP [49]. From the crude venom of Oxyopes kitabensis, residues [46g, 46h]. These peptides exhibit a wide range of Corzo et al. isolated five large, linear, cationic, amphipathic antibacterial and antifungal activities including Gram- AMP active against E. coli and S. aureus. These peptides are negative, Gram-positive, yeast and fungi, grouping them in the largest isolated and characterized AMP at present. Crude the AMP family. Some of these peptides have the ability to venom was fractioned in a RP-HPLC using a C18 column, interfere with cellular functions in the human innate immune fractions collected were further fractionated using cation system [51]. As AMPs are present in scorpion venoms, non- exchange HPLC with a sulfopropyl column and peptides antimicrobial peptides (NAMPs), without disulfide-bridges were finally purified by RP-HPLC using a C4 column. Dif- and a similar structure can be also be found also in these ferent gradients were used in RP-HPLC (Table 3). Peptides venoms, although the biological activities have not been de- molecular mass was examined using MALDI-TOF MS. In termined [51]. BmKbpp is an example of the AMPs´ with this case, amino acid sequence analyses were carried out antimicrobial, immune-regulatory, and bradykinin potentiat- using an automated gas-phase sequencer [46e]. From a dif- ing activities, isolated from Mesobutus martensii [51]. From ferent family, Yan and Adams identified two amphicathic - two different species of Androctonus (australis and helix AMPs from the venom of the wolf spider Lycosa caro- amoreuxi), two AMP were isolated and characterized [52]. linensis active against E. coli, B. thurigensis and C. albicans. From A. australis, Almaaytah et al. obtained the AMP using 24 Mini-Reviews in Organic Chemistry, 2014, Vol. 11, No. 1 Muñoz and Estrada-Gómez

RP-HPLC with a C5 column and a linear gradient of acetoni- ACKNOWLEDGEMENTS trile (Table 3), the identification and structural characteriza- tion were carried out using MALDI-TOF MS. Amino acid The authors are grateful with Sostenibilidad 2013-2014 analysis was performed using Edman degradation microse- Universidad de Antioquia. quencer (Procise). Both peptides were active against E. coli, S. aureus and C. albicans. From A. autralis, authors did not REFERENCES describe the isolation method, but they performed the AMP [1] WHO. Department of Essential Drugs and Medicines Policy. identification method using H-NMR spectroscopy, Double- http://www.who.int/en/ (Accessed October 10, 2012). quantum-filtered correlation spectroscopy (DQF-COSY), [2] (a) de Lima, D.C.; Alvarez Abreu, P.; de Freitas, C.C.; Santos, TOCSY and NOESY and reported the identification of a D.O.; Borges, R.O.; Dos Santos, T.C.; Mendes Cabral, L.; Rodrigues, C.R.; Castro, H.C. 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Received: March 01, 2013 Revised: April 18, 2013 Accepted: June 23, 2013