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700 Protein & Peptide Letters, 2012, 19, 700-707

Bioactive Peptides from Marine Organisms: A Short Overview

Fernando Lazcano-Prez, Sergio A. Romn-Gonzlez, Nuria Snchez-Puig* and Roberto Arregun-


Espinosa*

Instituto de Qumica, Universidad Nacional Autnoma de Mxico, Circuito Exterior, Ciudad Universitaria, Mxico
04510, D.F., Mxico

Abstract: Marine organisms are an immense source of new biologically active compounds. These compounds are unique
because the aqueous environment requires a high demand of specific and potent bioactive molecules. Diverse peptides
with a wide range of biological activities have been discovered, including antimicrobial, antitumoral, and antiviral activi-
ties and toxins amongst others. These proteins have been isolated from different phyla such as Porifera, Cnidaria, Nemer-
tina, Crustacea, Mollusca, Echinodermata and Craniata. Purification techniques used to isolate these peptides include clas-
sical chromatographic methods such as gel filtration, ionic exchange and reverse-phase HPLC. Multiple in vivo and in vi-
tro bioassays are coupled to the purification process to search for the biological activity of interest. The growing interest
to study marine natural products results from the discovery of novel pharmacological tools including potent anticancer
drugs now in clinical trials. This review presents examples of interesting peptides obtained from different marine organ-
isms that have medical relevance. It also presents some of the common methods used to isolate and characterize them.
Keywords: Marine toxins, reverse-phase HPLC, peptides, chromatography.

INTRODUCTION
Marine biodiversity is so vast that the majority of phyla
and more than 90% of all living classes of organisms are
found in the marine environment. In addition, the oceans
cover more than 70% of the Earths surface and thus consti-
tute an enormous wealth of natural products and new bio-
logically active compounds [1]. The chemical structure of
these compounds is very different from those obtained from
terrestrial and microbial systems. It is possible to find cyclic
and linear peptides and depsipetides (peptides in which one
or more of the amide bonds are replaced by ester bonds) con-
taining natural or non-natural amino acids (Figure 1). These
molecules can be synthesized by the organism itself or can
be obtained from marine microorganisms with whom they
have a symbiotic relationship [2]. Sessile marine inverte-
brates have evolved sophisticated chemical mechanisms that
they use for communication, defense, reproduction, and
regulation of calcium and sodium homeostasis or as antimi-
crobial agents. Their value as drugs is based on the fact that
they interact with proteins that have been conserved
throughout evolution and some of their human orthologues
may be involved in human disease processes, e.g., cell divi-
sion and apoptosis, or immune and inflammatory responses.
Several biological and pharmacological activities have been
reported for molecules isolated from marine species that Figure 1. Chemical structure of the Mirabamide-G depsipeptide
comprise antitumoral, antibacterial, antifungal, antiviral, from the marine sponge Stelletta clavosa [80]. Ester bond charac-
immunosuppressive, insecticidal, neurotoxic and cytotoxic teristic of a depsipeptide and other interesting chemical features
properties. The type of organisms from which these active such as the non-natural amino acids present in this compound are
compounds have been isolated includes marine invertebrates highlighted as follow: Cyan: 2,3-dihydroxy-2,6,8-trimethyldeca-
(4Z,6E)-dienoic acid (Dhtda) moiety, dark blue: 2,3 diamino buta-
noic acid (Dab), brown: 3-hydroxyleucine, pink: N-methylthreo-
*Address correspondence to this author at the Instituto de Qumica, Univer- nine, green: 
  
, yellow:   
 
sidad Nacional Autnoma de Mxico, Circuito Exterior, Ciudad Universi-
taria, Mxico 04510, D.F., Mxico; Tel: +52 55 56224468; Fax: +52 55 
        

  
56162217; E-mail: arrespin@unam.mx   

1875-5305/12 $58.00+.00 2012 Bentham Science Publishers


Bioactive Peptides from Marine Organisms Protein & Peptide Letters, 2012, Vol. 19, No. 7 701

such as sponges, mollusks, sea anemones, tunicates, poly- phymines, BE and A1E1 were isolated from the sponge
chaetes, echiuroid worms, crustaceans and to a lesser extent Homophymia sp. and elicited a potent cytotoxic activity with
from fish [3]. IC50 values in the nM range against a variety of human can-
cer cell lines [11].
According to Cannell [4], there are five main steps to
follow for the isolation and characterization of a bioactive Stylopeptide 2 is a proline-rich cyclodecapeptide that has
substance: (a) the source selection, (b) extraction and frac- been isolated from a cytotoxic extract of the Papua New
tionation, (c) separation and isolation of the molecules of Guinea marine sponge Stylotella sp. The structural assign-
interest and (d) the structural and functional characterization. ment was based on Collision Induced Dissociation (CID),
Peptides from animal tissues must be efficiently solubilized NMR spectroscopy and amino acid analysis. This peptide
in their biologically active form and recovered in high yields was initially purified from a dichloromethane-methanol ex-
avoiding a rapid degradation. The extraction of marine pep- tract by solvent partitioning. The dichloromethane-soluble
tides and proteins of interest from their natural source is usu- fraction that contained the stylopeptide 2 displayed activity
ally made in water or aqueous buffers in order to maintain against the P388 lymphocytic leukemia cells. Successive
their functionality. Fractionation is often done using a salt Sephadex LH-20 partition chromatography followed by vac-
(e.g. ammonium sulfate) or acetone. The purification steps uum-liquid chromatography, preparative thin-layer chroma-
usually include chromatographic methods like gel filtration, tography (TLC), and high-performance liquid chromatogra-
ion exchange and reverse-phase high performance liquid phy (HPLC) rendered the pure compound [12].
chromatography (RP-HPLC). This last being the method of
Three cyclic peptides, euryjanicins B, C and D, have
choice for the purification of peptides from their natural
been isolated from the Puerto Rican marine sponge Pro-
sources [5]. Finally, techniques like Edman degradation or
suberites laughlini. The structures were elucidated by
protease digestion followed by mass spectrometry are used
chemical degradation, ESI MS/MS and extensive 2D NMR
to identify the primary sequence of the peptide, while circu- methods [13]. To purify these peptides, the sample was ini-
lar dichroism and NMR provide information related to their
tially fractionated with ethyl acetate followed by 1H and 13C
secondary and tertiary structure respectively.
NMR to confirm the presence of peptides. The extract was
Before starting the isolation of novel bioactive molecules further subjected to normal-phase Silica gel column chroma-
it is very important to have a sensitive and reproducible se- tography using a mixture of chloroform and methanol previ-
lection system that allows the experimenter the identification ously saturated with NH3. Similar fractions were pooled to-
of the peptide of interest [6]. A good selection system is the gether based on their TLC, NMR, and biological activity
key to find new biologically active compounds that can be profiles. The first fraction contained a mixture of small pep-
used as pharmacological tools for elucidating physiological tides that were further fractionated using a silica gel column
mechanisms and new therapeutic agents. The biological as- chromatography with a mixture of hexane and ethyl acetate.
says used to follow the activity of the different fractions ob- Further purification of one the fractions by C-18 Silica gel
tained during the purification steps include a wide range of reverse-phase chromatography yielded pure euryjanicin A,
in vivo and in vitro experiments like hemolysis, citotoxicity, euryjanicin B, and the known cyclic octapeptide Dominicin.
antimicrobial, antitumoral and antiviral activities and elec- Pure euryjanicin C and D were obtained by subsequent re-
trophysiological experiments. verse-phase HPLC.
The native function of the different marine-derived com- Recently two cytotoxic peptides named Yakuamides A
pounds is not generally known, nor how the host organism and B were isolated from the marine sponge Ceratopsion sp.
produces them. This makes difficult finding an application They are linear peptides rich in dehydroamino acids and -
for them, but their evaluation in nontraditional disease tar- hydroxyamino acids with an inhibitory growth effect against
gets (i.e., other than cancer, inflammation, and infectious several human cancer cell lines [14].
diseases) could lead to the discovery of novel drugs.
New anabaenopeptin-like peptides, paltolides A-C, were
isolated from a deep-water marine sponge Theonella swin-
PEPTIDES FROM SEA SPONGES hoei. The methanol extract of the lyophilized sponge was
Sea sponges are a well-known source of interesting new partitioned between butanol and water and the organic layer
bioactive peptides. Structurally, they include linear peptides, was fractionated on Sephadex LH-20. Fractions containing
peptide lactones, cyclic peptides, and depsipeptides (a grow- peptides were purified by reverse-phase HPLC. The struc-
ing research area in several cancer cell lines [7]). Some of tures of paltolides A-C were determined by NMR and tan-
these depsipeptides contain non-proteinogenic amino acids dem MS techniques and belong to a rare subgroup of
or hydroxy acid residues, while others show minimal altera- sponge-derived anabaenopeptins that have in common a C-
tions from the common ribosomal peptides. Cyclodepsipep- terminal tryptophan residue linked to the epsilon-amine of a
tides produced by marine sponges have unique structures lysine bearing a D-configuration [15].
that comprise unusual amino acids and N-terminal
polyketide-derived moieties [8]. However, it is rather diffi- PEPTIDES FROM MOLLUSKS
cult to isolate sufficient quantities of these metabolites for
Cytotoxic cyclic peptides have also been found in mol-
pharmacological and toxicological testing [9]. Papuamides
lusks. Dolastatins are a group of cyclic and linear peptides
are a representative class of marine sponge cyclic depsipep- isolated from the sea hare Dollabella auricularia. Dolastatin
tides. Papuamide A was isolated from Theonella mirabilis
10 is a linear peptide with antitumoral activity against mur-
and T. swinhoei and protected T-acute lymphoblastic leuke-
ine leukemia P388 cells. It displayed unprecedented potency
mia cell cultures from infection by HIV [10]. Homo-
702 Protein & Peptide Letters, 2012, Vol. 19, No. 7 Lazcano-Prez et al.

in experimental antineoplastic and tubulin assembly systems. neural and muscular cells with subsequent paralysis of the
It is in Phase I clinical trials as anticancer agent for treatment organism. The principal targets of these toxins are sodium
of breast and liver cancers, solid tumors and leukemia [16]. channels (-, - and O-conotoxins), potassium channels (-
Kahalalide F is a cyclic depsipeptide discovered in the slug and A-conotoxins) [24], calcium channels (-conotoxins),
Elysia rufescens that showed in vitro and in vivo selectivity nicotinic acetylcholine receptors (-conotoxins) [20], nore-
for prostate-derived cell lines and tumors. This peptide is in pinephrine transporter inhibitors (-conotoxins), and antago-
phase I clinical trials in patients with androgen-independent nist of nicotinic acetylcholine receptors (-conotoxins) [20,
prostate cancer [17]. Conus mollusks contain a family of 25, 26]. Most of the conotoxins have post-translational modi-
linear peptides rich in disulfide bonds known as conotoxins. fications (PTMs) such as glycosylation, tryptophan broma-
tion, C-terminus amidation, glutamic acid -carboxylation,
The Conus Genus N-terminal glutamine cyclization and tyrosine sulfation [27]
(Table 1). Mass-spectrometry is widely used to assess the
The marine cone snails are a diverse group of animals different post-translational modifications present in the cono-
comprising 500 to 700 different species [18, 19]. They can toxins. The importance, however, of these modifications for
be divided in three groups according to their feeding habits: the toxins function is not clear yet. In some cases, synthetic
piscivorous (fish eaters), vermivorous (worm eaters) and conopeptides lacking the native modifications are less active
molluscivorous (mollusk eaters). They use a complex mix- or totally loose their activity [28], while other conopeptides
ture of biological active molecules for functions such as de- do not loose their activity at all [29]. In addition, the precise
fense against predators, paralyzing their prey to eat it or to mode of interaction between the post-translational modifica-
repel another competing cone for the same ecological niche. tion and the molecular target has not been yet elucidated. It
Conus toxins (or conotoxins) are short peptides (8-50 amino has been suggested that the similarities between the structure
acids) rich in cysteines. These conopeptides are organized in of serotonin with that of the bromotryptophan residue pre-
Superfamilies that originate from a handful of genes [20]. sent in -conotoxins could explain the activity of this peptide
Until now there have been described 13 superfamilies named against the serotonin receptor [30]. Due to their potency in
by capital letters. Additionally, each conopeptide is further blocking ion-channels, conotoxins could be used to treat
classified in families described by a Greek letter according to pathologies related to the malfunctioning of these channels.
their pharmacological target [21]. The sequence of each Ziconotide (Prialt) is one of the few conotoxins approved
toxin is highly variable and the only conserved region within for medical use [31-33]. It is a synthetic drug derived from
a superfamily corresponds to that of the cystine framework the -conotoxin M-VII-A of Conus magus that acts as a cal-
[22]. The framework is defined according to the cysteine cium antagonist and is used to treat chronic pain. Its analge-
arrangement along their sequence and, in some cases, ac- sic effect is 1000 more potent than morphine with the advan-
cording to their disulfide connectivity (Figure 2) [23]. tage of not being addictive [34, 35].
Conotoxins Molecular Targets To isolate conus toxins it is necessary to first remove the
venom duct of the animal and macerate it in a buffered solu-
The conotoxins affect the mechanism of action of many tion to extract all the peptides present in it. Removal of the
ion channels, transporters and membrane receptors [24]. venom duct means the death of the animal, reason why many
They affect the ion influx and outflow passing through the scientists around the world use milking of these animals
cell membrane. This in turn, results in the depolarization of

Figure 2. Schematic organization of Conus peptides rich in disulfide bonds depicting the gene superfamilies, cysteine arrangement and
pharmacological targets. A detailed list of the superfamilies and frameworks can be review in data deposited in the ConoServer. ? unknown
target.
Bioactive Peptides from Marine Organisms Protein & Peptide Letters, 2012, Vol. 19, No. 7 703

Table 1. Common Post-translational Modifications Present in Conotoxins and the Enzymes Responsible for it

Modification Peptide Sequence Enzyme Reference

[81]
Disulfide bond formation GI Disulfide isomerase
ECCNPACGRHYSC^

Hydroxylation of proline GIIIA RDCCTOOKKCKDRQCKOQRCCA^ Proline hydroxylase [82]

Hydroxylase with D- [83]


Hydroxylation of D-valine gld-V* APANS(dHyV)WS
amino acid specificity

Protein amidating [84]


Amidation of C-terminus SI ICCNPACGPKYSC^
monooxygenase

Carboxylation of glutamic acid Conantokin-G GELQVNQLIRKSN^ -Glutamate carboxylase [85]

Bromination of tryptophan Bromocontryphan GCOwEPWC^ Bromo peroxidase [86]

Isomerization of tryptophan Contryphan GCOwEPWC^ Tryptophan epimerase [87]

Cyclization of N-terminal Gln Tx10c ZTCCGYRMCVOC^ Glutaminyl cyclase [88]

Sulfation of tyrosine EpI GCCSDPRCNMNNPYC^ Tyrosyl sulfotranferase [89]

Polipeptide HexNAc [81]


O-glycosylation SIVA ZKSLVPSVITTCCGYDOGTMCOOCRCTNSC^
transferase

, -carboxyglutamate; Z, piroglutamic acid; Y, tyrosine sulfate; w, D-tryptophan; W, 6-L-bromotryptophan; O, 4-trans hydroxyproline; S, Hex3HexNAc2-Ser; ^ indicates ami-
dated C-terminus; dHyV, D--hydroxyvaline.

instead. In this technique the animal is incited to expel its


harpoon into a latex membrane where the toxins are col- Sea Anemones Toxins
lected [30, 36]. Subsequently the mixture is fractionated by Sea anemone neurotoxins are divided into sodium chan-
RP-HPLC in a C-18 column and the peptides eluted with a nel toxins, potassium channel toxins, acid-sensing ion chan-
linear gradient of acetonitrile, water and TFA [37-40]. The nel toxins and protease inhibitors. The first cnidarian Na+
peptides obtained from the fractionated venom are analyzed channel modulating toxins purified were ATXs I-III from
by mass spectrometry. This technique gives basic informa- Anemonia sulcata [43] and Anthopleurin A from An-
tion regarding the primary sequence, the post-translational thopleura xanthogrammica [44]. Sodium channel toxins are
modifications and even the cysteine pairing of known toxins subdivided into four types: type 1 and 2 toxins contain 46-49
[36]. Analysis of the conotoxin genes is not easy due to the amino acids, except for AeI from Actinia equina that is 54
low sequence conservation amongst them. To facilitate the amino acid long, and all of them possess three disulfide
classification of conotoxins, a specialized database named bridges. Type 3 toxins consist of 27-32 amino acid residues
ConoServer (www.conoserver.com) was built. This data- and may have three or four disulfide bridges. Type 4 toxins
base contains the nucleotide sequences of the different genes include Calitoxin I and Calitoxin II that were isolated from
that codify for the conotoxins together with the correspond- Calliactis parasitica and are constituted by 46 amino acid
ing primary sequence of the prepropeptides and mature pep- residues and three disulfide bridges.
tides. This program allows the comparison of any new se-
quence with those contained in the database using a Blast The first purified K+ channel blocker was BgK from
program to identify the superfamily of the toxin and its prob- Bunodosoma granulifera [45]. Since then, the pharmacologi-
able pharmacological effect [21]. cal properties and chemical structure of other sea anemones
have been described [46-50]. Potassium channel toxins have
been classified into three types: type 1 toxins block Kv1
CNIDARIAN POLYPEPTIDES (Shaker) potassium channels. They have 35-37 amino acid
residues and are cross-linked by three disulfide bridges. Type
The phylum Cnidaria is unique such that practically all of
its members are toxic. Cnidarian toxins are a rich source of 2 toxins, known as Kalicludines 1-3 (AsKC), have 58-59
amino acid residues and also exhibit blocking activity
polypeptides with a wide variety of biological activities such
against Kv1 potassium channels though with less potency
as pore-forming cytolisins, phopholipases, neurotoxins and
than type 1 toxins. AsKC 1, 2 and 3 share sequence identity
protease inhibitors. Sea anemones have been extensively
with Kunitz-type protease inhibitors so they also act as pro-
studied compared to other cnidarians due to the stability of
tease inhibitors. Type 3 toxins are represented by BDS-I and
their toxins [41]. These toxins selectively target ion channels
and provide potent molecular probes for ion channel struc- BDS-II from Anemonia sulcata that specifically block Kv3.4
channels and APETx1 from Anthopleura elegantissima a
ture and function [42].
selective blocker of human ether-a-go-go-related gene
704 Protein & Peptide Letters, 2012, Vol. 19, No. 7 Lazcano-Prez et al.

(HERG) potassium channels. APETx1 inhibits human acids was isolated and characterized from the hemocytes of
HERG channel currents in a voltage-dependent manner by the spider crab Hyas araneus. The peptide named Arasin 1
shifting the activation properties of HERG channel [51]. An has an N-terminal domain rich in proline and arginine and a
inhibitor of an acid-sensing ion channel, APETx2, has been C-terminal domain containing two disulfide bonds [63].
isolated from Anthopleura elegantissima [52]. It inhibits
only the ASIC-3 channel making it a promising tool to study ECHINODERMATA POLYPEPTIDES
the physiological involvement of it in neuronal excitability
and pain coding. Although echinoderms cause fewer envenomations and
poisonings compared to other marine animals like coelenter-
The methods used for the isolation of several sodium ates, some species have been studied for their toxicological
channel toxins from sea anemones use traditional chroma- and pharmacological interest [64]. Sea urchin toxins have
tographic methods. The first step generally uses a size exclu- been poorly studied, though some authors have isolated some
sion chromatography with a small pore resin such as Se- toxins from pedicellariae; the venomous apparatus in sea
phadex G-50 [51] combined in some cases with the use of a urchins [65-67]. Recently, two antibacterial peptides were
Serdolit AD-2 column after an acetone precipitation of the isolated from coelomocyte extracts of Strongylocentrotus
proteins [52, 53]. Gel filtration is usually followed by a droebachiensis [68].
cation exchange chromatography and a final purification by
RP-HPLC. Recently, four toxins toxic to crabs were purified The crown-of-thorns starfish Acanthaster planci is a
from the whole body extract of Stichodactyla haddoni using toxic organism, which inflicts noxious symptoms due to the
just two steps, a gel filtration on Sephadex G50 and RP- venomous spines on its surface. An anticoagulant factor
HPLC [54]. Two of these toxins are new structurally potas- named Plancinin was isolated from the crude venom of the
sium channel toxins different from those already reported spines. The venom was fractionated with ammonium sulfate
such that they are cross-linked by two disulfide bridges. The and then applied into a DEAE-cellulose column. The pure
only other sea anemone toxin with two disulfide bridges is protein was obtained by using a Sephadex G50 column
AmI from A. maculata that has four cysteine residues at the twice. Plancinin is a dimeric peptide linked by a disulfide
same positions as SHTX I and SHTX II [55]. bond of about 7500 Da in its native form [69]. Two phos-
pholipases (named AP-PLA2-I and II) with hemolytic activ-
ity in the presence of phosphatidylcholine were purified from
Cubozoan Toxins
the same starfish venom [70]. These toxins were purified
Hemolytic toxins with molecular weights of approxi- using several techniques that included a CM-cellulose col-
mately 45 kDa have been isolated from the jellyfish Caryb- umn followed by an ammonium sulfate precipitation, a
dea rastoni (CrTX-A and CrTX-B) and Carybdea alata phenyl sepharose CL-4B column and a sephacryl S-200 col-
(CaTX-A and CaTX-B) [56, 57]. The four toxins were iso- umn were used for toxin I, while a Superose 12 HR10/30
lated from the sonicated tentacles of the jellyfish. The hemo- column was used for toxin II. Both purified toxins were fi-
lysin CARTOX from Carybdea marsupialis with an appar- nally achieved by RP-HPLC chromatography. The molecular
ent molecular mass of about 102-107 kDa, was isolated ap- masses of native AP-PLA2-I and II were determined to be 28
plying the crude extract to a cellulose column [58]. Snchez- and 12 kDa, respectively. Both PLA2s gave a single band
Rodrguez and collaborators partially purified a neurotoxin corresponding to a molecular mass of 15 kDa on SDS-PAGE
and three cytolysins from the same species using classical in the presence of a reducing agent indicating that AP-PLA2-
methods [59]. I is a dimer composed of the same subunit, while AP-PLA2-
II is a monomer. Shiomi et al. [71] purified a basic glycopro-
Two homologous proteins from the highly toxic sea wasp
tein of 25 kDa determined by SDS disc electrophoresis using
Chironex fleckeri were isolated from the nematocyst extract
by SDS-PAGE, then transferred to PVDF membranes and CM-cellulose and Sephadex G-100 chromatography.
stained with Coomassie R-250. Protein bands (43 and 45
kDa) were excised from the membranes, air-dried and ana- PEPTIDES FROM NEMERTINES, ECHIUROIDS AND
lyzed by Edman degradation [23]. ANNELIDS
The mucus secreted by the marine worm Cerebratulus
BIOACTIVE PEPTIDES FROM CHELICERATA AND lacteus contains a family of polypeptide cytotoxins (A-I, A-
CRUSTACEA II, A-III and A-IV) and neurotoxins (B-I, B-II, B-III and B-
The hemolymph of marine invertebrates contains many IV) [72]. To purify the A toxins, a Sephadex G-50 chroma-
tography followed by CM-cellulose gradient chromatogra-
biologically active substances including antimicrobial pep-
phy was used. Two CM-cellulose gradient separations at
tides. These molecules have low molecular weights (less
different pH values were required to resolve cytolisins A-III
than 10 kDa) and generally form pores in membranes [60].
and A-IV. As for the B toxins [73], the mucus was extracted
These peptides have an amphipathic structure that facilitates
in acidic conditions and the toxic components were adsorbed
their ability to attach to, destabilize and/or penetrate the cy-
toplasmic membrane of microorganisms. The horseshoe crab in a carboxymethylcellulose resin for storage. Desorption
was accomplished by packing the CM-cellulose in a column
Lymulus polyphemus is the source of polyphemusins I and II,
and the components stepwise eluted with 0.1 M ammonium
two 18 residue peptides that inhibit the growth of Gram-
acetate (pH 6.5) buffer. The first purification step was a Se-
negative and Gram-positive bacteria and fungi. The hemo-
phadex G-50F column chromatography. The third fraction
lymph of Thalamita crenata and Charybdis lucifera also
contained toxins lethal to crayfish and it was separated by
contain peptides with antimicrobial activity [61, 62]. A
proline and arginine-rich anti-microbial peptide of 37 amino CM-cellulose gradient chromatography at pH 7.5.
Bioactive Peptides from Marine Organisms Protein & Peptide Letters, 2012, Vol. 19, No. 7 705

Ovchinnikova and co-workers purified two novel antimi- ACKNOWLEDGEMENTS


crobial peptides, arenicin-1 and -2, from the marine poly-
N. S-P acknowledges the financial support from UNAM-
chaetas Arenicola marina coelomocytes [74]. The two pep-
DGAPA project PAPIIT IN204010. R. A-E acknowledges
tides exhibited activity against Gram-positive and Gram-
the financial support from UNAM-DGAPA project PAPIIT
negative bacteria and fungi. The extract obtained from the
isolated coelomocytes was ultra-filtered and applied to a IN201511.
preparative continuous acid-urea PAG electrophoresis and
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Received: July 29, 2011 Revised: September 6, 2011 Accepted: February 11, 2012

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