Identification of Hemolytic and Phospholipase Activity in Crude Extracts from Sea Anemones by Straightforward Bioassays

Summary

Here, we describe a protocol to obtain crude venom extract from sea anemone and detect its hemolytic and phospholipase activity.

Abstract

Sea anemone venom composition includes polypeptide and non-proteins molecules. Cytolytic components have a high biotechnological and biomedical potential for designing new molecular tools. Sea anemone venom locates in glandular cells from ectoderm and sub-cellular structures called nematocysts, both of which are distributed throughout the sea anemone body. This characteristic implies challenges because the cells and nematocyst must be lysed to release the venom components with other non-toxic molecules. Therefore, first, the venom is derived from a crude extract (mixture of different and diverse molecules and tissue debris). The next step is to detect polypeptides with specific bioactivities. Here, we describe an efficient strategy to obtain the sea anemone crude extract and bioassay to identify the presence of cytolysins. The first step involves inexpensive and straightforward techniques (stirred and freeze-thaw cycle) to release cytolysins. We obtained the highest cytolytic activity and protein (~500 mg of protein from 20 g of dry weight). Next, the polypeptide complexity of the extract was analyzed by SDS-PAGE gel detecting proteins with molecular weights between 10 kDa and 250 kDa. In the hemolytic assay, we used sheep red blood cells and determined HU₅₀ (11.1 ± 0.3 µg/mL). In contrast, the presence of phospholipases in the crude extract was determined using egg yolk as a substrate in a solid medium with agarose. Overall, this study uses an efficient and inexpensive protocol to prepare the crude extract and applies replicable bioassays to identify cytolysins, molecules with biotechnological and biomedical interests.

Introduction

Marine animals are a rich source of biologically active compounds. In recent decades, the composition of sea anemone venom has attracted scientific attention since it comprises a diversity of polypeptides with hemolytic, cytotoxic, enzymatic (phospholipase, protease, chitinase), and neurotoxic activity and inhibitory effects on proteolytic activity¹. In addition, these polypeptides are potential sources for the development of molecular tools in biotechnological and therapeutical use^2,3.

There are few reports about sea anemone venom and its molecular component....

Protocol

The sea anemones were collected according to guidelines of the National Commission for Aquaculture, Fisheries, and Food of the Federal Government of Mexico (permit number PPF / DGOPTA 07332.250810.4060). Bioethics Committee of the Institute of Biotechnology, National Autonomous University of Mexico approved all the experiments with sea anemones. The sheep blood sample was purchased at the Center for Practical Teaching and Research in Animal Production and Health (CEPIPSA, National Autonomous University of Mexico).

1. Organism collection

1. Collect sea anemone A. dowii.
   NOTE: Here, the organisms were....

Results

The representative results of the protocol used to obtain the crude extract of sea anemone showed that combining two techniques (agitation and cycles of freezing and thawing) produced an efficient discharge of nematocysts, and the total amount of protein was 500 mg (8 mg/mL) (Figure 3).

The crude extract's protein complexity could be observed from 10 kDa and greater than 250 kDa through SDS-PAGE electrophoresis. In addition, cytolysins were detected in the mol.......

Discussion

The high demand for new compounds with applications in different fields of science and industry has led to the study of venom. Venom represents a rich source of molecules that serves as a template for generating new molecular tools. However, the complexity of these venoms requires the implementation and combination of various methods to obtain and study them.

Here, we show a method for obtaining and analyzing the venom of the sea anemone Anthopleura dowii, Verrill 1869, which can be u.......

Materials

Name	Company	Catalog Number	Comments
15 mL conical centrifuge tube	Corning	430766
2-Bromophenol blue	Sigma	B75808
2-mercaptoetanol	Sigma-Aldrich	M6250-100ML
50 mL conical centrifuge tubes	Corning	430828
Acetic Acid Glacial	J.T. Baker	9515-03
Acrylamide	Promega	V3115
Agarose	Promega	V3125
Bisacrylamide	Promega	V3143
Bovine Serum Albumin Fraction V	Sigma	A3059-100G
Bradford Protein Assays	Bio-Rad	5000006
Calcium chloride	Sigma-Aldrich	C3306
Cell culture plates 96 well, V-bottom	Corning	3894
Centrifuge	Eppendorf	5804R
Centrifuge tubes	Corning	CLS430829
ChemiDoc MP system	Bio-Rad	1708280
Citric acid	Sigma-Aldrich	251275
Clear flat.bottom 96-Well Plates	Thermo Scientific	3855
Coomassie Brilliant Blue G-250	Bio-Rad	#1610406
Coomassie brilliant blue R-250	Bio-Rad	1610400
Dextrose	J.T. Baker	1916-01
Ductless Enclosure	Labconco	Vertical	https://imagej.nih.gov/ij ImageJ 1.53c
Gel Doc EZ	Bio Rad.		Gel Documentation System
Glycerol	Sigma-Aldrich	G5516-4L
Hemocytometer	Marienfeld	650030
ImageJ (Software)	NIH, USA	Version 1.53c
Incubator 211	Labnet	I5211 DS
Methanol	J.T. Baker	9049-03
Mini-PROTEAN tetra cell	Bio-Rad	1658000EDU
Na2HPO4	J.T. Baker	3824-01
NaCl	J.T. Baker	3624-01
NaH2PO4.H2O	J.T. Baker	3818-05
Origin software		version 9	To design the plot with sigmoidal adjustments
Petridish	Falcon	351007
Pipetman kit	Gilson	F167380
Precast mini gel	BioRad	1658004
Prestained Protein Ladder	Thermo Scientific	26620
Protease Inhibitor Cocktail	Roche	11836153001
Protein Assay Dye Reagent Concentrate	Bio-Rad	5000006
Rhodamine 6G	Sigma-Aldrich	252433
SDS	Sigma-Aldrich	L4509
Sodium citrate dihydrate	JT Baker	3646-01
Spectrophotometer	THERMO SCIENTIFIC	G10S UV-VIS
Tris Base	Sigma-Aldrich	77-86-1
Volt Power Supply	Hoefer	PS300B