Name: identification of hemolytic and phospholipase activity in crude extracts from sea anemones by straightforward bioassays
Uploaded: 2022-03-29T14:00:00
Description: Watch this Scientific Journal Video about Identification of Hemolytic and Phospholipase Activity in Crude Extracts from Sea Anemones by Straightforward Bioassays at JoVE.com

This work was supported by Programa de Apoyo a Proyectos de Investigaci&#243;n e Innovaci&#243;n Tecnol&#243;gica (PAPIIT), with a grant number IT200819. The authors acknowledge to Tom Musselman, Rock Paper Editing, LLC, for checking the English grammar of this manuscript; and the technical assistance of Samanta Jim&#233;nez (CICESE, Ensenada), and Juan Manuel Barbosa Castillo (Instituto de Fisiolog&#237;a Celular, UNAM).&#160;We also thank to Dr Augusto C&#233;sar Lizarazo Chaparro (CEPIPSA) for obtaining sheep blood.&#160;We especially thank Dr Jos&#233; Saniger Blesa, ICAT-UNAM,&#160;&#160;for the facilities in his laboratory for the video recording.&#160;

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).
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<ol>
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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial,+antiprotozoal,+and+toxic+activities+of+Cnidarian+extracts+from+the+Mexican+Caribbean+Sea.">Antimicrobial, antiprotozoal, and toxic activities of Cnidarian extracts from the Mexican Caribbean Sea.</a> Pharmaceutical Biology. 45 (1), 37-43 (2007).</li><li>S&#225;nchez-Rodr&#237;guez, J., Cruz-Vazquez, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+biological+characterization+of+neurotoxic+compounds+from+the+sea+anemone+Lebrunia+danae+(Duchassaing+and+Michelotti,+1860).">Isolation and biological characterization of neurotoxic compounds from the sea anemone Lebrunia danae (Duchassaing and Michelotti, 1860).</a> Archives of Toxicology. 80 (7), 436-441 (2006).</li><li>de Oliveira, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caissarolysin+I+(Bcs+I),+a+new+hemolytic+toxin+from+the+Brazilian+sea+anemone+Bunodosoma+caissarum:+purification+and+biological+characterization.">Caissarolysin I (Bcs I), a new hemolytic toxin from the Brazilian sea anemone Bunodosoma caissarum: purification and biological characterization.</a> Biochimica Et Biophysica Acta. 1760 (3), 453-461 (2006).</li><li>Norton, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+characterisation+of+proteins+with+cardiac+stimulatory+and+haemolytic+activity+from+the+anemone+Actinia+tenebrosa.">Purification and characterisation of proteins with cardiac stimulatory and haemolytic activity from the anemone Actinia tenebrosa.</a> Toxicon: Official Journal of the International Society on Toxinology. 28 (1), 29-41 (1990).</li><li>Gondran, M., Eckeli, A. L., Migues, P. V., Gabilan, N. H., Rodrigues, A. L. 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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...

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 use2,3.
There are few reports about sea anemone venom and its molecular components due to the complexity of obtaining the venom, even isolation, and characterization of toxins. The extraction methods used in the reports involved lysis and emptying the contents of cells that are related and unrelated to the venom production1.
A particular characteristic in all cnidarians is the absence of a system for production and release of the venom centralized in a single anatomical region. Instead, the nematocysts are structures that keep the venom4,5. Other types of cells, called epidermal gland cells, also secrete toxins and are also distributed throughout the body of sea anemones6.
The first and most crucial challenge in obtaining the venom is the generation of an extract with sufficient manipulation in subsequent processes, without the inactivation or degradation of labile proteins. Next, the cells must be lysed, and the components-in this case, polypeptides must be efficiently and quickly extracted, avoiding proteolysis and hydrolysis while eliminating other cellular components7.
Different methods are used to obtain the crude extract of a sea anemone; some involve sacrificing the organism while others allow it to be kept alive. Methods that imply the use of the organism&#180;s whole body allow for the release of most toxins from the venom8, compared to methods that keep organisms alive, which extract only some components of the venom9. The preparation of an extract requires evaluating the presence and potency of a substance of interest through a specific bioassay, which includes strategies to observe the pharmacological effects by in vivo or in vitro methods10.
Sea anemone venom contains cytolytic polypeptides, pore-forming toxins (PFTs)11, and phospholipases12; these molecules are models in the study of protein-lipid interaction, molecular tools in cancer therapy, and biosensors based on nanopore3. The classification of sea anemone PFTs is carried out according to their size or molecular weight, from 5 kDa to 80 kDa. The 20 kDa PFT, the most studied and known as actinoporins11, is of particular interest for its biomedical potential in the development of molecular tools for possible applications as anticancer, antimicrobial, and nanopore-based biosensors. Another cytolysin, including phospholipases, specifically phospholipase A2 (PLA2)13, releases a fatty acid due and hydrolyzes phospholipids, destabilizing the cell membrane. Due to this mechanism of action, PLA2 promises to be an essential model for the study and applications in inflammatory diseases. It could serve as a model for studies of lipid behavior in the cell membrane14.
Here, we describe an efficient protocol for obtaining the crude extract from sea anemone Anthopleura dowii Verrill, 1869, and detecting hemolysins and phospholipases. Both are relevant toxins that could be used as a template to design new molecular tools.

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

identification of hemolytic and phospholipase activity in crude extracts from sea anemones by straightforward bioassays

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 &plusmn; 0.3 &#181;g/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.

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&#39;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...

Watch this Scientific Journal Video about Identification of Hemolytic and Phospholipase Activity in Crude Extracts from Sea Anemones by Straightforward Bioassays at JoVE.com

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

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

Sea anemone venom composition includes polypeptide and non-proteins molecules. Cytolytic components have a high biotechnological and biomedical potential ...

Sea anemone toxin interaction must use the least number of organisms and efficient protocols to obtain great diversity and abundance of the toxins and fast identification of a specific toxin. We combined two efficient techniques to prepare the crude extract with a high amount of protein and describe reproducible, specific, fast, and inexpensive assays to identify hemolysis and phospholic bases in the sea anemone venom. The method can be used in almost any example that is decided to identify one or both types of cytolysins.The method's efficiency is achieved by taking care of the times in each step while preparing the crude extract. The key to hemolysis is careful handling of the erythrocyte to avoid mechanical lysis, and in phospholysis assay, taking care of the temperature in the preparation of the plate. Demonstrating the procedure will be Dr.Santos Ramirez-Carreto, biologist Sandra Salazar Garcia, and biologist Giovanni Macias Martinez from my laboratory.Begin by cleaning the collected sea anemone A.doi with distilled water by hand to remove the big substrate particles adhering to the body. Immediately freeze the organisms at minus 80 degrees Celsius in an ultrafreezer for 72 hours. Then, place the organisms in special glasses for freeze drying with lyophilization conditions for 48 hours.For tissue hydration, reconstitute 20 grams dry weight of lyophilized organisms with 60 milliliters of 50-millimolar sodium phosphate buffer and one inhibitor cocktail tablet. In a magnetic stirrer, continuously stir the sample at around 800 RPM and four degrees Celsius for 12 hours. After freezing the sample at minus 20 degrees Celsius for 12 hours to induce cell lysis and nematocyst discharge, place the container in water at room temperature until it thaws up to 90%Repeat this procedure three times.To clarify the extract, centrifuge the sample at 25, 400 times G for 40 minutes at four degrees Celsius. Recover the supernatant and centrifuge it again. Repeat the step three to four times and recover the supernatant at last.The recovered supernatant or crude extract contains the venom components and other cell molecules such as lipids, carbohydrates and nucleic acids. Determine the protein concentration of the crude extract using a commercial Bradford colorimetric assay kit. Incubate the samples for five minutes at room temperature without shaking.Then, measure the absorbance at 595 nanometers. Plot the averages of the sample BSA absorbances and determine the equation of the graph where y is the absorbance, m is the slope of the line, x is the protein concentration, and b is the ordinate to the origin. To analyze the crude extract, add 30 micrograms of the crude extract to five microliters of the protein loading buffer to denature the proteins.The final volume must be less than 50 microliters. Run electrophoresis at 25 milliamperes constant for one hour and 30 minutes. To avoid the diffusion of the gel proteins, add the fixation solution to the proteins and incubate at room temperature for 40 minutes with shaking at 80 RPM.Then, decant the solution and add the protein crosslinking solution. Incubate for 30 minutes at room temperature with shaking at 80 RPM. Remove the solution and wash the gel with 100 milliliters of distilled water for five minutes.After that, remove the water and repeat this procedure four times. Carry out protein staining with 50 milliliters of Coomassie Brilliant Blue R-250 filtered solution stirring at 60 RPM in a laboratory rotary oscillator at room temperature for 15 minutes. Extract one milliliter of blood from the jugular vein of a sheep and remove the needle from the syringe.Immediately drain blood into a tube with 50 milliliters of Alsever solution and centrifuge at 804 times G for five minutes at four degrees Celsius. Discard the supernatant and add 30 milliliters of Alsever solution by sliding it along the tube's inner walls. Slowly resuspend the pellet using a Pasteur plastic pipette and centrifuge the solution again at 804 times G for five minutes at four degrees Celsius.Repeat this procedure until the supernatant is clarified. Maintain the final volume of the Alsever solution to achieve a concentration of 2 million cells per milliliter approximately. Then, use a Neubauer chamber or hemocytometer to count the cells.Incubate the solution mixtures for one hour at 37 degrees Celsius without shaking and then centrifuge the plate at 804 times G for five minutes at four degrees Celsius. Recover the supernatant in another 96-well plate with a flat bottom. If the crude extract contains cytolysins, erythrocytes will lyse and release hemoglobin.Read the absorbance at 415 nanometers and calculate the amount of extract that produces hemolysis in 50%of the erythrocytes. Increase the amount of sea anemone crude extract and design the plot with sigmoidal adjustments using appropriate software. Wash one chicken egg with 1%SDS in distilled water and separate the egg yolk from the egg white under sterile conditions.Prepare solutions A, B, C, and D.Add 500 microliters of solutions A and C to solution B and 100 microliters of solution D.Mix them and pour 25 milliliters into each Petri dish. Wait for the solution to solidify under sterile conditions and make wells of approximately two to three millimeters in diameter with a thin tube. Add a total of 20 microliters of phosphate buffer as a negative control in one well and 20 microliters of a determined phospholipase as a positive control in another well.Place different amounts of the crude extract protein 5, 15, 25, 35, and 45 micrograms in the remaining wells, each in a final volume of 20 microliters. Incubate at 37 degrees Celsius for 20 hours. 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.The nematocytes before and after stimuli are shown here. The nematocyst tubule is exposed in this image, indicating the toxin release. The electrophoretic profile of sea anemone crude extract is shown here.The sea anemone crude extract was analyzed by SDS-PAGE 15%polyacrylamide gel and showed protein from 10 kilodaltons to 250 kilodaltons of molecular weight. Here, lane 1 is the protein ladder and lane 2 is sea anemone venom. Cytolysins were detected in the molecular weight zone of 15 kilodaltons and 20 kilodaltons.Shown here is the hemolysis assay in erythrocytes from sheep. The hemolytic activity of the extract before and during the freezing and thawing cycles increased until 100%hemolysis was reached with 50 micrograms of the total crude extract protein in the last two cycles. The amount needed to lyse 50%sheep erythrocytes is 11.1 0.3 micrograms per milliliter.Phospholipase activity was detected by the formation of clear halos in the areas of the agar plate where the crude extract sample was applied. The results show the presence of phospholipase activity from 15 micrograms of total protein. The diameter of the halo increased in a dose-dependent manner;that is, if the amount of crude extract increased, the diameter of the halo increased.While attempting the procedure, make sure to induce nematocyst discharge, quantitate the total protein, analyze crude extract by SDS-PAGE electrophoresis, calculate the amount of crude extract protein to produce 50%of hemolysis, and use different amounts of protein in phospholipase assay. We can analyze the crude extract by proteomic technique to identify polypeptides with great relevance in biotechnology and biomedicine, even profile and characterize a unique molecule and then produce it in the laboratory for an application. We obtained part of the sequence of a performing protein with antitumor activity in the human obdurate lung carcinoma epithelial cells.Likewise, we identify the sequence of phospholipases of biotechnological interest.

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