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

这项工作得到了Apoyo a Proyectos de Investigación e Innovación Tecnológica（PAPIIT）的支持，赠款编号为IT200819。作者感谢Tom Musselman，Rock Paper Editing，LLC，检查了这份手稿的英语语法;以及萨曼塔·希门尼斯（CICESE，恩塞纳达）和胡安·曼努埃尔·巴尔博萨·卡斯蒂略（墨西哥国立自治大学塞卢拉研究所）的技术援助。我们还感谢Augusto César Lizarazo Chaparro博士（CEPIPSA）获得绵羊血液。我们特别感谢ICAT-UNAM的José Saniger Blesa博士，感谢他实验室的录像设施。

海葵是根据墨西哥联邦政府水产养殖、渔业和食品国家委员会的指导方针（许可证号PPF / DGOPTA 07332.250810.4060）收集的。墨西哥国立自治大学生物技术研究所生物伦理学委员会批准了海葵的所有实验。绵羊血液样本是在动物生产和健康实用教学和研究中心（CEPIPSA，墨西哥国立自治大学）购买的。
1. 生物体收集
<ol><li>收集海葵 A. dowii。 注：此处，这些生?...

<ol>
	<li>Fraz&#227;o, B., Vasconcelos, V., Antunes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sea+anemone+(Cnidaria,+Anthozoa,+Actiniaria)+toxins:+an+overview.">Sea anemone (Cnidaria, Anthozoa, Actiniaria) toxins: an overview.</a> Marine Drugs. 10 (8), 1812-1851 (2012).</li><li>Jayathilake, J. M. N. J., Gunathilake, K. V. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+preparation+of+biological+extracts+for+protein+purification.">Advances in preparation of biological extracts for protein purification.</a> Methods in Enzymology. 463, 285-303 (2009).</li><li>Bulati, M., 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=Partially+purified+extracts+of+sea+anemone+Anemonia+viridis+affect+the+growth+and+viability+of+selected+tumour+cell+lines.">Partially purified extracts of sea anemone Anemonia viridis affect the growth and viability of selected tumour cell lines.</a> BioMed Research International. 2016, 3849897 (2016).</li><li>Orts, D. J. B., 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=Biochemical+and+electrophysiological+characterization+of+two+sea+anemone+type+1+potassium+toxins+from+a+geographically+distant+population+of+Bunodosoma+caissarum.">Biochemical and electrophysiological characterization of two sea anemone type 1 potassium toxins from a geographically distant population of Bunodosoma caissarum.</a> Marine Drugs. 11 (3), 655-679 (2013).</li><li>Hader, D., Erzinger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bioassays: Advanced Methods and Applications. , (2017).</li><li>Anderluh, G., Macek, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytolytic+peptide+and+protein+toxins+from+sea+anemones+(Anthozoa:+Actiniaria).">Cytolytic peptide and protein toxins from sea anemones (Anthozoa: Actiniaria).</a> Toxicon: Official Journal of the International Society on Toxinology. 40 (2), 111-124 (2002).</li><li>Nevalainen, T. J., 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=Phospholipase+A2+in+cnidaria.">Phospholipase A2 in cnidaria.</a> Comparative Biochemistry and Physiology. Part B, Biochemistry &amp; Molecular Biology. 139 (4), 731-735 (2004).</li><li>Razpotnik, A., 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=A+new+phospholipase+A2+isolated+from+the+sea+anemone+Urticina+crassicornis+-+its+primary+structure+and+phylogenetic+classification.">A new phospholipase A2 isolated from the sea anemone Urticina crassicornis - its primary structure and phylogenetic classification.</a> The FEBS Journal. 277 (12), 2641-2653 (2010).</li><li>Dennis, E. A., Cao, J., Hsu, Y. -. H., Magrioti, V., Kokotos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipase+A2+enzymes:+Physical+structure,+biological+function,+disease+implication,+chemical+inhibition,+and+therapeutic+intervention.">Phospholipase A2 enzymes: Physical structure, biological function, disease implication, chemical inhibition, and therapeutic intervention.</a> Chemical Reviews. 111 (10), 6130-6185 (2011).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Analytical Biochemistry. 72, 248-254 (1976).</li><li>Kwon, Y. -. C., Jewett, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+preparation+methods+of+crude+extract+for+robust+cell-free+protein+synthesis.">High-throughput preparation methods of crude extract for robust cell-free protein synthesis.</a> Scientific Reports. 5, 8663 (2015).</li><li>Eno, A. E., Konya, R. S., Ibu, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+properties+of+a+venom+extract+from+the+sea+anemone,+Bunodosoma+cavernata.">Biological properties of a venom extract from the sea anemone, Bunodosoma cavernata.</a> Toxicon: Official Journal of the International Society on Toxinology. 36 (12), 2013-2020 (1998).</li><li>Morales-Landa, J. 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. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+crude+extract+from+the+sea+anemone,+Bunodosoma+caissarum+elicits+convulsions+in+mice:+possible+involvement+of+the+glutamatergic+system.">The crude extract from the sea anemone, Bunodosoma caissarum elicits convulsions in mice: possible involvement of the glutamatergic system.</a> Toxicon: Official Journal of the International Society on Toxinology. 40 (12), 1667-1674 (2002).</li><li>Dion, A. S., Pomenti, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ammoniacal+silver+staining+of+proteins:+mechanism+of+glutaraldehyde+enhancement.">Ammoniacal silver staining of proteins: mechanism of glutaraldehyde enhancement.</a> Analytical Biochemistry. 129 (2), 490-496 (1983).</li><li>Ram&#237;rez-Carreto, 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=Identification+of+a+pore-forming+protein+from+sea+anemone+Anthopleura+dowii+Verrill+(1869)+venom+by+mass+spectrometry.">Identification of a pore-forming protein from sea anemone Anthopleura dowii Verrill (1869) venom by mass spectrometry.</a> The Journal of Venomous Animals and Toxins Including Tropical Diseases. 25, 147418 (2019).</li><li>Nelson, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+lipids+of+sheep+red+blood+cells.+I.+Lipid+composition+in+low+and+high+potassium+red+cells.">Studies on the lipids of sheep red blood cells. I. Lipid composition in low and high potassium red cells.</a> Lipids. 2 (1), 64-71 (1967).</li><li>Ram&#237;rez-Carreto, 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=Transcriptomic+and+proteomic+analysis+of+the+tentacles+and+mucus+of+Anthopleura+dowii+Verrill,+1869.">Transcriptomic and proteomic analysis of the tentacles and mucus of Anthopleura dowii Verrill, 1869.</a> Marine Drugs. 17 (8), 436 (2019).</li></ol>

对新化合物的高需求在科学和工业的不同领域都有应用，这导致了对毒液的研究。毒液代表了丰富的分子来源，可作为生成新分子工具的模板。然而，这些毒液的复杂性需要实施和组合各种方法来获取和研究它们。
在这里，我们展示了一种获取和分析海葵 Anthopleura dowii的毒液的方法，Verrill 1869，可用于探索其他海葵物种的毒液，从冻干标本开始，然后粗提取。冻干步?...

海洋动物是生物活性化合物的丰富来源。近几十年来，海葵毒液的组成引起了科学的关注，因为它由多种多肽组成，具有溶血性，细胞毒性，酶促性（磷脂酶，蛋白酶，几丁质酶）和神经毒性活性和对蛋白水解活性的抑制作用1。此外，这些多肽是开发分子工具在生物技术和治疗用途中的潜在来源2，3。
关于海葵毒液及其分子成分的报道很少，因为获得毒液的复杂性，甚至分离和毒素的表征。报告中使用的提取方法涉及裂解和排空与毒液产生1相关且无关的细胞内容物。
所有蜈蝓病患者的一个特殊特征是缺乏集中在单个解剖区域的毒液生产和释放系统。相反，线虫囊是保持毒液4，5的结构。其他类型的细胞，称为表皮腺细胞，也分泌毒素，并且也分布在海葵体内6。
获得毒液的第一个也是最关键的挑战是在后续过程中产生具有充分操作的提取物，而不会灭活或降解不稳定的蛋白质。接下来，必须裂解细胞，并且组分 - 在这种情况下，必须有效和快速地提取多肽，避免蛋白水解和水解，同时消除其他细胞组分7。
使用不同的方法获得海葵的粗提取物;有些涉及牺牲生物体，而另一些则允许它保持活力。暗示使用生物体整个身体的方法允许从毒液8中释放大多数毒素，而保持生物体存活的方法仅提取毒液的某些成分9。提取物的制备需要通过特定的生物测定法评估目标物质的存在和效力，其中包括通过 体内 或 体外 方法观察药理作用的策略10。
海葵毒含有细胞溶解多肽，成孔毒素（PFT）11和磷脂酶12;这些分子是研究蛋白质 - 脂质相互作用，癌症治疗中的分子工具以及基于纳米孔3的生物传感器的模型。海葵PFT的分类是根据其大小或分子量进行的，从5 kDa到80 kDa。20 kDa PFT是研究最多和被称为放线菌素11的，因其在开发分子工具方面具有特别感兴趣的生物医学潜力，可用于抗癌，抗菌和基于纳米孔的生物传感器。另一种细胞溶解素，包括磷脂酶，特别是磷脂酶A2（PLA2）13，释放脂肪酸并水解磷脂，破坏细胞膜的稳定性。由于这种作用机制，PLA2有望成为炎症性疾病研究和应用的重要模型。它可以作为研究细胞膜14中脂质行为的模型。
在这里，我们描述了一种有效的方案，用于从海葵 Anthopleura dowii Verrill（1869）获得粗提取物，并检测溶血素和磷脂酶。两者都是相关的毒素，可以用作设计新分子工具的模板。

<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

海葵毒液组合物包括多肽和非蛋白质分子。细胞溶解成分在设计新的分子工具方面具有很高的生物技术和生物医学潜力。海葵毒液位于外胚层和称为线囊的亚细胞结构的腺细胞中，两者都分布在整个海葵体内。这一特征意味着挑战，因为细胞和线虫囊囊必须裂解以释放与其他无毒分子一起释放毒液成分。因此，首先，毒液来源于粗提取物（不同和不同的分子和组织碎片的混合物）。下一步是检测具有特定生物活性的多肽。在这里，我们描述了一种获得海葵粗提取物和生物测定的有效策略，以鉴定细胞溶解素的存在。第一步涉及廉价和直接的技术（搅拌和冻融循环）来释放细胞溶解素。我们获得了最高的细胞溶解活性和蛋白质（从20克干重中获得约500毫克蛋白质）。接下来，通过SDS-PAGE凝胶检测分子量在10 kDa和250 kDa之间的蛋白质来分析提取物的多肽复杂性。在溶血测定中，我们使用绵羊红细胞并测定HU50 （11.1±0.3μg/ mL）。相比之下，使用蛋黄作为底物在琼脂糖固体介质中测定粗提取物中磷脂酶的存在。总体而言，本研究使用高效且廉价的方案来制备粗提取物，并应用可复制的生物测定来鉴定细胞溶解素，具有生物技术和生物医学兴趣的分子。

用于获得海葵粗提取物的方案的代表性结果表明，结合两种技术（搅拌和冷冻和解冻循环）产生了线虫囊肿的有效排出，蛋白质总量为500mg（8mg / mL）（图3）。
粗提取物的蛋白质复杂性可以通过SDS-PAGE电泳从10 kDa和大于250 kDa观察到。此外，在15 kDa和20 kDa的分子量区检测到细胞溶解素，其分子量范围可能对应于磷脂酶13 或放线菌素<sup clas...

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

在这里，我们描述了一种从海葵中获得粗毒血清提取物并检测其溶血和磷脂酶活性的方案。

通过直接生物测定法鉴定海葵粗提取物中的溶血和磷脂酶活性

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

海葵毒素相互作用必须使用最少数量的生物体和有效的协议来获得毒素的巨大多样性和丰度，并快速鉴定特定毒素。我们结合了两种有效的技术来制备含有大量蛋白质的粗提取物，并描述了可重复的，特异性的，快速的和廉价的测定方法，以鉴定海葵毒液中的溶血和磷酸碱基。该方法可用于几乎任何决定鉴定一种或两种类型的细胞溶解素的示例。该方法的效率是通过在制备粗提取物时注意每个步骤的时间来实现的。溶血的关键是小心处理红细胞以避免机械裂解，并且在磷解测定中，注意制备板的温度。来自我实验室的Santos Ramirez-Carreto博士，生物学家Sandra Salazar Garcia和生物学家Giovanni Macias Martinez将演示该程序。首先用蒸馏水清洁收集的海葵A.doi，以去除粘附在身体上的大基质颗粒。立即将微生物在零下80摄氏度的超低温冰箱中冷冻72小时。然后，将生物体置于特殊的玻璃杯中，在冻干条件下冷冻干燥48小时。对于组织水合作用，用60毫升50毫摩尔磷酸钠缓冲液和一片抑制剂鸡尾酒片重重建20克干重的冻干生物。在磁力搅拌器中，以约800 RPM和4摄氏度连续搅拌样品12小时。将样品在零下20摄氏度冷冻12小时以诱导细胞裂解和线虫囊排出后，将容器置于室温下的水中直至解冻至90%重复此过程三次。为了澄清提取物，将样品在4摄氏度下以25，400倍G离心40分钟。回收上清液并再次离心。重复步骤三到四次，最后回收上清液。回收的上清液或粗提取物含有毒液成分和其他细胞分子，如脂质，碳水化合物和核酸。使用商业布拉德福德比色测定试剂盒测定粗提取物的蛋白质浓度。将样品在室温下孵育五分钟而不摇动。然后，测量595纳米处的吸光度。绘制样品BSA吸光度的平均值并确定图形的方程，其中y是吸光度，m是线的斜率，x是蛋白质浓度，b是原点的纵坐标。为了分析粗提取物，将30微克粗提取物加入5微升蛋白质上样缓冲液中以使蛋白质变性。最终体积必须小于 50 微升。以25毫安恒定值运行电泳1小时30分钟。为了避免凝胶蛋白的扩散，将固定溶液加入蛋白质中，并在室温下以80RPM振荡孵育40分钟。然后，倾析溶液并加入蛋白质交联溶液。在室温下孵育30分钟，以80RPM振荡。取出溶液，用100毫升蒸馏水洗涤凝胶五分钟。之后，取出水并重复此过程四次。用50毫升考马斯亮蓝R-250过滤溶液进行蛋白质染色，在室温下以60 RPM在实验室旋转振荡器中搅拌15分钟。从绵羊的颈静脉中提取一毫升血液，并从注射器中取出针头。立即将血液沥入装有50毫升Alsever溶液的管中，并在4摄氏度下以804倍G离心5分钟。弃去上清液，并通过沿管的内壁滑动上清液加入30毫升Alsever溶液。使用巴斯德塑料移液管缓慢重悬沉淀，并在4摄氏度下以804倍G再次离心溶液5分钟。重复此过程，直到上清液澄清。保持Alsever溶液的最终体积，以达到大约每毫升200万个细胞的浓度。然后，使用Neubauer室或血细胞计数器对细胞进行计数。将溶液混合物在37摄氏度下孵育一小时而不摇动，然后在4摄氏度下以804倍G离心板5分钟。在另一个具有平底的96孔板中回收上清液。如果粗提取物含有细胞溶解素，红细胞将裂解并释放血红蛋白。读取415纳米处的吸光度，并计算在50%的红细胞中产生溶血的提取物量。增加海葵原油提取量，并使用适当的软件进行乙状调整设计地块。在蒸馏水中用1%SDS清洗一个鸡蛋，并在无菌条件下将蛋黄与蛋清分开。准备溶液A，B，C和D.向溶液B和100微升溶液D中加入500微升溶液A和C.将它们混合并倒入每个培养皿中25毫升。等待溶液在无菌条件下固化，并用细管制作直径约2至3毫米的孔。在一个孔中加入总共20微升的磷酸盐缓冲液作为阴性对照，在另一个孔中加入20微升确定的磷脂酶作为阳性对照。将不同量的粗提取蛋白5，15，25，35和45微克放入剩余的孔中，每个孔的最终体积为20微升。在37摄氏度下孵育20小时。用于获得海葵粗提取物的方案的代表性结果表明，结合两种技术，搅拌和冷冻和解冻循环，产生了线虫囊肿的有效排出。这里显示了刺激前后的线粒细胞。该图像中线囊小管暴露在外，表明毒素释放。海葵粗提取物的电泳曲线如图所示。采用SDS-PAGE 15%聚丙烯酰胺凝胶对海葵粗提取物进行分析，显示蛋白质分子量从10千道尔顿到250千道尔顿。在这里，泳道1是蛋白质梯，泳道2是海葵毒液。在15千道尔顿和20千道尔顿的分子量区检测到细胞溶解素。这里显示的是绵羊红细胞的溶血试验。在冷冻和解冻周期之前和期间，提取物的溶血活性增加，直到在最后两个周期中用50微克总粗提取物蛋白达到100%溶血。裂解50%羊红细胞所需的量为每毫升11.1 0.3微克。通过在应用粗提取物样品的琼脂板区域形成透明光晕来检测磷脂酶活性。结果显示15微克总蛋白存在磷脂酶活性。光环的直径以剂量依赖性的方式增加;也就是说，如果粗提取物的量增加，光环的直径增加。在尝试该过程时，确保诱导线虫囊团排出，定量总蛋白，通过SDS-PAGE电泳分析粗提取物，计算粗提取蛋白的量以产生50%的溶血，并在磷脂酶测定中使用不同量的蛋白质。我们可以通过蛋白质组学技术分析粗提取物，以鉴定在生物技术和生物医学中具有重要意义的多肽，甚至可以分析和表征独特的分子，然后在实验室中生产它以进行应用。我们获得了在人类顽固性肺癌上皮细胞中具有抗肿瘤活性的执行蛋白的部分序列。同样，我们确定了生物技术感兴趣的磷脂酶序列。

Research

JoVE Journal

Biochemistry

Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

小分子结合蛋白的鉴定在天然细胞环境由活细胞光亲和标记

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several ...

科研

生物化学

Defining Substrate Specificities for Lipase and Phospholipase Candidates

定义底物特异性的脂肪酶和磷脂酶考生

Microorganisms produce a wide spectrum of (phospho)lipases that are secreted in order to make external substrates available for the organism. ...

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

RNA 结合区质量谱鉴定的样品制备

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

Preparation of Keratin Hydrolysate from Chicken Feathers and Its Application in Cosmetics

鸡羽毛角蛋白水解物的制备及其在化妆品中的应用

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for ...

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

拟南芥线粒体的分离和呼吸测量

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

The Determination of Protease Specificity in Mouse Tissue Extracts by MALDI-TOF Mass Spectrometry: Manipulating PH to Cause Specificity Changes

MALDI 质谱法测定小鼠组织提取物蛋白酶特异性: 操作 PH 值导致特异性改变

Proteases have several biological functions, including protein activation/inactivation and food digestion. Identifying protease specificity is important ...

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

用体外激酶法鉴定细胞周期蛋白依赖性激酶1特异磷酸化部位

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; ...

The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation

利用生物测定引导分馏法鉴定海洋鳗信息素

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and ...

Detection of Phospholipase C Activity in the Brain Homogenate from the Honeybee

蜜蜂匀浆中磷脂酶 C 活性的检测

The honeybee is a model organism for evaluating complex behaviors and higher brain function, such as learning, memory, and division of labor. The mushroom ...