Identification of Hemolytic and Phospholipase Activity in Crude Extracts from Sea Anemones by Straightforward Bioassays
Por:
Ramirez-Carreto, Santos, Salazar-Garcia, I, Sandra, Martínez G.M., Rodriguez-Almazan, Claudia
Publicada:
1 mar 2022
Resumen:
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 nontoxic 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 (similar to 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 mu 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.
Filiaciones:
Ramirez-Carreto, Santos:
Instituto de Biotecnologia, Universidad Nacional Autonoma de Mexico, Mexico
Univ Nacl Autonoma Mexico, Inst Biotecnol, Mexico City, DF, Mexico
Salazar-Garcia, Sandra, I , Univ Nacl Autonoma Mexico, Inst Biotecnol, Mexico City, DF, Mexico
Salazar-Garcia, I, Sandra:
Instituto de Biotecnologia, Universidad Nacional Autonoma de Mexico, Mexico
Univ Nacl Autonoma Mexico, Inst Biotecnol, Mexico City, DF, Mexico
Martínez G.M.:
Instituto de Biotecnologia, Universidad Nacional Autonoma de Mexico, Mexico
Rodriguez-Almazan, Claudia:
Instituto de Biotecnologia, Universidad Nacional Autonoma de Mexico, Mexico
Departamento de Micro y Nanotecnologías, Instituto de Ciencias Aplicadas y Tecnología, Universidad Nacional Autónoma de México, Mexico
Univ Nacl Autonoma Mexico, Inst Biotecnol, Mexico City, DF, Mexico
Univ Nacl Autonoma Mexico, Inst Ciencias Aplicadas & Tecnol, Dept Micro & Nanotecnol, Mexico City, DF, Mexico
Salazar-Garcia, Sandra, I , Univ Nacl Autonoma Mexico, Inst Biotecnol, Mexico City, DF, Mexico
Univ Nacl Autonoma Mexico, Dept Micro & Nanotecnol, Inst Ciencias Aplicadas & Tecnol, Mexico City, DF, Mexico
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