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

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.