Name: A Liposome Membrane Permeability Assay for Investigating the Effects of Phosphatidylinositol Phosphate Groups on Membranotropic Action of Venom PLA2 (Video) | JoVE Journal
Uploaded: 2025-09-26T14:00:00.000+00:00
Duration: 10 min 31 s
Description: Learn a sensitive liposome membrane permeability assay for studying phospholipase A2 effects on model membranes. This cost-effective method enhances resolution in membrane biophysics and toxin interaction research.

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5 minutes

The studies in this article have been supported by the start-up grant from the Beijing Chaoyang KaiWen Academy (E.S.G.) and by the SEPA Award, NIH 5R25MD019151-03 (R.K.D.). The work pertaining to the preparation of HDP-2 was supported by a grant from the Russian Science Foundation No. 24-15-00280 (Y.N.U.).&#160;The authors would like to acknowledge Dr. Maryam Barancheshmeh in writing the script and developing the video on the application of molecular docking simulation, which was prepared subsequent to the publication of this research article.

1. In silico simulation of HDP-2P interaction with PI and phosphoinositides using molecular modeling software
<ol>
	<li>Software installation and preparation
	<ol>
		<li>Install the referenced chemical drawing editor for constructing ligand molecules as shown in Supplemental File 1-Supplemental Figure S1A.</li>
		<li>Install the molecular modeling software for molecular docking of ligand to receptor.</li>
		<li>Install the Automated Docking Tools for ligand and receptor preparation.</li>
		<li>As the molecular modeling simulation software depends on the Python environment, install Python.</li>
	</ol>
	</li>
	<li>Ligand preparation
	<ol>
		<li>First, use the chemical drawing editor to build the atomic coordinates for molecular structures of PI, PI-4-P, PI-4,5-P2, cardiolipin, and phosphatidylserine. 
		&#8203;NOTE: The example protocol in the following steps of the ligand preparation describes the steps only for PI.</li>
		<li>Build the molecular structure of PI using the chemical drawing editor as illustrated in Supplemental File 1-Supplemental Figure S1B and save the file in PDB format as shown in Supplemental File 1-Supplemental Figure S2.</li>
		<li>Open the PDB file in the automated docking tool software as shown in Supplemental File 1-Supplemental Figure S3A.</li>
		<li>Add hydrogen atoms as shown in Supplemental File 1-Supplemental Figure S3B and select either polar or all hydrogen atoms as shown in Supplemental File 1-Supplemental Figure S4A. 
		NOTE: After completion of PI docking with HDP-2P, select only polar hydrogen atoms to simplify visualization of polar or hydrogen bonds.</li>
		<li>Set the PI molecule as the ligand for the automated docking tool software as shown in Supplemental File 1-Supplemental Figure S4B.</li>
		<li>Save the ligand as a PDBQT file as shown in Supplemental File 1-Supplemental Figure S5A. Make sure to make all bonds rotatable.</li>
	</ol>
	</li>
	<li>Receptor preparation
	<ol>
		<li>Download the PDB file of phospholipase A2 HDP-2 (PDB code 2I0U), consisting of two monomers, one is acidic, enzymatically inactive, A, and the other is basic, enzymatically active, E. To delete enzymatically inactive monomer A, import the 2I0U file to the molecular modeling software (Supplemental File 1-Supplemental Figure S5B), select all atoms of monomer A (Supplemental File 1-Supplemental Figure S6A), then go to Edit and delete all atoms of monomer A (Supplemental File 1-Supplemental Figure S6B). Keep enzymatically active monomer E, which is now designated as the receptor.</li>
		<li>Open the HDP-2P receptor PDB file in the automated docking tool software as shown in Supplemental File 1-Supplemental Figure S7.</li>
		<li>Remove all water molecules as shown in Supplemental File 1-Supplemental Figure S8A.</li>
		<li>Add hydrogen atoms as shown in Supplemental File 1-Supplemental Figure S8B. Please note that after completion of the HDP-2P docking with PI, only polar hydrogens have to be selected to simplify visualization of polar and hydrogen bonds.</li>
		<li>Set the HDP-2P as the receptor, as shown in Supplemental File 1-Supplemental Figure S9A, and save it as a PDBQT file. Ensure the receptor is set without rotatable bonds.</li>
	</ol>
	</li>
	<li>Docking parameter setup
	<ol>
		<li>Open both the ligand and the receptor PDBQT files.</li>
		<li>Enter the grid box setup interface, as shown in Supplemental File 1-Supplemental Figure S9B, and define the grid box size (size x = 44.644; size y = 74.939; size z = 43.050) and center (center x = 25.626; center y = 9.582; center z = 41.825) coordinates, which fully enclose the entire surface of the receptor molecule as shown in Supplemental File 1-Supplemental Figure S10A.</li>
		<li>Close the grid box setup interface with the current settings as shown in Supplemental File 1-Supplemental Figure S10B and save the docking parameters in a config.txt file as shown in Supplemental File 1-Supplemental Figure S11A.</li>
	</ol>
	</li>
	<li>Running the molecular docking software
	<ol>
		<li>Launch the molecular docking software by using the config.txt file as shown in Supplemental File 1-Supplemental Figure S11B.</li>
		<li>Execute the docking calculation (~30-90 min; Supplemental File 1-Supplemental Figure S12).</li>
		<li>Check the docking_log.txt file to review the docking scores (binding affinity).</li>
		<li>Select the docking result with the lowest energy conformation.</li>
	</ol>
	</li>
	<li>Analysis of docking data
	<ol>
		<li>Use the automated docking tool software to open the docking result PDBQT file.</li>
		<li>Analyze the ligand-receptor binding sites, focusing on the receptor&#39;s active center and intermolecular interactions, including ionic, ion-polar, polar, and hydrogen bonding interactions. Make a table for PI and phosphoinositide, which interacts with the receptor&#39;s active site, to describe binding site affinities in kcal/mol, charged and polar moieties in PI/phosphoinositide polar head, and HDP-2P residues involved in intermolecular bonding, and types of bonds as shown in Table 1 and Table 2. Repeat docking for each receptor-ligand pair 3x to make sure that there is no deviation in binding sites, binding affinities, charged and polar moieties of PI/phosphoinositide and HDP-2P residues, and the type of bonds.</li>
	</ol>
	</li>
</ol>
2. Hydrolytic activity of HDP-2P in PC liposomes enriched with PI, PI-4-P, PI-4,5-P2 , PS, or CL monitored by using the coenzyme A acylated with free fatty acids
NOTE: Chloroform, methanol, NH3, and CuSO4 are hazardous. Always use nitrile gloves, goggles, and lab coat. Handle all protocols withthese reagents under the fume hood. Store chloroform in an amber glass bottle. Store methanol in flammable cabinets away from oxidizers. Keep methanol away from flames and sparks. Label all waste containers for these reagents and dispose according to the local hazardous waste disposal regulations.
<ol>
	<li>Prepare 100 mL of buffer solution for the HDP-2P-catalyzed phospholipid hydrolysis by dissolving 5.55 mg of CaCl2 in 0.10 M Tris-HCl buffer (pH 7.4).</li>
	<li>Prepare 50 mL of 1.0 M EDTA stock solution by dissolving 7.31 g of EDTA in deionized distilled water (ddH20).</li>
	<li>Prepare 0.54 mM HDP-2P stock solution in 0.10 M Tris-HCl buffer (pH 7.4). Molar mass of HDP-2P is 13.827 kDa.</li>
	<li>Prepare the following stock solutions of phospholipids at concentration 10.0 M in chloroform/methanol mixture (2 to 1 by volume): egg yolk L-&#945;-phosphatidylcholine (PC), bovine heart cardiolipin (CL), bovine brain L-&#945;-phosphatidyl-L-serine (PS), PI, PI-4-P, and PI-4,5-P2. Molar masses of PC, CL, PS, PI, PI-4-P, and PI-4,5-P2 are 768 Da, 1,466 Da, 792 Da, 867 Da, 962 Da, and 1,057 Da, respectively, as further described below.</li>
	<li>Prepare five samples of PC liposomes. To prepare a sample of PC liposomes, add 75 &#181;L of chloroform/methanol stock solution of PC to a glass test tube and dry the lipids at 25 &#176;C under 10 Pa vacuum for 1 h until a lipid film is formed. Hydrate the lipid film for 1 h with 15 mL of buffer solution for phospholipid hydrolysis at 25 &#176;C in a thermostat. Then, insert the titanium pistol of a 100-150 W ultrasonic dispenser into the lipid suspension in the glass test tube placed in an ice bath and sonicate the suspension at 22 kHz frequency for 10 min, not exceeding 50% of the maximum power. To remove titanium &#39;dust&#39; particles, centrifuge liposomes at 200 &#215; g (1,270 rpm with a 10 cm rotor) for 10 min at room temperature.</li>
	<li>Prepare samples of PC liposomes enriched with either CL, PS, PI, PI-4-P, or PI-4,5-P2 at the PC and other lipid molar ratio 4 to 1 by mixing chloroform/methanolphospholipid stock solutions-60 &#181;L of PC with 15 &#181;L of either CL, PS, PI, PI-4-P, or PI-4,5-P2-in a glass tube. Then, dry the lipids in a 10 Pa vacuum or for 3 h at room temperature, hydrate the lipid film in buffer solution for phospholipid hydrolysis, and sonicate with ultrasonic waves as described above. 
	NOTE: For each type of liposomes, prepare five samples.</li>
	<li>Place 1.0 mL from five samples from each type of liposomes described above to separate tubes and add 10 &#181;L of colorless coenzyme-A solution from the NEFA kit to each tube and then add 0.00, 0.93, 1.86, 2.79, or 3.72 &#181;L of 0.54 mM HDP-2P stock solution to separate tubes to obtain in liposome samples the HDP-2P/phospholipid molar ratios of 0.000, 0.001, 0.002, 0.003, and 0.004, respectively.</li>
	<li>Immediately after adding HDP-2P to the liposomes, incubate liposome samples for 15 min at 37 &#176;C in a thermostat and then, stop phospholipid hydrolysis induced by HDP-2P by adding 100 &#181;L of stock EDTA solution. After that, vortex the samples for 1 min.</li>
	<li>Quantify the hydrolytic activity of HDP-2P by measuring the optical density of liposomal samples at 550 nm using a spectrophotometer, which would measure the extent that colorless coenzyme-A solution turns purple when it gets acylated with free fatty acids.</li>
	<li>Repeat the above procedure two more times so that the average optical density values are derived from three independent trials. 
	NOTE: Each data point obtained in this experimental trial is the average value of the optical density. Ideally, the standard deviation of experimental data points should lie within &#177;5% of the average.</li>
</ol>
3. Effects of HDP-2P on permeability of PC liposomal membranes enriched with PI, PI-4-P, PI-4,5-P2 , PS, or CL measured by a ligand substitution reaction in CuSO&#8203;4 solution involving a NH3 ligand trapped inside the liposomes
NOTE: Chloroform, methanol, NH3, and CuSO4 are hazardous. Always use nitrile gloves, goggles, and lab coat. Handle all protocols with these reagents under the fume hood. Store chloroform in an amber glass bottle. Store methanol in flammable cabinets away from oxidizers. Keep methanol away from flames or sparks. Label all waste containers for these reagents and dispose according to the local hazardous waste disposal regulations.
<ol>
	<li>Prepare 20 mL of 0.03 M stock solutions for each of six phospholipids - PC, CL, PS, PI, PI-4-P, PI-4,5-P2- in chloroform/methanol (2 to 1 volume). Molecular masses of PC, CL, PS, PI, PI-4-P, PI-4,5-P2 are 768, 1466, 792, 867, 962, and 1057 Da, respectively.</li>
	<li>Prepare 100 mL of 0.10 M Tris-HCl (pH 7.4) buffer with at a final concentration of 0.05 M CuSO4.</li>
	<li>Prepare 4 mL of 1.5 &#215; 10-3 M HDP-2P stock in 0.05 M CuSO4, 0.10 M Tris-HCl (pH 7.4) buffer. Molar mass of HDP-2P is 13.827 kDa.</li>
	<li>To prepare 100 mL of 0.5 M NH3 solution, use 25% NH3 solution (density 905 g/L30, equal to 13.30 M [905 g/L &#215; 0.25 M &#247; 17.00 g/L = 13.30 M]). Thus, to prepare 100 mL of 0.5 M NH3, use 3.76 mL of 25% NH3 solution: (0.5 M &#215; 1,000 mL &#247; 13.30 M) &#247; 10 = 3.76 mL of 25% NH3 solution. Place 3.76 mL of 25% NH3 solution in a 100 mL volumetric flask and bring the volume to 100 mL with 0.10 M Tris-HCl (pH 7.4) buffer.</li>
	<li>To prepare liposomes with NH3 in the inner volume of liposomes, place 12.5 mL of 0.03 M stock phospholipid solution in chloroform/methanol in a tube and dry phospholipids in 10 Pa vacuum or for 3 h at room temperature. Hydrate the phospholipid film for 1 h with 12.5 mL of 0.5 M NH3, 0.10 M Tris-HCl (pH 7.4) at 25 &#176;C in a thermostat; then sonicate the lipid suspension at 22 kHz frequency as described in step 2.5 to produce sonicated liposomes. 
	NOTE: In the solution with sonicated liposomes, NH3 is located inside and outside the liposomes.</li>
	<li>To eliminate NH3 outside the liposomes, dialyze the liposomes using a dialysis membrane (molecular cut-off 1.0 kDa) for 5 hat room temperature against 0.10 M Tris-HCl (pH 7.4) using a magnetic stirrer. 
	NOTE: Dialysis for 5 h was sufficient to completely remove NH3, as the addition of 0.5 mL of dialyzed liposomes to 0.5 mL of 0.10 M Tris-HCl, 0.05 M CuSO4 buffer yielded the same optical density at 600 nm as that of liposomes prepared without NH3 and mixed with the same buffer.</li>
	<li>After dialysis, centrifuge the liposomes at 200 &#215; g for 90 min as described in step 2.5 to pellet the liposomes. Discard the supernatant and resuspend the liposomes with 5.0 mL of 0.10 M Tris-HCl, 0.05 M CuSO4 (pH 7.4) buffer, and divide the resuspended liposomes into five samples of 1.0 mL each. Add to these five 1.0 mL samples separately 80, 60, 40, 20, and 0 &#181;L of 0.10 M Tris-HCl, 0.05 M CuSO4 (pH 7.4) buffer and then 0, 20, 40, 60, 80 &#181;L of HDP-2P stock buffer to adjust in the liposome samples the HDP-2P/phospholipid molar ratios 0.000, 0.001, 0.002, 0.003, and 0.004, respectively.</li>
	<li>Incubate the liposome samples for 30 min in a thermostat at 37 &#176;C; then, measure the optical density of the liposome samples at 600 nm using a spectrophotometer. Follow the steps described in section 3 for all six liposome samples and repeat 3x to ensure triplicate readings of optical density for each data point. 
	NOTE: The standard deviation of experimental data points should always be within &#177;5% of the average.</li>
</ol>

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<li>Zhang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Naja+mosambica+mossambica+cobra+cardiotoxin+targets+mitochondria+to+disrupt+mitochondrial+membrane+structure+and+function%5BTitle%5D)+AND+%22Toxins%22%5BJournal%5D)">Naja mosambica mossambica cobra cardiotoxin targets mitochondria to disrupt mitochondrial membrane structure and function.</a> Toxins. 11, 152(2019).</li>
<li>Gasanov, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Naja+naja+oxiana+cobra+venom+cytotoxins+CTI+and+CTII+disrupt+mitochondrial+membrane+integrity%3A+implications+for+basic+three-fingered+cytotoxins%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Naja naja oxiana cobra venom cytotoxins CTI and CTII disrupt mitochondrial membrane integrity: implications for basic three-fingered cytotoxins.</a> PLoS One. 10 (6), 1-28 (2015).</li>
<li>Akosah, Y., Yang, J., Pavlov, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inorganic+polyphosphate+and+ion+transport+across+biological+membranes%5BTitle%5D)+AND+%22Biochem+Soc+Trans%22%5BJournal%5D)">Inorganic polyphosphate and ion transport across biological membranes.</a> Biochem Soc Trans. 52 (2), 671-679 (2024).</li>
<li>Weast, R. C. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aWeast%2C+RC+%22CRC+Handbook+of+Chemistry+and+Physics%22">CRC Handbook of Chemistry and Physics</a>. , 62nd ed, CRC Press. (1982).</li>
<li>Lemmon, M. A., Ferguson, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Signal-dependent+membrane+targeting+by+pleckstrin+homology+%28PH%29+domains%5BTitle%5D)+AND+%22Biochem+J%22%5BJournal%5D)">Signal-dependent membrane targeting by pleckstrin homology (PH) domains.</a> Biochem J. 350 (Pt 1), 1-18 (2000).</li>
<li>Ford, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Curvature+of+clathrin-coated+pits+driven+by+epsin%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Curvature of clathrin-coated pits driven by epsin.</a> Nature. 419 (6905), 361-366 (2002).</li>
<li>Khan, S. A., Ilies, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+phospholipase+A2+superfamily%3A+structure%2C+isozymes%2C+catalysis%2C+physiologic+and+pathologic+roles%5BTitle%5D)+AND+%22Int+J+Mol+Sci%22%5BJournal%5D)">The phospholipase A2 superfamily: structure, isozymes, catalysis, physiologic and pathologic roles.</a> Int J Mol Sci. 24 (2), 1353(2023).</li>
<li>De Oliveira, A. L. N., Lacerda, M. T., Ramos, M. J., Pedro, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Viper+venom+phospholipase+A2+database%3A+the+structural+and+functional+anatomy+of+a+primary+toxin+in+envenomation%5BTitle%5D)+AND+%22Toxins%22%5BJournal%5D)">Viper venom phospholipase A2 database: the structural and functional anatomy of a primary toxin in envenomation.</a> Toxins. 16 (2), 71(2024).</li>
<li>Hayashi, D., Dennis, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differentiating+human+phospholipase+A2%27s+activity+toward+phosphatidylinositol%2C+phosphatidylinositol+phosphate+and+phosphatidylinositol+bisphosphate%5BTitle%5D)+AND+%22Biochim+Biophys+Acta+Mol+Cell+Biol+Lipids%22%5BJournal%5D)">Differentiating human phospholipase A2's activity toward phosphatidylinositol, phosphatidylinositol phosphate and phosphatidylinositol bisphosphate.</a> Biochim Biophys Acta Mol Cell Biol Lipids. 1869, 159527(2024).</li>
<li>Alekseeva, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heterodimeric+V.+nikolskii+phospholipases+A2+induce+aggregation+of+the+lipid+bilayer%5BTitle%5D)+AND+%22Toxicon%22%5BJournal%5D)">Heterodimeric V. nikolskii phospholipases A2 induce aggregation of the lipid bilayer.</a> Toxicon. 133, 169-179 (2017).</li>
<li>Burke, J. E., Dennis, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phospholipase+A2+structure%2Ffunction%2C+mechanism%2C+and+signaling%5BTitle%5D)+AND+%22J+Lipid+Res%22%5BJournal%5D)">Phospholipase A2 structure/function, mechanism, and signaling.</a> J Lipid Res. 50, S237-S242 (2009).</li>
<li>Dennis, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phospholipase+A2+enzymes%3A+physical+structure%2C+biological+function%2C+disease+implication%2C+chemical+inhibition%2C+and+therapeutic+intervention%5BTitle%5D)+AND+%22Chem+Rev%22%5BJournal%5D)">Phospholipase A2 enzymes: physical structure, biological function, disease implication, chemical inhibition, and therapeutic intervention.</a> Chem Rev. 111 (10), 6130-6185 (2011).</li>
<li>Gasanov, S. E., Kim, A. A., Yaguzhinski, L. S., Dagda, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Non-bilayer+structures+in+mitochondrial+membranes+regulate+ATP+synthase+activity%5BTitle%5D)+AND+%22Biochim+Biophys+Acta+Biomembr%22%5BJournal%5D)">Non-bilayer structures in mitochondrial membranes regulate ATP synthase activity.</a> Biochim Biophys Acta Biomembr. 1860, 586-599 (2018).</li>
<li>Aripov, T. F., Gasanov, S. E., Salakhutdinov, B. A., Rozenshtein, I. A., Kamaev, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Central+Asian+cobra+venom+cytotoxins-induced+aggregation%2C+permeability+and+fusion+of+liposomes%5BTitle%5D)+AND+%22Gen+Physiol+Biophys%22%5BJournal%5D)">Central Asian cobra venom cytotoxins-induced aggregation, permeability and fusion of liposomes.</a> Gen Physiol Biophys. 8, 459-474 (1989).</li>
</ol>

The authors have no conflicts of interest to disclose.

In this study, we investigated whether phosphate groups covalently bound to the inositol ring in phosphatidylinositol (PI) can prevent the degradation of phospholipid membranes by phospholipase A2 (PLA2) enzyme. To assess the progression of phospholipid membrane degradation caused by the hydrolytic activity of PLA2, we designed a simple yet highly sensitive and accurate method to measure changes in membrane permeability, which should increase as structural degradation progresses.
The method is based on a ligand substitution reaction involving the hexaaquacopper(II) complex, [Cu(H2O)6]2+, suspended in the aqueous environment outside phospholipid liposomes, while ammonia (NH3) is encapsulated inside the liposomes. Ammonia has a higher affinity for Cu2+ than water molecules, but NH3 must permeate through the liposomal membrane to access the external aqueous phase, where it substitutes H2O molecules in [Cu(H2O)6]2+. If the structural integrity of the liposomal membrane is compromised due to PLA2-catalyzed phospholipid hydrolysis, increased membrane permeability to NH3 will facilitate ligand substitution, leading to a color change from the pale blue of [Cu(H2O)6]2+ to the dark blue of [Cu(H2O)2(NH3)4]2+. We have monitored this transformation quantitatively by employing spectrophotometry to record the change in optical density at 600 nm.
We have previously used 1H-NMR with paramagnetic ion ferrocyanide, [Fe(CN)6]3-, to study changes in membrane permeability affected by HDP-2P24 and by cobra venom cardiotoxins24,25,26,27,28,39. We have also used a range of fluorescent and phosphorescent probes to study the effects of cobra venom cardiotoxins on the permeability of liposomal membranes20,23,40. However, ferrocyanide and fluorescent/phosphorescent probes are large non-physiological molecular entities that penetrate lipid membranes when membrane integrity is significantly damaged. In contrast, our novel ligand substitution assay utilizes NH3&#160;-- a small, natural cellular metabolite capable of permeating lipid membranes at subtle lipid-packing perturbations. This enhanced sensitivity of our novel membrane permeability method is evidenced by its ability to detect changes in membrane permeability at an HDP-2P/lipid molar ratio of 0.001, compared to 0.004 required for &#185;H-NMR/ferrocyanide detection23.
In the same study, we used erythrosine phosphorescence quenching by ferrocene to examine the effects of HDP-2P on intermembrane lipid exchange, assessed by the change in the ratio of erythrosine lifetime in test samples (&#964;n) to that in the control sample (&#964;0)23. At an HDP-2P/lipid molar ratio of 0.004, the &#964;n/&#964;0 value decreased to 0.423, indicating a state of membrane fluidity permeable to divalent cations24. Thus, our NH3-based ligand substitution assay detects membrane permeability at an HDP-2P/lipid molar ratio of 0.001, whereas conventional assays (1H-NMR/ferrocyanide and phosphorescence probe quenching) detect membrane permeability only at a molar ratio of 0.004. This demonstrates that our NH3-based assay is four times more sensitive than conventional assays.
The exceptional sensitivity of our NH3-based assay yielded permeability data that closely correlated with hydrolytic activity measurements, with only one apparent discrepancy: PC+PI-4-P liposomes showed insignificant hydrolysis without permeability changes, suggesting that minimal local lysophospholipid/fatty acid production does not affect the global lipid packing, which does not influence membrane structural integrity and permeability. Thus, this study demonstrated that the ligand substitution reaction in a complex ion system, involving a ligand encapsulated within the inner volume of liposomes, produced results that excellently aligned with the hydrolytic activity data of HDP-2P. These findings, in turn, were in strong agreement with in-silico studies on the binding of PI and phosphoinositides to the molecular surface of HDP-2P.
Overall, the AutoDock simulation in the present study revealed that PI binds to the active center at four out of nine identified binding sites, whereas PI-4-P binds to the active center at only one of the nine predicted sites. The hydrolytic activity of HDP-2P in PC+PI-4-P liposomes was approximately 5x lower than in PC+PI liposomes. Furthermore, in-silico studies predicted no binding of PI-4,5-P2 to the active center of HDP-2P. Consistently, PLA2 activity assays and ligand substitution reaction experiments showed that PI-4,5-P2 completely inhibited both phospholipid hydrolysis and the increase in membrane permeability, while PI-4-P showed less, non-significant hydrolytic activity and entirely prevented membrane permeability changes. These observations are in good agreement with in vitro activity studies of human cytosolic, calcium-independent, and secreted PLA2 enzymes toward PI, PI-4-P, and PI-4,5-P235. These studies showed that only Group IVA cytosolic PLA2 exhibited relatively high catalytic activity toward PI and PI-4-P, while its activity toward PI-4,5-P2 was barely detectable. All other tested PLA2 enzymes showed no activity toward PI-4,5-P235. This lack of activity has been attributed to the highly hydrophilic phosphate groups on the inositol ring, which interfere with productive interactions between the membrane surface and PLA2 enzymes35. We suggest that the outward extension of phosphate groups on the inositol ring into the solution and their ability to form ion-polar and hydrogen bonds with residues of HDP-2P&#39;s groove-like cavity likely prevents HDP-2P from approaching the membrane surface closely enough to exert its hydrolytic activity, thereby stabilizing the membrane and reducing degradation of phospholipid membranes by HDP-2P which can be detrimental to cells by activating cell death pathways. This mechanism seems to align with the potential protective role of phosphoinositides in disease conditions involving aberrant enzyme activity.
The strong correlation between results obtained from membrane permeability assays, hydrolytic activity measurements, and in silico molecular docking studies highlights the reliability of the ligand substitution reaction method. This approach, utilizing a ligand encapsulated within liposomes, proves to be an accurate and effective tool for assessing membrane permeability changes caused by structural perturbations in the stable bilayer organization of phospholipids. While we acknowledge that molecular docking studies using AutoDock presented in this paper do not provide a thorough understanding of how phospholipids can interact with HDP-2P in a dynamic and longitudinal manner, molecular dynamics studies need to be performed in the future to identify additional molecular mechanisms of how HDP-2 can permeabilize phospholipid bilayers composed of various phospholipids. It is important to note that performing molecular dynamics studies requires high computational resources, such as high-performance computers and clusters, which are not available at many academic institutions that have limited resources and research funding. Finally, we acknowledge the use of a rigid receptor(HDP-2P) in the present techniques paper and the lack of a solvent/membrane environment. However, we want to emphasize that performing molecular dockings of HDP-2P with various phospholipids using a rigid receptor is the conventional approach to do initial docking studies to identify a first pass of docked candidate structures which can be followed up in the future with a flexible receptor once the critical amino acid residues within the catalytic side that bind to substrates are identified to focus on molecular regions that can be rendered flexible in follow-up molecular docking studies and in a solvated environment. Hence, leveraging collaborations with other institutions that have more resources can be a way to help corroborate some of the initial findings provided by AutoDock and identify additional molecular mechanisms of substrate-enzyme interactions.
In conclusion, the findings of this study demonstrate that the ligand substitution reaction method, utilizing a ligand encapsulated within liposomes, can be effectively applied to studies of membrane permeability in various membrane-mediated processes. These include ligand-receptor interactions, lipid-protein interactions, intermembrane exchange, membrane fusion and fission, and neuromuscular contractions, among others. Furthermore, this approach holds significant potential for investigating membranotropic pharmaceuticals. At the same time, we recognize that while novel methods in model membranes are invaluable for dissecting specific mechanisms of membrane interactions, their inherent simplifications limit their ability to fully replicate the structural, compositional, and functional complexity of biological membranes in vivo. It is therefore essential to interpret data from model systems with caution and to validate findings using cellular or organismal models wherever possible.

Phosphatidylinositol (PI) is an anionic phospholipid consisting of a phosphatidic acid backbone linked to inositol, a hexahydroxy six-carbon ring, via a phosphate group. Inositol can be reversibly converted into phosphoinositides by phosphorylation at the 2, 3, 4, 5, and 6 carbon positions1. PI and phosphoinositides are found in plasma membranes and organelles of various cells across animals, plants, and microorganisms. Seventy years after their discovery by Mable and Lowell Hokin2,3,4, PI and phosphoinositides remain the subject of extensive research aimed at understanding their molecular mechanisms and their relevance to human health5,6,7.
Apart from brain tissue, phosphoinositides are typically present at low concentrations, accounting for about 0.5 to 1% of the total lipids in the inner leaflet of the plasma membrane. The published data indicate that PI is unlikely to serve a major structural role under physiological conditions1. However, during disease states, the levels of PI and phosphoinositides increase, and they flip-flop to the outer leaflet of the plasma membrane, indicating that these phospholipids may be linked to disease resistance1. Although it has long been thought that PI plays a less significant physiological role compared to phosphoinositides, emerging evidence highlights the importance of PI-4-P and PI-4,5-P2 in various physiological processes1. These processes include intracellular signaling, intercellular signal transduction8,9,10, membrane trafficking11,12,13, modulation of gene expression1, as well as cell proliferation14 and survival1. Despite this, the molecular mechanisms by which PI and phosphoinositides drive these processes remain poorly understood.
PI and phosphoinositides are abundant in brain tissue cells, where they constitute approximately 10% of the total phospholipids, though their levels may transiently increase in the early stages of brain diseases suggesting that phosphoinositides may counteract the development of neurological pathologies1 such as multiple sclerosis and Alzheimer's disease triggered by axonal demyelination caused by aberrant PLA2s activity15,16,17. These enzymes belong to the various classes and groups including group IIA secretory phospholipase A2 (sPLA2)15. Elevated levels of this group of enzymes are found in the cerebrospinal fluid of patients with multiple sclerosis and Alzheimer's, where they contribute to the breakdown of myelin membranes, leading to demyelination15. It has also been reported that sPLA2s from snake venoms inhibit transmission at neuromuscular synapses18,19, causing central nervous system disorders and adversely affecting the peripheral neuromuscular system20.
In this study, we investigated whether PI and phosphoinositides can protect model phospholipid membranes from the effects of aberrantly expressed sPLA2 enzymes. Model membranes were made of phosphatidylcholine (PC), the most abundant phospholipid in all mammalian cell membranes21, enriched with a 20% molar ratio of either PI, PI-4-P, or PI-4,5-P2. To mimic the effects of aberrantly expressed sPLA2 enzymes on model membranes, we used the basic subunit HDP-2P of the group IIA secretory viper venom neurotoxic PLA2 enzyme22, whose hydrolytic activity has been previously studied on model myelin membranes23.
For this work, we developed a novel approach to investigate changes in the permeability of liposomal membranes affected by HDP-2P. The novel method is based on a ligand substitution reaction in CuSO4 solution outside the liposomes, involving an NH3 ligand trapped inside the liposomes. If HDP-2P-induced membrane permeability allows NH3 leakage from liposomes, the released ammonia will quantitatively substitute four water molecules in hydrated copper ions via the reaction: [Cu(H2O)6]2+ (aq) + 4NH3 (aq) <img alt="Static equilibrium; ΣFx=0; vector diagram; forces, balance, physics, analysis." src="/files/ftp_upload/68594/68594eq1.jpg" style="margin: auto;" data-altupdatedat="1783396841000" data-alt="Equation 1"> [Cu(H2O)2(NH3)4]2+ (aq) + 4H2O (l). This ligand exchange produces a characteristic increase in solution optical density measurable by spectrophotometry (λ = 600 nm), serving as our quantitative readout for membrane permeability changes. Our ligand substitution-based approach demonstrates superior sensitivity for assessing membrane permeability compared to conventional techniques like 1H-NMR23,24,25,26,27,28 and ion transport assays29, while offering the additional advantage of not requiring expensive instrumentation. This simple and cost-effective method, applied in conjunction with HDP-2P hydrolytic activity measurements and the in silico simulation of PI, PI-4-P, and PI-4,5-P2 binding to the molecular surface of HDP-2P, allowed us to conclude that the phosphates of the inositol ring in PI abolish the disruptive effects of HDP-2P on PC membranes enriched with PI-4-P or PI-4,5-P2. Should this finding be confirmed in further studies, the ability of the inositol ring phosphates in PI to protect against the harmful effects of abnormally acting cellular proteins, potentially contributing to membrane stability in disease conditions involving aberrant protein activity, may pave the way for the development of novel pharmaceuticals for the treatment of neurological disorders such as Alzheimer's disease and multiple sclerosis.

<table><tbody><tr><td>Basic subunit HDP-2P of V. nikolskii viper venom PLA2, HDP-2, 98.5% purity&amp;nbsp;</td><td>Institute of Bioorganic Chemistry, Moscow</td><td></td><td>HDP-2 isolated according to potocol in PMID:18083205. HDP-2P purified from HDP-2 by DEAE Sephadex A-50&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Cardiolipin (Heart, Bovine) (sodium salt),&gt;99%</td><td>Macklin</td><td>C916138</td><td></td></tr><tr><td>Coenzyme A, FUJIFILM Wako&amp;rsquo;s NEFA kit&amp;nbsp;</td><td>Lexington, MA, USA</td><td></td><td></td></tr><tr><td>Dialysis tubing cellulose membrane molecular weight cut-off 1.0 kDa</td><td>Sigma Aldrich Trading Co. Ltd., Shanghai, China&amp;nbsp;</td><td></td><td></td></tr><tr><td>Digitally controlled water-tank thermostat&amp;nbsp;</td><td>YoungGuangMing Medical Instruments Ltd. Co., Beijing, China&amp;nbsp;</td><td></td><td></td></tr><tr><td>Electronic high precision analytical balance FA2004&amp;nbsp;</td><td>Shanghai Maiyi Ltd. Co., China</td><td></td><td></td></tr><tr><td>JingHua 752 UV-Visible range spectrophotometer&amp;nbsp;</td><td>Shanghai Jinghua Instruments Co., Ltd., Shanghai, China</td><td></td><td></td></tr><tr><td>L-&amp;alpha;-phosphatidylcholine (Egg, Chicken),&amp;ge;95%</td><td>Macklin</td><td>L799415</td><td></td></tr><tr><td>L-&amp;alpha;-phosphatidylinositol (Liver, Bovine) (sodium salt),&amp;ge;99%</td><td>Macklin</td><td>L780292</td><td></td></tr><tr><td>L-&amp;alpha;-phosphatidylinositol-4,5-bisphosphate (Brain, Porcine) (ammonium salt),&amp;ge;99%</td><td>Macklin</td><td>L996223</td><td></td></tr><tr><td>L-&amp;alpha;-phosphatidylinositol-4-phosphate (Brain, Porcine) (ammonium salt), 99%</td><td>Macklin</td><td>L970250</td><td></td></tr><tr><td>L-&amp;alpha;-phosphatidylserine (Brain, Porcine) (sodium salt),&gt;99%</td><td>Macklin</td><td>L864755</td><td></td></tr><tr><td>Magnetic Stirrer JB-3&amp;nbsp;</td><td>Shanghai Leici JB-3</td><td></td><td></td></tr><tr><td>pH Meter PHS-3C&amp;nbsp;</td><td>Shanghai INESA Scientific Instruments Co., Ltd., China</td><td></td><td></td></tr><tr><td>TGL-16B microcentrifuge&amp;nbsp;</td><td>Shanghai 3 Anting Co., Shanghai, China</td><td></td><td></td></tr><tr><td>Ultrasonic Dispenser Yt-JY96-IIN&amp;nbsp;</td><td>Shanghai Yetuo technology Co., Ltd., China</td><td></td><td></td></tr><tr><td>Vacuum Pump TW-1M</td><td>Tingwei Co. Ltd., Wenling, China</td><td></td><td></td></tr></tbody></table>

a liposome membrane permeability assay for investigating the effects of phosphatidylinositol phosphate groups on membranotropic action of venom pla2

A Liposome Membrane Permeability Assay for Investigating the Effects of Phosphatidylinositol Phosphate Groups on Membranotropic Action of Venom PLA2

We developed a novel, cost-effective, and highly sensitive assay for assessing liposomal membrane permeability based on ligand substitution reactions involving ligands encapsulated in the inner volume of liposomes, which we combined with a hydrolytic activity assay (using coenzyme A acylation) and molecular modeling simulation to investigate whether phosphates of the inositol ring in phosphatidylinositol protect membranes from the damaging effects of aberrantly expressed enzymes. Aberrant expression of phospholipases A2 (PLA2) leads to demyelination of axons that triggers the development of multiple sclerosis and Alzheimer&#39;s disease. To mimic the effects of aberrantly expressed PLA2, we used the basic subunit of viper venom PLA2, HDP-2P. The HDP-2P was tested on phosphatidylcholine (PC) liposomes enriched with phosphatidylinositol, phosphatidylinositol-4-phosphate (PI-4-P), or phosphatidylinositol-4,5-diphosphate (PI-4,5-P2). While HDP-2P showed minimal hydrolytic activity and only slightly increased permeability in PC liposomes, enrichment of PC liposomes with PI enhanced both hydrolysis and permeability. Remarkably, PI-4-P and PI-4,5-P2&#160;dramatically inhibited HDP-2P&#39;s hydrolytic activity and completely reduced permeability. Molecular modeling simulations revealed that phosphates of the inositol ring in phosphatidylinositol sterically block phospholipid access to HDP-2P&#39;s active site, preventing hydrolysis. Both in vitro and in silico data suggest that phosphorylated phosphatidylinositols are associated with reduced PLA2&#160;activity, indicating a potential mechanism for mitigating excessive PLA2 activity -- presumably as a means of protecting cell membranes from degeneration.

We present a cost-effective method for assessing liposomal membrane permeability with superior sensitivity and resolution over conventional techniques. Using a novel encapsulated ligand substitution reaction, this technique detects phospholipase A2-induced changes in model membranes. Easily integrated with standard analyses, this accessible approach enhances resolution for studying membrane biophysics and toxin interactions.

We developed a novel, cost-effective, and highly sensitive assay for assessing liposomal membrane permeability based on ligand substitution reactions ...

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Research

JoVE Journal

Biochemistry

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Single-molecule Super-resolution Imaging of Phosphatidylinositol 4,5-bisphosphate in the Plasma Membrane with Novel Fluorescent Probes

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

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

Learn a sensitive liposome membrane permeability assay for studying phospholipase A2 effects on model membranes. This cost-effective method enhances resolution in membrane biophysics and toxin interaction research.

A Liposome Membrane Permeability Assay for Investigating the Effects of Phosphatidylinositol Phosphate Groups on Membranotropic Action of Venom PLA2 (Video) | JoVE Journal

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