Permeability experiments

Experiment 4: Exploring slower moving particles

Remember that in Experiment 3, we saw water moving across because of the osmotic pressure difference between the non-penetrating (non-permeating) NaCl outside the cell and the non-permeating proteins and other large molecules inside the cell.

In 100% Physiological Saline (which is 150 mM NaCl), the osmotic pressure of water in the presence of the non-permeating NaCl outside the cell equals the osmotic pressure of water in the presence of the non-permeating proteins and other large molecules inside the cell.

So then there is no net water movement.

In all the other saline solutions in Experiments 2 and 3, we reduced the [NaCl] by replacing some of the NaCl with water. Water is permeating, so the osmotic pressure was now higher for water to flood into the cell.

Water can flood into the cell because of its access to the aquaporin channels that allow it to get into the cell without having to cross through the lipid barrier.

What if we replaced only some of the NaCl with water and some of the NaCl with something else which, like water, can penetrate BUT does not have special channels through which the molecules can zip across the membrane? Instead, the molecule has to cross through the lipid bilayer of the cell membrane - which it can do because it can dissolve in lipid, unlike water. That means it too will cross over as will the water, but not as fast as the water.

Therefore lysis will occur, but slower.

That's what we will study now. And since we are using particles that can get across only through the cell membrane, we can then study how the properties of a molecule - namely, mass and lipid solubility - affect how quickly it can dissolve in and cross over through the lipid membrane.

We might expect that a large lipid-soluble molecule will get across more slowly than a small lipid-soluble molecule - and we will test that in Experiment 4.1.

And we might expect that a highly lipid-soluble molecule will get across more quickly than a poorly lipid-soluble molecule - and we will test that in Experiment 4.2.

4.1. Effect of a penetrating solute's size on rate of water entry

Instructions

1. Add a solute and some saline solution to the test tube.
2. Add a few drops of blood.
3. In the table below, record how long lysis takes to occur for each solution.
4. Once you've completed trial 1, press "Simulate remaining trials" below to automatically fill out the remaining values.

Simulation

To interpret the results you obtained in this experiment, you need to know the size of the penetrating solutes used here.

Their molecular weights are:

• Ethylene Glycol = 62 Daltons
• Di-Ethylene Glycol = 106 Daltons

Both have very similar lipid solubilities, determined from the Partition Coefficient (P_oct:water), a measure of solubility in oil or ether versus water and therefore a measure of how easily they penetrate a lipid membrane. Log P_oct:water for Ethylene Glycol = -1.36 and for Di-Ethylene Glycol = -1.32. The more positive the value, the higher the solubility of the substance in oil relative to water. The two values are very similar, meaning that Lipid solubility is not a confounding factor for this experiment.

4.2. Effect of a penetrating solute's lipid solubility on rate of water entry

Instructions:

1. Add the Propylene Glycol and saline solution to the test tube.
2. Add a few drops of blood.
3. In the table below, record how long lysis takes to occur for each solution.
4. Once you've completed trial 1, press "Simulate remaining trials" below to automatically fill out the remaining values.

Measurements for Ethylene Glycol are automatically populated from the previous experiment.

To interpret the results you need to know the lipid solubility of the two solutes considered here (Ethylene Glycol and Propylene Glycol). This will be taken from a standard measure known as the Partition Coefficient, a measure of solubility in oil or ether versus water and therefore a measure of how easily they penetrate a lipid membrane.

Log P_oct:water: Propylene Glycol = -0.92 and Ethylene Glycol = -1.36. (The more positive the value, the higher the solubility of the substance in oil relative to water.)

The two have very similar molecular weights (Ethylene Glycol = 62 and Propylene Glycol = 76), so the data are not confounded by that property.