Permeability experiments

Experiment 5: Exploring facilitated transport

Experiment 5 consists of two experiments – Experiments 5.1 and 5.2. In Experiment 5.1 the objective is to examine how membrane transporters can facilitate the speed of penetration of solutes into a cell and In Experiment 5.2 the objective is to examine how such facilitated diffusion interacts with solute size.

Again, as in Experiment 4, we will replace the water in the external solution with some other molecule that will penetrate but either through a facilitated diffusion process, which allows it to go across the membrane faster than having to dissolve in and cross through the lipid bilayer, or by passive diffusion in which the molecule has to dissolve in and cross through the lipid bilayer

5.1. Effect of facilitated diffusion on rate of water entry

In Experiment 5.1 you will compare the speed of lysis when water is replaced with either Methyl Urea or with Ethylene Glycol.

Methyl Urea and Ethylene Glycol have very similar MWs and Partition Coefficients, a measure of their solubility in oil or ether versus water and therefore a measure of how easily they penetrate a lipid membrane.

Molecular Weight: Methyl Urea = 74 daltons, Ethylene Glycol = 62 daltons;

Partition Coefficients (Log P oct:water): Methyl Urea = -1.76 and Ethylene Glycol = -1.36. (The more positive the value, the higher the partition coefficient, and the higher the solubility of the substance in oil relative to water.)

However, mammalian RBCs have a urea transporter in their membranes and this is responsible for >70% of the permeation of Methyl Urea through the RBC membrane. Ethylene Glycol has no such transporter and also cannot avail itself of the Urea transporter. This will allow you to compare these two compounds to determine the effect of transport facilitation on the diffusion rate.

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.

Note: If lysis doesn't occur within 90 seconds, we assume it will never occur, and you should enter a value of 90 seconds for that trial.

5.2. Effect of varying mass on two molecules with facilitated diffusion

In Experiment 5.2, we will compare two molecules that have facilitated diffusion processes to assist them to get across the cell membrane. These molecules are Methyl Urea and Glucose both using facilitated diffusion to penetrate through the RBC membrane. However, they vary markedly in size: Molecular Weight: Methyl Urea = 74 daltons, Glucose = 180 daltons;

This will allow you to determine the effect of size on transport via facilitated diffusion.

Instructions:

1. Add the Glucose 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.

Note: If lysis doesn't occur within 90 seconds, we assume it will never occur, and you should enter a value of 90 seconds for that trial.

Measurements for Urea are automatically populated from the previous experiment.