Diffusion Through a Membrane State Lab

Part 1 – Build a model cell and demonstrate diffusion across a selectively permeable membrane

*Binds to helical structure of starch (iodines are the faded purple spheres above), turning the substance or solution a blue/black, but only binds because structure is helical. Therefore, will it bind to glucose? explain

Lugol’s solution (essentially iodine)

No, it isn’t helical and therefore the solution will not turn blue-black.

Diffusion Through a Membrane State Lab

Part 1 – Build a model cell and demonstrate diffusion across a selectively permeable membrane

Conclusion: Lugol’s solution is a starch indicator

Lugol’s solution (essentially iodine)

What is lugol’s iodine solution?

Diffusion Through a Membrane State Lab

Part 1 – Build a model cell and demonstrate diffusion across a selectively permeable membrane

Dialysis tubing (see below)

1. Making the model “cell”

A synthetic membrane that has microscopic pores (holes) that only let molecules of a certain size or smaller to pass. The one we are using only allows monomers or smaller molecules to pass.

Diffusion Through a Membrane State Lab

Part 1 – Build a model cell and demonstrate diffusion across a selectively permeable membrane

Dialysis tubing

If you soak it in water, you will be able to open up the tubing to put solution inside. Before you can do this, however, you must…

1. Making the model “cell”

A synthetic membrane that has microscopic pores (holes) that only let molecules of a certain size or smaller to pass. The one we are using only allows monomers or smaller molecules to pass.

Diffusion Through a Membrane State Lab

Part 1 – Build a model cell and demonstrate diffusion across a selectively permeable membrane

Dialysis tubing

1. Making the model “cell”

A synthetic membrane that has microscopic pores (holes) that only let molecules of a certain size or smaller to pass. The one we are using only allows monomers or smaller molecules to pass.

Diffusion Through a Membrane State Lab

Part 1 – Build a model cell and demonstrate diffusion across a selectively permeable membrane

Dialysis tubing

Artificial Cell

Then put glucose and starch solution in bag

Glu

cose

, starch

, w

ate

r

1. Making the model “cell”

A synthetic membrane that has microscopic pores (holes) that only let molecules of a certain size or smaller to pass. The one we are using only allows monomers or smaller molecules to pass.

Diffusion Through a Membrane State Lab

Part 1 – Build a model cell and demonstrate diffusion across a selectively permeable membrane

2. Place the model cell into a solution containing lugol’s (iodine).

Artificial Cell

Glu

cose

, starch

, w

ate

r

Predict what should happen:1. The glucose should diffuse out down its concentration gradient and the iodine should diffuse in down its concentration gradient since both can pass through the membrane pores. The starch should stay in – too large to pass through pores. Water should diffuse in and out – direction will depend on how much solute you dissolved in the water.2. How will you observe that the iodine went in?

I-

I-

I-

I-

I-

I-

I-The outside should remain amber (the color of lugols) since there is no starch present to react with it.

4. How do we observe that glucose diffused out?

The iodine should bind to the helical starch structure causing the solution inside the dialysis tubing to turn blue-black.3. How will you observe that the starch did

not diffuse out?

Diffusion Through a Membrane State Lab

Part 1 – Build a model cell and demonstrate diffusion across a selectively permeable membrane

Use Benedict’s solution- Benedict’s is a molecule that reflects blue light that will react with monosaccharides, lactose and maltose and convert into a molecule that reflects yellow/orange light. The reaction is endergonic. How we can we get it to happen?

2. MUST HEAT!!1. Add a few drops of benedict’s to the solution

3. Turns orange if monosaccharides, lactose or maltose is present

You must heat the Benedicts in the presence of the sugars. Input ENERGY!

Diffusion Through a Membrane State Lab

Part 1 – Build a model cell and demonstrate diffusion across a selectively permeable membrane

2. Place the model cell into a solution containing lugol’s (iodine).

Artificial Cell

Glu

cose

, starch

, w

ate

r

How do we observe that glucose diffused out?

Take a sample of the outer solution into a clean test tube, add a few drops of benedicts, and heat it up. If it turns orange then it diffused out.

I-

I-

I-

I-

I-

I-

I-

Time to do it!

Diffusion Through a Membrane State Lab

Part 1 – Build a model cell and demonstrate diffusion across a selectively permeable membrane

Artificial Cell

Glu

cose

, starch

, w

ate

r

Take a sample of the outer solution into a clean test tube, add a few drops of benedicts, and heat it up. If it turns orange then it diffused out.

I-

I-

I-

I-

I-

I-

I-

Time to do it!

Q. You have a solution containing 1mM catalase. The pH of the solution is around 6.8 and the salt (NaCl) concentration is around 400 mM (milli Molar). However, you have previously determined that this protein functions best at a pH of 5.7 and a salt concentration of 250 mM. Explain how you can get your protein into the desired conditions without changing the concentration of the enzyme much.

Diffusion Through a Membrane State Lab

Part 1 – Build a model cell and demonstrate diffusion across a selectively permeable membrane

Q. You have a solution containing 1mM catalase. The pH of the solution is around 6.8 and the salt (NaCl) concentration is around 400 mM (milli Molar). However, you have previously determined that this protein functions best at a pH of 5.7 and a salt concentration of 250 mM. Explain how you can get your protein into the desired conditions without changing the concentration of the enzyme much.

Diffusion Through a Membrane State Lab

Part 1 – Build a model cell and demonstrate diffusion across a selectively permeable membrane

Cata

lase

solu

tion

with

pH

6.8

and

40

0m

M N

aC

l

A. Use a dialysis tube. Put your protein solution into the dialysis membrane and tie it off. Place the membrane into the desired solution. Salt will diffuse out and H+ will diffuse in. Protein will stay inside as it is too large to fit through pores…

This is called Buffer Exchange

Q. You have a solution containing 1mM catalase. The pH of the solution is around 6.8 and the salt (NaCl) concentration is around 400 mM (milliMolar). However, you have previously determined that this protein functions best at a pH of 5.7 and a salt concentration of 250 mM. Explain how you can get your protein into the desired conditions without changing the concentration of the enzyme much.

Aqueous solution with pH 5.7 and 250 mM NaCl

Diffusion Through a Membrane State Lab

Part 2 – Demonstrate diffusion of water across a real membrane (osmosis)

Red onion cells in water (turgid)

You want to observe the effect of salt on osmosis across the plasma membrane of red onion cells under the light microscope. How will you do this? You look at the cells in water first. This is your…______________.

What will you do next?

Add salt to the solution that the cells are in. What would you expect to happen and why?

control group

All of the cells should be _____________.

turgid

Diffusion Through a Membrane State Lab

Part 2 – Demonstrate diffusion of water across a real membrane (osmosis)

Red onion cells in water (turgid)plasmolysis

Osmosis (diffusion of water) should occur from inside the cell [high] to outside [low] since the salt CANNOT cross the membrane and will cause the water concentration outside the cell to now lower than inside…you should observe plasmolysis. How do we make these cells turgid again?

Add salt (NaCl)

Diffusion Through a Membrane State Lab

Part 2 – Demonstrate diffusion of water across a real membrane (osmosis)

Red onion cells in water (turgid)plasmolysis

Put them back into tap water (get rid of the salt).

Remove salt (NaCl)

Diffusion Through a Membrane State Lab

Part 2 – Demonstrate diffusion of water across a real membrane (osmosis)

Red onion cells in water (turgid)plasmolysis

Remember: This is a real cell membrane (phospholipid bilayer with integral membrane protein), which will only allow small hydrophobic molecules to pass (you should know why). Sodium chloride (Na+, Cl-) is charged and hence hydrophilic.

Remove salt (NaCl)