Exercise 3: Movement Across Cell Membranes Reading: Silverthorn 4 th ed, pg. 132-136, 153 159; Silverthorn 5 th ed, pg. 136 140. A selectively permeable barrier is one of the defining features of a living cell. The cell membrane and the associated transport proteins found in the membrane are responsible for regulating the movement of hundreds, if not thousands, of different types of molecules into and out of the cell. All molecular motion is influenced by diffusion, which is the tendency for particles to spread from higher concentrations to lower concentrations until they are evenly distributed, or reach equilibrium. This movement towards equilibrium is the driving force behind a majority of physiological processes, from neuronal impulses to renal function. Today we will investigate the movement of several different types of molecules across a cell membrane, including water, and we will examine the physical properties of these different molecules to see how they influence this movement. Today s Objectives 1. Observe the movement of water across a membrane in model cells (decalcified eggs) and examine the environmental conditions that determine the direction of osmosis. 2. Compare the rate of osmosis when the concentration gradient varies. 3. Observe the effect of molecular size on the movement of solutes across a membrane. 4. Observe the effect of polarity on the movement of solutes across a membrane. Osmosis When a selectively permeable membrane can inhibit the movement of some types of solutes and a concentration gradient exists, water will diffuse towards the higher solute concentration to equalize the concentration on both sides of the membrane. If you have a hard time remembering which way water moves in the presence of an osmotic imbalance (concentration gradient), just remember that SOLUTES SUCK! Water will always be drawn towards more concentrated solutes. A solution can be described by its tonicity. Tonicity describes how a solution affects cell volume. A hypotonic solution will cause a cell to stretch and swell as water enters because it has a lower solute concentration (hypo = below) than a cell. A hypertonic solution will draw water out of a cell and make it shrink because it has a
higher relative solute concentration(hyper = above). An isotonic solution produces no change cell volume because there is no difference in concentration (iso = same); an isotonic solution is said to be in osmotic equilibrium with the cell. You have observed this phenomenon when your fingers get wrinkled after soaking in bath water, a hypotonic solution. Your skin wrinkles because the skin cells swell with water and your skin becomes too large to fit smoothly on your finger tips. Conversely, your skin may feel dry and tight after a day swimming in the ocean, a hypertonic solution, as the salt from the sea water draws water out of your skin cells. In the following experiment, you will be using decalcified eggs as model cells. The eggs have been treated with vinegar to remove the calcium from the shell, leaving behind a membrane that is permeable to water (solvent), but not to other molecules (solutes). Materials: 5 decalcified eggs 5 weigh boats, one for each egg 3 Beakers or plastic containers with solutions A, B, & C 2 Beakers or plastic containers with solutions 1 and 2 Paper towel Gram scale Procedure IA: Determining the Tonicity of Extracellular Fluid: 1. Fill three beakers with enough of solution A, B, or C to cover an egg, about 300 ml. 2. Obtain three decalcified eggs. Gently dry and weigh each egg before immersing it in Solution A, B, or C. Dry the egg by gently rolling it on a paper towel. Do not dry the egg for too long because the paper towel will begin to draw out water from inside the egg and will change the weight of the egg. Record the weight of each egg in Table 1. 3. Let the three eggs soak in solutions A, B, and C for 20 minutes. Go on to Procedure IB while you are waiting. The soak time should be at least 20 minutes, but can be longer if it is more convenient. 4. After at least 20 minutes, dry and weigh each egg and record your results. Use a "+" sign to indicate an increase in weight and a " " sign to indicate a decrease in weight. 5. The change in weight reflects the movement of water into or out of the egg. Based on the movement of water, determine if the Solutions A, B, and C are hypotonic, isotonic, or hypertonic.
Egg in Solution A Egg in Solution B Egg in Solution C Table 1. Weight of Eggs and Tonicity of Solutions A, B, and C Weight Before Soaking (g) Weight After Soaking (g) Difference in Weight (g) Tonicity of Solution (hyper-, hypo-, or iso-) Questions: 1. Compare the presoak weight for eggs A, B, and C with their weights after the 20 minute soak. What is the tonicity of each solution? 2. Explain the physiological cause of the change in the weight of each egg. 3. What physical conditions are required to cause the water to move in a particular direction, into or out of the egg? 4. Explain why osmotic homeostasis must be closely regulated for the all the body fluid compartments. Procedure IB: The rate of osmosis is dependent on the difference in solute concentration across the membrane. Water will diffuse more quickly if the concentration gradient is steeper (the difference in concentration is greater). In this experiment, you will be soaking your eggs in two different hypotonic solutions and measuring the rates of osmosis. 1. Gently dry and weigh the last two eggs. Record the results in Table 2 as your zero time points. 2. Immerse one egg in solution 1 and the other in solution 2. Dry and reweigh each egg after two minutes. Return the eggs to their solutions after weighing. Reweigh the eggs every 2 minutes for about a half hour. 3. Plot your results on graph paper (not binder paper!). Plot the data for both solutions on the same graph using different symbols or colors. You do not need to begin your Y-axis at zero. 4. Compare the rate of water movement into each egg by calculating the slope of each line using the formula below, where Y is the change in the egg weight and X is the change in the time. slope = Y X
Table 2. Weight of Egg every 2 minutes in Solutions 1 and 2 Solution 1 Solution 2 Time (minutes) Egg Weight (grams) Change in Wt (Relative to 0 Time Point) Egg Weight (grams) Change in Wt (Relative to 0 Time Point) 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Questions 5. How does the rate of osmosis differ for the two solutions? 6. Which solution is more hypotonic, solution 1 or 2? Membrane Permeability We have just observed how osmosis can change the volume of a "cell" depending on the tonicity of the external environment. Water moves until either osmotic equilibrium is achieved or osmotic pressure prevents further osmosis from occurring.
The diffusion of solutes into or out of a cell also influences osmosis. Diffusion of a solute will occur if 1) there is a concentration gradient and 2) if a solute is penetrating, or able to cross the membrane. As a penetrating solute enters a cell, water follows the solute to maintain osmotic balance and the volume of the cell increases. If a solute is nonpenetrating, or unable to cross the membrane, then osmolarity does not change and osmosis does not occur. Figure 1 illustrates this principle. Penetration, however, is dependent on the permeability of the cell membrane to a particular solute, which is in turn dependent on the physical characteristics of that solute. In this part of today's exercise, we will examine how solute size and solute polarity influence membrane permeability. Figure 1. A cell is placed in an isotonic solution (a). Cell volume does not change initially (b), but the penetrating solute diffuses into the cell and changes the intracellular osmolarity (c). Water follows the penetrating solute to maintain osmotic balance, increasing cell volume (d). The Effect of Molecular Size In order to study the effect of molecular size on membrane permeability, you will place red blood cells into isosmotic solutions of alcohols of increasing molecular size. If an alcohol molecule is able to penetrate the membrane, diffusion will occur due to the concentration gradient and water will follow the alcohol molecule. The volume of the cell will increase until hemolysis occurs (the cell bursts open). The amount of time required for hemolysis to occur depends on how easily the solute can penetrate the cell membrane. Since these solutions are isosmotic, no lysis will occur if the alcohol molecules are unable to cross the membrane.
We will use three different alcohols: ethylene glycol, glycerol, and ribose (Figure 2). They differ in size, having 2,3 and 5 carbon atoms respectively. Any difference in observed hemolysis time must be due to differences in size. A Note to Clarify the Difference Between Isosmotic and Isotonic: The suffix "osmotic" is used to describe the relative concentration of a solution compared to a cell when the solute is penetrating, or able to enter the cell. The suffix "tonic" is used when a solute is nonpenetrating. More about this later. Materials and equipment Isosmotic Alcohol solutions (0.3M): Glycerol; Ethylene Glycol; and Ribose. Horse blood diluted with saline. Mix well before taking a sample! Disposable latex or nitrile gloves Procedure IIA Ethylene Glycol Glycerol Ribose Figure 2. Molecules of about the same polarity (one OH per carbon), but increasing size (number of carbon atoms). 1. Obtain one tube each containing 3 ml of Glycerol, Ethylene Glycol and Ribose solutions. 2. To each tube add two drops of horse blood. Mix by gently finger vortexing (instructor will demonstrate). Record the time the blood is added in the second column of Table 3. 3. The tubes should be cloudy immediately after the addition of blood. As hemolysis occurs, the solutions will become transparent. 4. Record the time when the tubes become transparent in the third column of Table 3. 5. Calculate the "Hemolysis Time" by subtracting the time the blood was added from the time the tube became transparent and record the number of minutes and seconds
Table 3. Effect of Molecular Size Data Molecule Time blood was added Time solution became transparent Hemolysis Time Carbon Atoms (size) Ethylene Glycol 2 Glycerol 3 Ribose 5 The Effect of Molecular Polarity Polarity is a chemical property that affects a molecule's solubility. Remember from our review of chemistry that polar molecules are more water soluble because water is itself a polar molecule. The uneven distribution of electrons create some areas on a molecule that are more negative which are balanced by other areas on a molecule that are more positive. The more negatively charged areas tend to associate with the H side of H 2 O, while the more positively charged areas tend to associate with the OH side of H 2 O. What does water solubility have to do with membrane permeability? Remember that cell membranes are composed of a lipid bilayer. A lipid bilayer is more permeable to hydrophobic, or fat soluble molecules. Conversely, a lipid bilayer is less permeable to hydrophilic, or water soluble molecules. Molecules with OH groups, Propyl Alcohol Glycerol like alcohols, tend to be polar because oxygen is such an electronegative atom. Figure 3. Molecules of about the same size (3 The number of OH groups on a molecule affects the degree of polarity a molecule will exhibit. Examine the structures of Propyl Alcohol and Glycerol in Figure 3. Note that both have a backbone of three carbon atoms and about the same overall size. The main difference between them is the number of polar OH groups: Propyl Alcohol has only one (C 3 H 7 OH), while Glycerol has three (C 3 H 5 OH 3 ), one for each carbon atom. This makes Glycerol a much more polar molecule than Propyl Alcohol. One would predict that cell membranes would be less permeable to more polar or hydrophilic molecules. These molecules will have a harder time crossing the membrane and have a longer hemolysis time.
Procedure IIB 1. Obtain two test tubes. Add 3 ml of Propyl alcohol to one and 3ml of Glycerol to the other. 2. To each tube add two drops of horse blood and mix by finger vortexing. Record the time the blood is added in Table 4. 3. The tubes should be cloudy immediately after the addition of blood. As hemolysis occurs, the solutions will become transparent. 4. Record the time when the tubes become transparent. 5. Calculate the "Hemolysis Time" by subtracting the time the blood was added from the time the tube became transparent and record the number of minutes. Table 4. Effect of Molecular Polarity Data Molecule Time blood was added Time solution became transparent Hemolysis Time Polar OH Groups Propyl Alcohol 1 Glycerol 3 Questions: 6. How do ethylene glycol, glycerol, and ribose molecules differ from each other? 7. How do propyl alcohol and glycerol differ from each other? 8. What is the purpose of having these solutions isosmotic? 9. What causes a blood solution to go from cloudy to clear? 10. Based on your data, how does a molecule s size affect its ability to cross a cell membrane? 11. Based on your data, how does a molecule s polarity affect its ability to cross a cell membrane?