Experiment Membrane Transport Objectives Referring to energy, what two ways can substances enter a cell?

What is active transport? What is passive transport? How is osmosis related to diffusion? How can we demonstrate active transport? How can we demonstrate Brownian movement? How can we demonstrate diffusion (2 ways)? How can we demonstrate osmosis (3 ways)? In terms of relationships between substances, how can we define hypertonic, isotoni c, and hypotonic? What is the relationship between the size of a molecule and its rate of diffusio n? ____________________________________________________________________________ Supplies Materials Needed

Active Transport: Bakers Yeast 0.75% Na2CO3 0.02% Neutral Red Erlenmeyer Flasks Flame or Hot Plate Brownian Movement: India Ink (Carmine Dye or Whole Milk may be substituted) Slides and Cover Slips Diffusion: Beaker of Distilled Water 1.5% Agar-agar (or Gelatin) in petri plate Potassium Permanganate (KMnO4) Crystals Potassium Dichromate (K2Cr2O7) Crystals Methylene Blue Crystals Metric Ruler (to measure in mm) OsmosisThistle Tube: Thistle Tube Osmometer

Salt, Sugar Distilled Water Scale Artificial Cell: Dialysis Tubing or Sacs Sugar String Beakers Distilled Water Scale Osmosis and Red Blood Cells: Distilled Water; Salt Solutions: 12-15% Salt Salt Solutions: 0.85% Salt (This is human, isotonic saline.) Blood (Human or Animal): Lancets and Alcohol if Human Blood is used Slides and Cover Slips Microscope ____________________________________________________________________________ Key Terms Active Transport Brownian Movement Cell Physiology Dialysis Diffusion Gradient Hypertonic Hypotonic Isotonic Membrane Transport Osmosis Passive Transport ____________________________________________________________________________ Introduction The individual cell is a dynamic microcosm, demonstrating in min iature all the processes and events that occur in the macrocosm, making the appa rent function of the whole organism the actual function of many individual cells working in unison. It is important that we understand how the individual cell, the microcos m, functions so that we can more fully understand how the organism as a whole, t he macrocosm, function. For instance, we can say that if an action potential (or nerve impulse) is to be generated in a part of the nervous system, a certain el ectrical stimulation must be present and certain ions must be moving through app ropriate channels. What we sometimes forget is that these eventsin this case the electrical stimulation and the ionic movementoccur at the cellular level. What se ems to be happening in the organism is happening only because the events are occ urring at the level of the individual cell. We could use similar analogies for e very system in the body. In lecture you examined the molecular intricate of the phospholipid bila yer known as the cell membrane, and you became aware that the cell membrane is s electively permeable, meaning that only certain substances can enter and leave t

he cell by freely crossing the membrane. You know, for instance, that the membrane is replete with channels, gate s, and carrier molecules that either facilitate, inhibit, or repel assorted ions and molecules as they randomly approach the demarcation barrier. This demarcati on barrier, the cell membrane, is functional in maintaining cellular integrity. You also know that since the cell is microcosm, ions and molecules must cross the barrier, both as nutrients entering the cell and as wastes leaving the cell. In cell physiology, we examine how events occur within the cell. Cellular functions follow the basic principles of physiology. Many of these functions do not lend themselves to easy demonstration, particularly this early in an introd uctory course. However, at this point we can demonstrate a number of functions d irectly related to membrane transport. And membrane transport is one of the main keys to cell physiology when we study the nervous system and the digestive syst em.) ____________________________________________________________________________ Preparation A. I. Background and Protocol

Explanation of Terms

1. Be certain you have a working knowledge of the following terms: Membrane Transport Active Transport Passive Transport Diffusion Osmosis Brownian Movement Gradient Membrane transport is any process, active or passive, by which a substance moves from one side of a membrane to the other side of the membrane. We have already established that the membrane acts as a barrier that can facilitate, inhibit, or repel substances, and yet certain substances must cross the barrier. Active transport requires cellular energy because the substance traversi ng the barrier either cannot do so without a push or cannot do so in sufficient quantity to maintain cellular integrity. Active transport is absolutely essentia l in maintaining the functioning organism. Active transport is accomplished via ionic pumps. Manu of our carrier systems work because of active transport. Gradi ents, which enable work to be done, are maintained because of active transport. Active transport can best be demonstrated by visualizing some very common events , both in the laboratory and in daily life. Passive transport requires no input of cellular energy. Several categori es of passive transport can be identified. Nevertheless, the most common readily identifiable form of passive transport is diffusion, the net movement of molecu les from an area of higher concentration to an area of lower concentration. Osmo sis is form of diffusion. In the human system, osmosis is the diffusion of water . Passive transport can be easily demonstrated by both diffusion and osmosis exp eriments. Brownian movement, while technically not diffusion, is covered here with the passive transport experiments because Brownian movement demonstrates molecu lar motion. It is precisely because of this innate movement that molecules are a ble to move passively from one point to the next pointincluding from one side of a membrane to the other side. A gradient is any difference in intensity between two sides of a demarca tion. For instance, a concentration gradient would be a difference in concentrat ions of a substance on each side of a demarcation. A physical barrier may or may not be present. We can also have temperature gradients, electrical gradients, f low gradients, and pressure gradients.

B.

General Procedural Points

Your instructor may set up parts of this experiment as a demonstra-tion. If so, answer the questions from your observations. 1. Work with a partner in Part II.

2. Divide the work between you and your partner in Part III so that each of you is doing one of the experiments. 3. If you use human blood, READ THE CAUTIONS GIVEN IN Anatomy of the Blood AND Blood Testing Procedures. ____________________________________________________________________________ Experimentation II. Active Transport

Active transport is the energy-requiring movement of a substance across a barrie r or a gradient. This transport could not take place without the input of energy . In the biological world, energy is supplied by the living cell. We can observe the principles of active transport by examining whether or not the uptake of a substance will take place in living and/or nonliving cells. Neutral red is a dye that is actively transported into the living yeast cell. Neutral red is red in an acidic solution and yellow in a basic solution. A sodium carbonate (Na2CO3) solution is basic. A. Experiment #1: Active Transport

1. Obtain two Erlenmeyer flasks, 2 g yeast, 50 ml solution carbonate, and 5 0 ml neutral red. 2. Into Flask #1 place half the yeast and half the sodium carbonate. Mix we ll and heat gently. Boil the solution for 2 or 3 minutes. When the solution is c ool, add about half the neutral red. The solution should be yellow. 3. To Flask #2 add the remaining Na2CO3 and neutral red. Gently swirl in th e remaining yeast and watch for a color change. Concept Check 1 Jot down your observations and discuss with your partner the differences in colo r between Flask #1 and Flask #2. B. Additional Observations

Concept Check 2 Discuss with your partner the involved in windmills and water pumps. What is the role of energy in these apparatuses? How can you relate these functions to the role of active transport in the cells? III. Passive Transport (Diffusion)

Passive transport is the movement of a substance across a membrane or a gradient without the expenditure of cellular energy. We have several experiments that de

monstrate passive transport. A. Experiment #2: Brownian Movement

All molecules with a temperature above absolute zero are in a state of constant motion. Because of this motion, molecules collide regularly and randomly. The ma nifestation of these collisions is called Brownian movement. Some disagreement e xists as to the exact nature of Brownian movement. Nevertheless, for our purpose s, the exact results are the same and we can see Brownian movement whenever we h ave a liquid mixture composed of differently sized molecules. Brownian movement is not actually a part of diffusion, but if the molecu les were not in motion, they could not diffuse across a membrane or a gradient. Thus, in order to understand diffusion we should gain some insight into molecula r movement. India ink is a suspension of carbon in water, along with ethyl alcohol a nd acetone. (Carmine dye and whole milk, the alternate substances for this exper iment, are also molecular suspensions.) 1. Obtain a slide, cover slip, and microscope.

2. Place a drop of India ink on a slide with a small drop of water. Cover carefully with a cover slip and allow the solution to rest for 2 or 3 minutes. 3. Observe the suspension under both low and high power. If possible, also observe the suspension under oil. Keep the light as low as possible so you manit ian maximum contrast. If your suspension is too dark, you may wish to start over using a larger drop of water. 4. Sketch what you see.

5.

Describe Brownian movement based on your observations.

B.

Experiment #3: Diffusion in Liquids

Diffusionthe net movement of molecules from an area of higher concentration to an area of lower concentrationcan be demonstrated in two different ways. 1. . Obtain a beaker of water and a crystal of potassium permanganate (KMnO4)

2. Drop the crystal into the water and watch the diffusion process. Use Figure 1 to sketch the initial diffusion path. Note the time and observe the beaker at 5-minute intervals. Note the time when you think diffusion is complete. How long did it take?

Figure 1 Diffusion in Water.

Table 1 Petri Plate Diffusion Time Time 15 min 30 min 45 min 60 min 75 min 90 min ____ KMnO4 K2Cr2O7 Methylene Blue

C.

Experiment #4: Diffusion In Solids

1. Obtain an agar-agar or gelatine petri plate and ruler. Also obtain equal crystals of potassium permanganate (KMnO4), potassium dichromate (K2Cr2O7), and methylene blue. 2. Construct an imaginary equilateral triangle on the petri plate and put o ne crustal at each angle, per Figure 2. At 15-minute intervals measure the radiu s (in millimeters) that each substance has diffused from the original crystal ma rk. Use Table 1 to record these distances. Figure 2 Petri Plate.

Concept Check 3 The molecular weight for KMnO4 is 158, for K2Cr2O7 is 294, and for methylene blu e is 320. In general terms, what is the relationship between the distance travel ed and the molecular weight of each crystal? [Challenge: Although your measurements are probably not exact enough to derive a mathematical formula, do you see that the diffusion rate of any substance might well be inversely proportional to the square root of the molecular weight?]

D.

Osmosis Overview

Osmosis is the movement of water from an area of higher water concentration, acr oss a selectively permeable membrane, to an area of lower water concentration. T hat is, from an area of lower solute concentration to an area of higher solute c oncentration. Movement is always toward equilibrium. Dilution is toward equilibr ium. Dialysis is the movement of a nonwater particle across a barrier. This m ovement is based on the size of the particle in relation to the size of the pore s within the barrier. In the living system this barrier is the cell membrane. We can perform three experiments to demonstrate the principles of osmosi s and dialysis. Work with a partner. Table 2 Artificial Cell Results ____ Concept Check 4 Explain your results by answering these questions: At what height did each solut ion stop rising? Then what happened? What differences did you notice between the sugar and salt osmometers? How do you explain those differences? If you had tas ted the water at the end of the experiment, what would you have noticed? How wou ld you explain this?

E. Experiment #6: Osmosis (Artificial Cell) This experiment is outlined in Figure 4 and Table 2 Figure 4 Artificial Cells.

1. Obtain 5 beakers, 5 artificial cells (dialysis tubing or sacs), string, sugar, scale. 2. Make 3 sugar solutions, one 40%, and one 10%. Set up 5 artificial cells.

Into cell #1 put some of the 40% solution, into cell #2 the 20% solution, into cell #3 the 10% solution, and into cell #4 and cell #5 put distilled water. 3. Take the 5 beakers. Into beaker #1 and #2 put distilled water, into #3 p ut 10% sugar, into #4 put 20% sugar, and into #5 put 40% sugar. Weigh each artif icial cell and place it into its corresponding beaker. 4. At the end of the laboratory periodor later in the day, depending in the directions from your instructorremove the cells from the beakers, wipe the cells with a paper towel, and reweigh. Calculate the percent differences in weight for each cell. Chart you results on Table2. What type of differences do you see in the different cells? How would you explain these differences? Weight of Start Weight at Finish #1 #2 #3 #4 #5 Sac40% Sugar BeakerWater Sac20% Sugar BeakerWater Sac10% Sugar Beaker10% Sugar SacWater Beaker20% Sugar SacWater Beaker40% Sugar

5. Below is a partially constructed bar graph. Complete this bar graph to s how the relative changes in the weights of the sacs. 6. Repeat #1-4 above using time as a variable. Use the space at the end of this experiment to construct a graph demonstrating your findings.

F.

Experiment #7: Osmosis (Red Blood Cells)

A solute is any substance dissolved in any solvent. In biology, the solute is us ually ionic, molecular, or particulate. The solvent is generally water or waterbased. Hypertonic, isotonic, and hypotonic are terms used to describe the relati onship between the solute concentrations on two sides of a membrane or gradient. Keep in mind that these are relationship terms. We shouldnt simply state, Solutio n X is hypertonic. To show the relationship, we should say, Solution X is hyperton ic to Y. Hyper-means greater (more, larger), so a hypertonic solution has a highe r particulate concentration than the substance with which it is being compared. Iso- means same, so the two isotonic solutions would be of the same concentratio n. Hypo- means beneath (less, below), so a hypotonic solution has a lower partic ulate concentration than the substance with which it is being compared. When particulate movement occurs between two concentrations, the net mov ement is always toward equilibrium. Note the words net movement. If pore size is a dequate, some movement against equilibrium does occur because molecules are in c onstant motion. We are concerned here with net movement. We are also concerned h ere only with passive movement. (Occasionally, cell membrane integrity or active transport mechanisms may influence net movement. We are not considering those a spects of movement here.) Note: Diseases such as cholera disrupt osmotic equilibrium. The cholera toxin causes changes in the membrane of the cells of intestinal epithelium. The se cells lose fluid to the lumen. Because the intestinal cells have become more

hypertonic to their environment, water from the interstitium enters these cells. This water is also lost to the lumen. Because of the increased hypertonicity of the interstitium, water leaves adjoining cells and enters the interstitium and eventually the epithelial cells. Dehydration progresses rapidly. Diarrhea is a c haracteristic of cholera because of the massive water loss. The large intestine (which is basically unaffected by the cholera toxin) cannot reabsorb the water a s rapidly as it is lost from the small intestine. I. PREPARATION OF A STOCK BLOOD SUSPENSION Equipment: ovine blood isotonic saline (0.15M NaCl; 0.9% NaCl is another way of expressing the same con centration) 1 box of parafilm 1 pipette (10cc) 1 pipette suction pump 1 test tube 1 Pasteur pipette Each of the experiments in this lab requires a few drops of a stock suspension o f ovine red blood cells, prepared as follows: 1. Put 10 ml of isotonic saline solution (0.9 % NaCl) in a clean test tube. 2. Add about 10 drops of ovine blood. 3. Seal the opening of the test tube with parafilm. 4. Mix thoroughly by putting your finger over the end of the tube and inverting twice gently. II. DEMONSTRATION OF OSMOSIS The effects of osmosis can be observed when red blood cells are exposed to salt solutions at concentrations different from that of blood plasma. Hemolysis or c renation results when water molecules enter or leave the cell. EXERCISE A Equipment: stock suspension of blood prepared in I. distilled water isotonic saline (0.15M NaCl)) 2 pipettes (10cc) 1 Pasteur pipette 1 pipette suction pump 2 test tubes 1. Add 5 drops of stock blood suspension to each of 2 test tubes containing the following solutions: i) 5 ml distilled water ii) 5 ml isotonic NaCl. 2. Observe the print on this page through each of the test tubes. The solution of isotonic saline and blood is turbid and the printed characters a ppear blurry: this indicates that there is no hemolysis of the red blood cells ( the print seen through the solution in the test tube appears blurry because the red blood cells are intact and thus diffract the light).

The solution of distilled water and blood is clear and the printed characters ca n be seen very clearly: this indicates that hemolysis of the red blood cells has occurred (the print seen through the solution in the test tube appears very cle ar because the red blood cells have exploded and thus do not diffract the light anymore). Note: Keep these tubes for future reference: they will be your control tubes fo r the exercises B & C. III. EFFECT OF MOLECULAR SIZE ON PERMEABILITY OF THE CELL MEMBRANE The cell membrane is differentially permeable. Ionized solutes such Cl--, in sp ite of their small molecular size, do not pass through rapidly. Moreover, activ e transport pumps for Na+ and Ca++ remove these ions from cytoplasm as fast as t hey enter cells. The cell membrane is more permeable to some of the unionized p olar solutes used in this experiment. EXERCISE B Equipment: stock blood suspension distilled water 0.3 M urea 0.3 M ethylene glycol 0.3 M glycerol 0.3 M glucose 5 test tubes 5 pipettes 1 pipette suction pump 1 timer Relevant properties and formulas for the solutes used in this experiment are giv en in Table 1. Note that molecular size (diameter) roughly parallels molecular weight. All of these compounds are polar since they have either OH (hydroxyl) or NH2 (amino) groups. 1. 2. Put 5 ml of 0.3 M urea in a test tube. Add 5 drops of red cell suspension and mix gently.

3. As the first drop of blood reaches the solution, start the timer (or not e the exact time if you prefer using your watch). Determine the time required for hemolysis to occur. You do this by holding the t ube in front of the print on this page; as hemolysis occurs, the print will appe ar more and more clear. The hemolysis time is the time required for the solution in the tube to go from turbid to clear (and the print to go from blurry to clear). You should stop your timer when the print seen through the test tube containing urea is as clear as the print seen through the control tube containing distilled water. 4. 5. Repeat the procedure using ethylene glycol, glycerol and glucose. Record hemolysis times in Table 2 and on the blackboard.

Table 1: Properties and Formulae of Solutes and Solvents SUBSTANCE MOLECULAR WEIGHT (g) MOLECULAR DIAMETER (A) FORMULA urea 60 3.6 H2N C=O H2N ethylene glycol 62 ~3.6 CH2OH CH2OH glycerol 92 6.2 CH2OH CHOH CH2OH glucose 180 8.6 CHO CHOH CHOH CHOH CHOH CH2OH Table 2: Your results. SUBSTANCE urea Ethylene glycol glycerol TIME TO HEMOLYSIS

glucose

IV. EFFECT OF LIPID SOLUBILITY ON PERMEABILITY OF THE CELL MEMBRANE This experiment examines the relation between lipid solubility and cell membrane permeability. Recall the structure of the plasma membrane. Ions and water solu ble molecules must pass through pores in the membrane because they cannot dissol ve in the central lipid layer of the membrane. On the other hand, lipid soluble molecules can dissolve in the lipid portion of the membrane and diffuse across e asily. Many organic molecules contain both non polar bonds, which confer lipid solubili ty, and polar bonds, which confer water solubility. Hydroxyl ( OH) and amino ( NH2) groups promote water solubility by virtue of the polar bonds between hydrog en and oxygen (in OH groups) or between hydrogen and nitrogen (in NH2 groups). On the other hand, carbon carbon or carbon oxygen bonds are nonpolar. When mole cules contain bonds of both types they are soluble (to a greater or lesser exten t) both in water and in lipids. The more polar bonds present, the greater is th e water solubility, and the less soluble the compound is in lipids. EXERCISE C Equipment: stock 0.3 M 0.3 M 0.3 M

blood suspension glycerol triacetin diacetin

3 3 1 1

test tubes pipettes pipette suction pump timer

The partition coefficient of a compound is a measure of its relative solubility in water and lipids. Partition Coefficient = Solubility in Olive Oil / Solubility in Water As the partition coefficient increases, fat solubility increases and water solub ility decreases. Note in Table 3 that the partition coefficient increases as th e number of polar hydroxyl groups decreases. Determine times to hemolysis for each solution, following the procedure outlined for EXERCISE B. (Repeat glycerol test do not use previous results). Record hemolysis times in Table 4 and on the blackboard. Table 3: Partition coefficients and formulas. SUBSTANCE MOLECULAR WEIGHT (g) PARTITION COEFFICIENT glycerol 92 0.00001 CH2OH CHOH CH2OH triacetin 218 0.01 CH2-O-COCH3 CHOH CH2OH diacetin 176 0.1 CH2-O-COCH3 CHOH CH2-O-COCH3 FORMULA

Table 4: Your results. SUBSTANCE glycerol TIME TO HEMOLYSIS

Monoacetin

diacetin

V. OBSERVATION OF OSMOSIS UNDER MICROSCOPE EXERCISE D Equipment: ovine blood distilled water isotonic saline (0.15M NaCl) 0.3 M NaCl 1 box of Kimwipes 3 microscope slides and cover slips 1 microscope 4 Pasteur pipettes masking tape 1. Label 3 microscope slides: "dist. water", "saline" and "0.3 M NaCl".

2. Label the 4 Pasteur pipettes: "dist. water", "saline", "0.3 M NaCl" an d "blood". 3. Dilute the stock ovine blood 2:1 with isotonic saline (10 drops isotonic saline to 5 drops of blood). 4. Place a drop of isotonic saline on the appropriate microscope slide. Ad d a small drop of the diluted ovine blood on the top of the drop of isotonic sal ine. Add a cover slip and examine immediately under the microscope. In the center of the slide, the red blood cells are piled up several layers thic k and thus it is very hard to determine the shape of individual red blood cells. Make sure that you look where the layer of blood is thinner and try to find a s pot where you see individual cells clearly. Use high power and reduced light int ensity. Sketch outlines of cells. 5. Repeat this procedure twice, but use distilled water and 0.3M NaCl inste ad of isotonic saline. CLEAN-UP See At the beginning of the lab on first page. Leave your space clean and tidy. WORKSHEET Name: Instructor: Partners Name (if applicable): Additional Activities 1. Try any of the diffusion experiments again. This time using exact measur ements. Compute the exact rate of diffusion for each substance. Use a variety of mathematical and/or statistical formulas to discover whether or not other relat ionships exist. 2. Design additional experiments to demonstrate movement across a membraneei ther active or passive. 3. Refer to the experiment on red blood cell osmosis. What would you need t o know if you wanted to determine the relative amount of water moving across the cell membrane? Answer to Selected Concept Check Questions 1. In flask #2 the neutral red must have been transported into the living y east cell. In Flask #1 the yeast cells were killed so no transport could occur. 2. These pumps correspond to the sodium/potassium pump in the cell membrane . The sodium/potassium pump actively pumps three sodium ions out of the cell for every two potassium ions pumped into the cell, thus maintaining the membrane po tential. You may wish to figure out other macroscopic situations where active en ergy input is required. 3. The potassium permanganate should have traveled about twice as far as th e potassium chromate. The potassium chromate should have traveled just a bit fur ID No.: Course / Section: Date:

ther than the methylene blue. 4. Water will continue to enter the thistle tube containing the sugar. Even tually (perhaps not until the next day) the sugar water may spill out the top of the tube because equilibrium is never reached. The sugar molecule is too large too pass through the membrane so the water in the beaker will remain pure. Water will enter the salt thistle tube and you will note an initial rise in the water lever, perhaps reaching half way up the tube. Then the water level will st art to drop. The sodium and chloride ions of the salt are small enough to cross the membrane and enter the beaker. The water in the beaker will eventually be sa lty to the taste. Equilibrium will eventually be reached.

Name: General Questions

Date:

Use your own paper in necessary. 1. Define active and passive movement across a cell membrane.

2. Explain what happened to the yeast solutions. Why was the solution in Fl ask #2 a different color than the solution in Flask #1? 3. What caused the Brownian movement you observed?

4. Explain what happened when you dropped the potassium permanganate (KMnO4 ) Crystals into the water. 5. If you diffusion rates do not match the expected diffusion rates, how mi ght you explain the discrepancy? 6. How was the thistle tube osmometer experiment related to our definition of osmosis? 7. In which of the experiments did osmosis occur? Dialysis? In which direct ion did these events occur? 8. Check your lecture text and find out what effective osmotic pressure is? How does it relate to cellular osmosis? 9. Explain hypertonic, isotonic, and hypotonic in terms of the red blood ce ll experiment. 10. List several factors (other than the ones we demonstrated by these exper iments) that might influence the rate of either active or passive transport. 11. Why are sugar and salt used for food preservations?