SOLANO, JYAN DESSE M. A-2L
January 31, 2013 Maricar S. Niez Exercise 5 Donnan Equilibrium and Active Transport
Guide Questions
Donnan Equilibrium: 1. What was the purpose of placing gelatin inside the artificial membrane? membrane? Since the experiment shows diffusion and osmosis works in an artificial plasma membrane, the gelatin placed placed inside simulates the the cytoplasm of a cell. The gelatin has a fairly high concentration of water, but it contains other components, so it isn't 100% water. The dialyzing bag bag depicts the semi-permeability of cell membranes while the gelatin represents the gelatinous form of the cytoplasm. +
2. Compare the H concentration before and after immersion in acidified dH 2O. Explain these results based on the principle of Donnan equilibrium. The initial pH of the solution inside the dialyzing bag was was 5.6. After 90 minutes, the pH of the solution lowered to 3.9, similar to the pH of the environment (acidified water). + This confirms that the H concentration inside inside the dialyzing bag increased. According to Donnan equilibrium, when two solutions of different strengths are separated by a permeable membrane, their concentration on both sides of the membrane equalizes due to diffusion of the solutes as well as the solvent. 3. In the unavailability of a pH meter, what appropriate alternative can be used to detect pH changes? changes? Explain. If pH meter is not available, pH paper can be used as an alternative to detect changes in pH. pH paper is used to test the acid levels in liquids. liquids. Color changes changes would occur occur if a solution is neutral like water, water, or a base. However the downside downside of pH paper is that that pH detection is subjective. There is a picture guide that would would help in determining the pH of the solution when pH paper is dipped dipped and the color changes. A person using it may consider the pH of of a solution to be 7, but but it should have been pH 6 or pH 8. Another alternative would be the use of Litmus paper. paper. Litmus paper is the most recognized member of chemical indicators. indicators. Like the pH paper, paper, it changes color when exposed exposed to an acidic or basic solution. Blue litmus turns red in acidic conditions and red litmus turns blue in basic conditions. conditions. Yet like the pH paper, it is also also not precise in determining exact pH. 4. Design another experiment/methodology to demonstrate Donnan equilibrium.
The schematic diagram below shows the mechanism of Donnan equilibrium. It compares two types of artificial cells, having same membrane permeability, but different contents – one is distilled water only and the other is a protein-containing fluid. This experiment will demonstrate how molecules traverse a membrane, depending on its molecular constitution. This experiment will transpire for more than 24 hours, so transport can be scrutinized aptly, after which pH will be determined in both artificial membranes to statistically determine significant differences.
Covered polyethylene containers will be filled with dialysis solution (has levels of minerals like K+ and Ca2+) at 37-38oC.
After 17 hours, dialysis will be terminated and 1-3 samples will be taken from the dialysis solution and 1 from the contents of each dialysis unit using syringes.
Each obtained contents will be placed on clean sterile tubes
Containers will be mounted on a shaking device inside an incubator and pH of dialysis solution will be adjusted by adding apt amounts of concentrated HCl.
Then the bags will be sealed and shaking platform will be set in motion.
The pH of the samples will be measured using a pH meter or a pH paper.
The bags will be subsequently filled with 12mL of protein-containing fluid. One dialysis unit will be always filled with dH20.
It can be observed that after the experiment, concentrations between dH20 and dialysis solution is equal, while concentrations between dialysis solution and bag with proteiin-fluid tend be unequal.
Then the actual ‘dialysis units’ will be placed at the top of the cover so that the bags hang down into the dialysis solution.
Active Transport: 5. What was the purpose of using Ringer’s solution as medium in the experiment? Ringer’s solution normally contains potassium chloride, calcium chloride, sodium chloride, and sodium bicarbonate. Other additions include chemical fuel sources for cells, including ATP and dextrose, as well as antibiotics and antifungals. This solution is dissolved in water for the purpose of creating an isotonic solution. Also according to Francis and Pumphrey’s experiment about active transport, they foun d out that oxygenfree Ringer solution causes an irreversible disappearance of the potential difference across the skin in the course of 20 –40 min.
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6. Discuss in detail the mechanism of Na -K transport. Write down pertinent equations and diagrams. The process of moving sodium and potassium ions across the cell membrane is an active transport process involving the hydrolysis of ATP to provide the necessary energy. It + + involves an enzyme referred to as Na /K -ATPase. This process is responsible for + + maintaining the large excess of Na outside the cell and the large excess of K ions on + the inside. It accomplishes the transport of 3 Na to the outside of the cell and the + transport of 2 K ions to the inside. This unbalanced charge transfer contributes to the separation of charge across the membrane. This pump is called a P-type ion pump because the ATP interactions phosphorylate the transport protein and causes a change + + in its conformation. The diagram below shows the detailed mechanism of Na -K transport.
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7. Propose a possible mechanism for the active transport of Na and K across the toad/frog skin bag considering that these ions must pass through the epithelial cells and not just across a membrane. The phenomenon of transport of ions, sugars, amino acids, etc. across frog skin and other epithelial systems has been commonly interpreted on the basis of the membrane
pump theory, according to which asymmetry in solute distribution as well as transport into and out of all living cells results from the permeability properties and "pump" + activities of the cell membrane. The transport of Na by frog skin involves migration of + Na along an array of fixed negative sites on the outside surface of the epithelial cells of the frog. 8. If the skin bag is turned proper side out, will this difference in the side of the skin exposed to the medium alter the results? How and why? Yes, there would be a great difference in the results when different side of the skin is exposed to the medium. According to the "two-membrane theory" by KoefoedJohnson, which concluded that the surface facing the outside has a selective high Na+ permeability and the surface facing the inside has a selective high K+ permeability. Due to this well-known "two-membrane" theory the inner membrane has essentially the same properties seen in most living cells including the possession of the Na-K pump in the form of K, Na-activated ATPase. It is the membrane facing the outside solution that is unusual and functions as the seat of transport regulation through the control of Na+ permeability. Effect of Cyanide: 9. Explain the change in the weight of the bag in the presence of sodium cyanide. Discuss how cyanide affects active transport. Sodium cyanide is an inorganic compound with the formula NaCN. This is a highly toxic and colorless inorganic salt derived from neutralization reactions involving the weak acid hydrogen cyanide. Sodium cyanide prevents respiration so any energy requiring processes, such as active transport, that occur will be hindered. 10. Compare the average and partial rates of active transport (R and R pi ) of the bag immersed in Ringer’s solution alone with that immersed in Ringer’s solution with NaCN. Account for any difference. The average and partial rates of active transport in the experiment where the bag is immersed in Ringer’s solution alone were negative. This is because active transport is occurring in the system. Molecules inside the frog skin bag tend to move outside, following the membrane potential gradient of the solution. Like the Na-K pump, molecules inside the bag go outside the solution as molecules outside tend to diffuse in the bag. On the other hand, the bag immersed in Ringer’s solution with sodium cyanide tend to have a positive values in R and Rpi since weight of the bag tend to increase as time it is immersed increases. This is because the solution outside has NaCN, which stops active transport by preventing permeability property of the membrane. Thus it prevents molecules inside the bag to go out; subsequently piling molecules up inside since molecules outside diffuse inside the bag.
