Lab Practical Study Guide
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Lab Practical Study Guide

Course Number: BISC 220, Spring 2009

College/University: USC

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LabPracticalStudyGuide Lab1:Macromolecules 4 classes of macromolecules: carbohydrates, proteins, lipids, and nucleic acids (monomers are monosaccharides, amino acids, fatty acids, nucleotides) Exercise 1: Carbohydrates: made of C, H, and O o Monosaccharides include glucose, fructose, galactose, ribose o Disaccharides Maltose = glucose + glucose Lactose = glucose + galactose Sucrose = glucose + fructose o...

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4 LabPracticalStudyGuide Lab1:Macromolecules classes of macromolecules: carbohydrates, proteins, lipids, and nucleic acids (monomers are monosaccharides, amino acids, fatty acids, nucleotides) Exercise 1: Carbohydrates: made of C, H, and O o Monosaccharides include glucose, fructose, galactose, ribose o Disaccharides Maltose = glucose + glucose Lactose = glucose + galactose Sucrose = glucose + fructose o Polysaccharides include starch (energy storage in plants), cellulose (structural), and glycogen (energy storage in animals) Starch is used for storing glucose in plants that was created by photosynthesis. Examples of food containing starch are potatoes and root veggies. It is a polymer of glucose with 1-4 glycosidic bond (linear amylose) and the occasional 1-6 bond (branched amylopectin) Cellulose is composed of 1-4 glycosidic linkages o Activity A Benedicts test (detects monosaccharides and disaccharidesbut not sucrose) Positive: colored precipitate (yellow, green, orange, or red) Negative: clear blue Must be mixed with a substance and then heated in boiling water to yield a result When mixed with glucose, it forms a red precipitate (positive) Limitations of these tests are that they are qualitative, not quantitative (dont tell us any information about how concentrated the substance is) o Activity B Iodine test: mix a few drops of I2KI solution with the sample (detects starch) Positive: dark blue Negative: yellow o Acticity C Hydrolysis: a chemical reaction used to break down polymers; a chemical bond is cleaved by the addition of H2O Two different experiments were run: hydrolysis of sucrose (Sucrose + HCl) and hydrolysis of starch (Starch + HCl) Sucrose hydrolyzes (into glucose and fructose) much faster than starch hydrolyzes, because starch is a polysaccharide Exercise 2: Lipids: diverse class of hydrophobic molecules o Fatty acids are primary made of carbon and hydrogen o Can be structural (membrane lipids) or store energy (triacylglycerols, sterols, vitamins) o Can be saturated (all single bondssolid at room temp) or unsaturated (at least one double bondliquid at room temp) o Energy storages molecules store ATP (seeds, adipose tissue) o Paper test: if it is transparent, there are lipids present. If it is opaque, there are no lipids present. Oil tested positive. Exercise 3: Proteins: a polymer of amino acids linked by a peptide bond o There are about 20 different amino acids o Biuret test detects peptide bonds: add to sample and wait for color change. Positive: pink (smaller proteins) or violet (larger proteins) Negative: blue Exercise 4: Macromolecules in food o Activity A: Separation of butter Butter is an emulsion: the lipids occur in very small droplets dispersed throughout the water-soluble portion (carbs and proteins) Clarification: the lipid is separated from the water-soluble protein part (used for cooking, clarified butter can fry at higher temps, keeps longer) Results Whole butter tested positive for Benedicts (sugar), Paper (lipid), and Biuret (protein). Biuret test is only slightly positive, this is because its hard to get the protein to mix with the reagent until the butter is clarified. Clarified butter (upper layer) tested positive for Sugar and Lipids Clarified butter (lower layer) tested positive for Sugar and Proteins Clarified butter lacks the taste, meaning protein molecules must be responsible for the butter taste You cannot clarify margarine because there are no proteins in the margarine o Activity B: Tests with Food Banana tested positive for sugar (orange), starch, lipid Coconut tested positive for starch, lipid, protein Milk tested positive for sugar (orange), lipid, and protein Peanut tested positive for protein Potato tested positive for sugar (green), starch, and lipid Potato has starch but does NOT taste sweet, only sucrose and fructose are sugars that taste sweet Lab2:Enzymes Enzyme: a biological catalyst that speeds up a reaction without being used up or altered (usually proteins) o Digestive enzymes: proteases, amylase, lipases o They increase the rate of reactions but do NOT change the final result of the reaction o Enzymes lower the activation energy (Ea) of a reaction. However, there is no difference in free energy between catalyzed and uncatalyzed reactions. o Rate of reaction can depend on: temperature, pH, concentration of enzymes/substrates, concentration of cofactors, time of incubation, activator and inhibitors Substrate: the material with which the catalyst reacts; modified during the reaction to form a new product Active Site: part of an enzyme that binds to the substrate, location where catalysis takes place. The enzyme-substrate complex remains connected until reaction is complete Cofactors: non-protein substances that usually bind to the active site on the enzyme and are essential for the enzymes to work (e.g. metal ionsCatechol Oxidase requires copper as a cofactor) o Coenzymes: organic cofactors (e.g. vitamins like riboflavin, thiamine, etc.) Activators: chemicals that must bind for the enzyme to be active Inhibitors: chemicals that shut off enzyme activity o Competitive inhibition: a molecule that is structurally similar to the substrate for a particular reaction competes for a position at the active site on the enzyme Adding additional substrate may reverse the inhibition reaction o Noncompetitive inhibition: the inhibitor binds to a part of the enzyme that is NOT the active site the catalytic properties of the enzyme are lost. It either: Physically blocks access to the active site OR Causes a conformational change in the protein, thus inactivating the active site The effect of temperature on enzyme activity o Low temperature low enzyme activity o High temperature disrupts H-bonds (shape of protein is denatured) The effect of pH on enzymatic activity o Changes in pH affect the shape of the enzyme and charges within the active site o pH optimum varies between enzymes. Pepsin = pH 2. Most enzymes = pH 7-9. Activity 1: Catechol Oxidase o Catechol Oxidase catalyzes the oxidation of catechol, a phenol found in plants Requires copper as a cofactor o Converts catechol to benzoquinone, a pigment that is responsible for the browning of fruits when exposed to oxygen. Water is a product of the reaction. o A dark brown color indicated the production of benzoquinone due to catechol oxidase activity o Canned pineapple is boiled so it denatures the protein (gelatin) when they are mixed. If fresh pineapple is used for the salad instead, the enzyme catalyzes a reaction a water is a product turns it into a runny mess. Activity 2: Inhibiting the Action of Catechol Oxidase o PTU (Phenylthiourea) is an inhibitor of catechol oxidase. Is it competitive or noncompetitive? o PTU is a noncompetitive inhibitor because addition of more substrate still led to the same reaction (no additional products were produced). o Competitive inhibition: increased substrate increased reaction rate. Noncompetitive inhibition: increased substrate no increase in reaction rate. Activities 3-5: Influence of Concentration, pH, and Temperature on the Activity of Amylase o Amylase is found in the saliva of many animals and is responsible for the digestion of starch into maltose. Starch (but not maltose) turns dark blue when mixed with Iodine reagent. Thus the rate of disappearance of starch allows for a quantitative measure of reaction rate. o Porcine pancreatic amylase was used in the experiments o Higher concentrations of amylase produced faster reaction rates (fastest was 50 secs at .5%) o pH 7 and 8 were optimal for amylase reaction rate (rate was 50 seconds). o A temperature of 22 degrees C was optimal for amylase reaction rate Lab3:Cell Physiology Eukaryotic cell membranes are fluid mosaics of lipids and proteins o As temperatures cool, membranes switch from a fluid state to a solid state o The temperature at which a membrane solidifies depends on the types of lipids o Membranes with unsaturated fatty acids are more fluid than those with saturated fatty acids Membrane structure results in selective permeability (size, polarity, etc. determine if it will pass through) o Passive Transport requires no input of energy from the cell Simple diffusion: the movement of molecules from an area of high concentration to an area of low concentration diffusion (e.g. of oxygen across the lipid bilayer) Osmosis: diffusion of water (small, polar molecule) across a selectively permeable membrane. Aquaporins are water channel proteins. Facilitated Diffusion: passive diffusion with the help of a transport protein Channel proteins Carrier proteins (clam-shell design) o Active Transport: ATP-driven, e.g. Na+-K+ pump o Bulk Transport: endocytosis (phago-, pino-, receptor mediated) and exocytosis Tonicity: a measure of osmotic pressure. Osmosis is driven by solutes affecting the concentration of water. Think about the solute concentration in the fluid surrounding the cell. o Hypertonic: has a higher solute concentration than the cell (but less H2O) o Hypotonic: has a lower solute concentration than the cell (but more H2O) o Isotonic: has the same solute concentration as the cell (same H2O concentration) Animal Cells: Animals and other organisms without rigid cell walls living in hypertonic or hypotonic environments must have special adaptations for osmoregulation o Hypotonic solution: water enters the cell, causing it to lyse (burst) o Isotonic solution: normal condition for the cell o Hypertonic solution: water leaves the cell, causing it to crenate (shrivel) Plant Cells: fare best in a hypotonic environment where they are turgid (firm) o Hypotonic solution: turgid, normal condition o Isotonic solution: flaccid o Hypertonic solution: plasmolysed (plasma membrane pulls shrivels and pulls away from cell wall, but remains attached) Activity 1: Diffusion through agar gel o Two chemicals, potassium permanganate and methylene blue, were placed in two separate wells of a Petri dish containing agar. Every 15 minutes for 1 hour, the distance they diffused was measured. o Potassium permanganate diffused through the agar at a faster rate than methylene blue. It diffused faster because it has a smaller molecular weight. o The rate of diffusion potassium : methylene blue was 4:1. Activity 2: Diffusion through dialysis tubing o Two solutions will be separated by an artificial membrane; membrane is selectively permeable o Sugar was small enough to pass through the membrane, but starch was too large (iodine test was negative) Activity 3: Observing Osmosis in Potato Cubes (cells = 0.9% solute) o Potato cubes were weighed and each was deposited into a solution for 1 hour. The three solutions were 0.9% saline (isotonic), DI water (hypotonic), and 2.5% saline (hypertonic). o Water entered the potato cube in the DI water, so it gained weight. o Water left the potato cube in the 2.5% saline solution, so it lost weight. o Water entered and left the potato in 0.9% saline solution at equal rates, so it stayed the same. Activity 4: Observing Osmosis in Red Blood Cells from SHEEP! (cells = 0.9% solute) o Normal blood cells (in isotonic solution) have a biconcave shape; they look like a jelly doughnut slightly pushed in at the center o Blood cells in a hypotonic solution burst. Hemolysis is the bursting of red blood cells when water moves into the cell. o Blood cells in a hypertonic crenate (shrivel) as water moves out. They look like prickly stars. Lab4:Photosynthesis Photosynthesis overview o 6 CO2 + 12 H2O (light and chlorophyll) 6 O2 + C6H12O6 + 6 H2O o Autotrophs: self-feeders; photosynthetic organisms that can synthesize organic nutrients (glucose) from inorganic materials (sunlight, carbon dioxide) Chlorophyll: the green pigment that captures light energy and converts it into the chemical energy of organic molecules (present in plants, plantlike protests, and cyanobacteria) o 2 Stages of Photosynthesis Light reaction (light-dependent reaction) Light is converted to chemical energy Water is split and oxygen is released as a product. 2 H+ and 2e- are also products; they are used to form NADPH and ATP via light energy IN: light, H2O, NADP+, ADP + P OUT: O2, NADPH, ATP Takes place in the thylakoid Dark reaction (light-independent reaction, a.k.a. Calvin cycle) CO2 from atmosphere reacts with ribulose biphosphate (RuBP), a 5-carbon sugar, to make glucose. ATP and NADPH are used in the process, and converted back to ADP and NADP+, which can be re-used in the light reaction of photosynthesis. IN: ATP, NADPH, CO2, RuBP, H OUT: G3P (sugar), NADP+, ADP + P Takes place in the stroma o Sugar products supply plant body with chemical energy (glucose) and carbon skeletons for other molecules. Excess glucose is stored as starch (glucose polymer) in the chloroplasts, or in roots, seeds, and fruits. o Pigments Chlorophyll a: absorbs red and blue/violet (reflects blue/green) Accessory pigments Chlorophyll b: absorbs blue and orange (reflects olive green) Carotenoids o Xanthophylls (yellow) o Carotenes (orange or yellow) Activity 4 (Benedicts and Iodine tests) o Tests the hypothesis: Chlorphyll is not necessary for photosynthesis o In the green Coleus leaf tissue, starch and sugar were both present o In the non-green Coleus leaf tissue, sugar was present but starch was not (sugar was transported from the photosynthetic regions of the plant) o The hypothesis was NOT supported. There was no chlorophyll and no sugar in the white parts of the plant, therefore photosynthesis did not take place in these parts. Activity 5 o Paper Chromatography: chloroplast pigments are separated (extracted) by their solubility in the non-polar solvent. The solvent used is acetone and petroleum ether mixture. o Molecular size and polarity of pigments determine the distance a pigment travels. The more soluble (non-polar) the component is, the further it goes. o Procedure: paint a line of spinach pigment solution, dip in solvent, and wait for pigments to separate out by solubility until solvent is 2 cm from the top. Calculate each pigments Rf (ratio factor) = distance moved by pigment (solute) / distance moved by solvent o Results: Chlorphyll b (yellow-green) was most polar, then chlorophyll a (blue-green), then xanthophylls, then carotene (yellow). o Not all of these pigments are visible in a health green leaf, because the leaf is performing photosynthesis, therefore the dominant pigment is chlorophyll. However, the yellow pigments are seen in autumn, because there is less light (not as much chlorphyll), so accessory pigments are able to show through. o Three parts of the spectrum responsible for most of photosynthesis: violet, blue, and red Chlorophyll a absorbs red and violet Chlorophyll b absorbs blue and orange Lab5:Cellular Respiration andFermentation Respiration: cellular processes which transfer energy in glucose bonds to bonds in ATP Redox reactions: Oxidation is the loss of electrons. Reduction is the gain of electrons NAD+: coenzyme responsible for transferring electrons in glycolysis and fermentation Glycolysis: glucose is broken down to form 2 pyruvates (takes place in the cytosol) o Reduction of NAD+ to NADH o Net gain of 2 ATP Aerobic Respiration: Glycolysis, Krebs cycle (mitochondria), Electron transport chain (mitochondria) o Oxygen is the final electron acceptor o Can make up to 38 ATP o Products are CO2, H2O, ATP Fermentation: glycolysis, then conversion of pyruvate to ethanol or lactate (takes place in cytosol) o Anaerobicno oxygen is used o 2 ATP are produced o Purpose is regeneration of NAD+ for continued glycolysis o 2 types: Lactic Acid Fermentation: Pyruvate is reduced and NADH is oxidized NAD+ and lactate Products are lactate, ATP Alcoholic Fermentation: Pyruvate releases a CO2 and forms acetaldehyde. Then acetaldehyde is reduced and NADH is oxidized NAD+ and EtOH (ethanol) Products are ethanol, CO2, ATP Experiment A: Yeast and alcoholic fermentation o Does the concentration of yeast affect the rate of fermentation? o Measure the output of CO2 (record fluid level in the graduated container every 2 mins for 20) o Use 5% Bakers Yeast (Saccharomyces cerevisiae), 20% glucose, DI water, incubate at 37C o Tubes 1 and 2 are negative controls (no yeast or no glucose); Tubes 3 and 4 are experimental o Results: higher yeast concentrations lead to faster rates of fermentation. Experiment B: The effect of temperature on yeast fermentation o Hypothesis: There will be an optimal temperature for yeast fermentation. As temperature increases, fermentation rate will also increase until this optimal temp. is reached. o Tube 4 (68C) was the optimal temperature for yeast fermentation

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