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BIOL 130L Exam Review - Well-Organized

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University of Waterloo
Dragana Miskovic

Identification of Some Macromolecules Treatment controls; positive and negative controls of specific samples that are used to check if the experimental conditions were set properly and/or if the experimental procedures were followed, so that all changes could be compared to the actual experimental results Time points; if time is a variable, there must be a control for every time point in the experiment Four Major Biological Macromolecules Carbohydrates (monosaccharides and polysaccharides)  Ex. Starch, glycogen, cellulose (all complex carbohydrates) a) Starch - Storage polysaccharides in plants - Composed of glucose monomers linked by glycosidic bonds - Combination of amylose and amylopectin polymers i. Amylose - Unbranched helical structure - Glucose units joined by α (1→4) linkages - Reacts with iodine to give blue colour (Iodine Test) ii. Amylopectin - Straight, branched sections - Glucose units joined by α (1→6) linkages b) Glycogen - Larger than starch - Similar to amylopectin but is more highly branched - Multibranched components give red-brown colour (Iodine Test) c) Cellulose - Used as structural components in plants - Cannot be digested by mammals  Ex. Reducing sugars, simple sugars (all basic carbohydrates) a) Straight chain form - Have an aldehyde group, making them aldose sugars - Aldehyde groups react to Benedict’s Test and develops a coloured precipitate when boiled b) Ring form Proteins  Made of peptide chains a) Peptide chains - Consist of amino acids connected by peptide bonds - React with Cu++ and alkali to produce a purple colour (Biuret Test) Lipids  Amphipathic a) Hydrophilic head b) Hydrophobic tail Nucleic Acids a) DNA - Double-stranded b) RNA - Single-stranded Iodine Test  Add a drop of iodine to a sample  Detects the presence of starch and glycogen a) Starch - Solution will turn blue-black - Blue-black caused by reaction between amylose and iodine - Iodine atoms align in the centre of the helical structure of amylose - Transfer of charge between starch and iodine cause spacing between energy levels - The spacing absorbs visible light selectively and give off blue-black colour b) Glycogen - Solution will turn red-brown - Red-brown caused by reaction between amylopectin and iodine - Since amylopectin is highly branched, it only binds to a small amount of iodine atoms, resulting in a red-brown colour Benedict’s Test  Add Benedict’s solution (blue) to a sample, boil  Detects the presence of reducing sugars a) Straight chain form - Has an aldehyde at the end of the chain - The aldehyde group reacts with the blue cupric ion of Benedict’s solution and reduces the cupric ions to cuprous ions - The cuprous ions synthesize with oxygen to form a precipitate, Cu O 2  The colour of the precipitate reflects the concentration of Cu O2formed: (low concentration) yellow → green → red → brown (high concentration) Biuret Test  Add NaOH to a sample, mix, add 1% CuSO 4  Detects the presence of proteins a) Proteins - Made of peptide chains - Peptide chains react with cupric ions and alkali to produce a violet colour - A single cupric ion reacts with 4 to 6 peptide bonds  The intensity of the colour produced is proportional to the amount of peptide bonds participating in the reaction Isolation of Some Macromolecules Centrifugation; a fractionation technique in which centrifugal force sediments suspended particles to the bottom of the tube. The sediment is known as a precipitate or pellet, and the remaining liquid above the sediment is known as the supernatant. The speed and time of a centrifugal run separate particles of different sizes or properties from the same suspension  Grind the yeast with sand to rupture the cell walls and cell membranes a) Cell Walls - Contain glucan (a cellulose-type polysaccharide) b) Cytoplasm - Contain glycogen, proteins, and nucleic acids  Add 5% TCA and grind a) Soluble in TCA will mix with solution - Starch - glycogen b) Insoluble in TCA will remain in suspension - Proteins - Nucleic acids  Centrifuge - The supernatants contain the carbohydrates/polysaccharides - The precipitate/pellets contain proteins and nucleic acids  Discard supernatants - Precipitate/pellets containing proteins and nucleic acids remain  Add 3% NaCl solution a) Soluble in NaCl will mix with solution - Nucleic acids b) Insoluble in NaCl will remain in suspension - Proteins Steps with the Protein and Nucleic Acid Fraction  Boiling the tubes will coagulate the suspended protein molecules  Centrifuge - The supernatants contain the nucleic acids - The precipitate/pellets contain the proteins  Decant supernatants into beaker Steps with the Nucleic Acid Fraction  Add ethanol to beaker of nucleic acids and chill in ice to force nucleic acids out of solution - Nucleic acids are insoluble in ethanol and will precipitate out of the solution to form a suspension  Centrifuge to form pellets of nucleic acids  Discard supernatants  Add H 2O t4 resuspend pellet and distribute into tubes - Nucleic acids are soluble in H 2O 4  Boiling in acid is a hydrolyzing process; it will break up the nucleic acids into its nucleotide subunits, and eventually into the base and sugar and phosphoric acid subunits [HYDROLYZED NUCLEIC ACID]  Add Ba(OH) t2 neutralize the contents (use red litmus paper to confirm neutralization)  Sulfuric acid and barium hydroxide will react to produce a salt that is insoluble and will precipitate out, leaving a salt-free solution of nucleic acids Steps with the Protein Fraction  Add pancreatic enzyme to resuspend the pellet and to hydrolyze proteins into their amino acid subunits [HYDROLYZED PROTEIN]  Add phosphate buffer to resuspend the pellet [UNHYDROLYZED PROTEIN] Characterization of Some Macromolecules Chromatography; a technique that separates mixtures into their individual components a) Stationary phase is the matrix - The matrix is an inert substance like cellulose or paper chromatography b) Mobile phase is the solvent  Separation depends on the relative tendencies of molecules in a mixture to associate more strongly with one or the other phase (eg. Opposite charges attract and same charges repel, soluble or insoluble)  The matrix is partially immersed in the mobile phase so that the application is above the level of solvent  The matrix absorbs the solvent  The solvent dissolves the mixture and allows he components to migrate along the matrix - Substances soluble with the solvent would move as quickly as the solvent and no separation would occur - Substances insoluble with the solvent would not migrate along the matrix at all  Chromatography is terminated when the solvent has almost reached the other end of the matrix  The leading edge of the solvent must be marked before the completed chromatogram is dried  Rf(the relative mobility factor) is the distance travelled by the substance over the distance travelled by the solvent Separation and Identification of Proteins  Solvent used: formic acid  8 known amino acids and 1 unknown  Comparing the relative mobility factor of the unknown amino acid to the relative mobility factor of the known amino acids will help identify the unknown amino acid Separation and Identification of Nucleic Acids  Solvent used: acetic acid  3 known nitrogenous bases and 1 solution of nitrogenous bases  Comparing the relative mobility factor of the solution of nitrogenous bases to the relative mobility factor of the known nitrogenous bases will help identify which nitrogenous base if more dominant in the solution Spectroscopy Enzymes Enzymes; proteins that catalyze biological reactions. Their specificity is determined by the number and order of their amino acids, and by their three-dimensional configuration of the protein itself  Enzymes combine with a substrate (the reactant) to form a substrate-enzyme complex which then breaks down into the unaltered enzyme and a product  That is: Substrate + Enzyme → Substrate-Enzyme Complex → Product + Enzyme  Factors that affect the rate of reaction: temperature, pH, enzyme concentration, substrate concentration, product concentration, energy of activation, etc  The direction of the reaction depends on the conditions under which the reaction is taking place Salivary Amylase  A digestive enzyme found in saliva a) Enzyme acts on starch molecules - breaks off maltose molecules from the ends of the starch chains - uses a water molecule to break the starch chain (hydrolysis)  Concentrations of salivary amylase used: 1%, 2%, 5%, 10% a) Do Iodine Test to ensure no presence of starch or glycogen b) Do Benedict’s Test to ensure no presence of reducing sugars  Used 1% starch suspension (contains 0.25% NaCl; the chloride ions activate salivary amylase)  Used McIlvaine’s buffer to maintain optimal pH for salivary amylase activity  Immersed tubes in water bath at 37⁰C (optimal temperature for salivary amylase activity)  Carry out iodine test for each concentration of salivary amylase at different time intervals until end-point is reached (when test becomes negative; indicating that starch is broken down) Phosphorylase a) An enzyme that acts on starch by breaking off glucose molecules - Ruptures glucose-glucose bonds by consuming HPO instea4 of water, hence requires phosphoric acid and is done by phosphorylase (phosphorylysis)  (Glucose) n HPO ↔ 4Glucose) + Glun-1e-1-phosphate a) Phosphorylysis is the forward reaction - HPO d4sassociates into hydrogen and phosphate - The energy released in the rupture of the glucose-glucose bond is used to create the glucose-phosphate bond in glucose-1-phosphate, hence no energy is released from the system b) Synthesis is the reverse reaction - The enzyme attaches the glucose molecule to a pre-existing starch primer (too short to give a positive iodine test) - Molar concentration of starch does not change, but grows longer - During synthesis, glucose must be present in the form of glucose-1-phosphate  Water is the medium in which the reaction takes place but is neither a product nor a reactant  The water concentration will not affect the direction of the reaction, as it did with salivary amylase *hydrolysis Osmosis Diffusion; molecules of a substance moving from an area of high concentration to an area of low concentration of the same substance  The cell acts as a permeable membrane that allows water to pass through freely in both directions Osmosis; movement of water through the cell membrane Isotonic; the concentration of solutes inside the cell and outside the cell are the same. In such an environment, the concentration of water on both sides of the membrane is identical. Therefore, there is no net movement of water in or out of the cell Hypertonic; the concentration of solutes outside of the cell is greater than the concentration of solutes inside the cell. In such an environment, the concentration of water is greater in the cell than outside. Therefore, the net movement of water will be out of the cytoplasm until equilibrium is reached Hypotonic; the concentration of solutes outside of the cell is less than the concentration of solutes inside the cell. In such an environment, the concentration of water is greater outside the cell than inside. Therefore, the net movement of water will be into the cytoplasm until equilibrium is reached. Part I. Osmosis in a Model System  The cellophane of the dialysis tubing resembles the action of a cell membrane in the sense that allows water molecules to pass freely through the membrane, but does not allow solute molecules to pass through  Water movement in or out of the “cells” were monitored by weighing the “cells”  Weighed each bag every 5 minutes to see any changes in water movement Part III. Osmosis in Animal Cells a) Isotonic environment - no net movement of water across membrane b) Hypotonic environment - water enters the cell - the cell swells and eventually bursts c) Hypertonic environment - Water moves out of the cell - The cell shrinks and the membrane retracts Part IV. Osmosis in Plant Cells  Plants cells have a cell wall, that is permeable to water, outside of the cell membrane  Most plant cells have a large vacuole within the cytoplasm a) Isotonic environment - the vacuole is filled with sap b) Hypotonic environment - the force exerted by the cell wall preven
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