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Biology 130L Exam notes

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Heidi Engelhardt

Summary for Quiz Monday, May 30, 2005 12:13 AM Title Identification of Some Macromolecules Gist of Experiment Use different tests to check for the existence of macromolecules in various substances o Iodine test checks for starch and/or glycogen o Benedict's test checks for reducing sugars o Biuret test checks for protein Notes on Underlying Theory Introduction The most abundant elements in living material are: o Carbon o Hydrogen o Oxygen o Nitrogen o Sulfur o Phosphorus There are 4 major types of biological macromolecules: o Carbohydrates Monosaccharides (i.e. glucose, fructose) Disaccharides (i.e. sucrose) Polysaccharides (i.e. starch, glycogen) o Lipids o Proteins o Nucleic acids Tests Iodine test o Information on starch: It is a polysaccharide used by plants to store glucose Glucose is held together with glycosidic bonds It is a mixture of 2 different polymers: amylose and amylopectin Amylose It is unbranched and helical molecule The glucose is joined by alpha 1 -> 4 linkages Amylopectin It is straight and highly branched The glucose is joined by alpha 1 -> 6 linkages o Information on glycogen: It is a polysaccharide used by animals to store glucose Glucose is held together with glycosidic bonds It is heavier than starch It is similar to amylopectin in overall structure, but is more highly branched o How does the test work? Iodine solution is usually pale yellow It turns blue-black in the presence of starch because of the amylose It turns red-brown in the presence of glycogen because of the multi-branched components Benedict's test o Information on sugar: All sugars can exist as straight chains or in ring form The straight-chain forms are called aldose sugars They have a terminal aldehyde group (C single-bonded to H, double- bonded to O) o How does the test work? Blue cupric ions (Cu ) in Benedict's solution are reduced to cuprous ions (Cu )+ by the free aldehyde groups, and we get a precipitate of cuprous oxide (Cu O): + - - + - 2 4Cu + 2OH + 2e -> 2Cu O + 2H2+ 2e The amount of cuprous oxide formed is proportional to the concentration of free aldehyde groups The color of the precipitate varies depending on this as well (blue -> green -> orange -> red -> brown) Ketose sugars (i.e. non-aldose, which means non-straight chain, which means no free aldehyde group) can ALSO reduce Benedict's solution This happens because the basic conditions of the experiment isomerize a ketose to an aldose, and then the reduction happens with an aldose Biuret test o Information on proteins: They are composed of amino acids, which are connected by peptide bonds A peptide bond is the carboxyl group of one amino acid covalently linked to the alpha-amino group of the next amino acid o How does the test work? Biuret solution is a solution of sodium hydroxide (NaOH) and copper sulfate (CuSO4) Under alkaline conditions (caused by the NaOH), the peptide bonds within proteins react with the Cu++ ions to form a purple complex So we identify the presence of protein by looking for this purple color Summary for Quiz Monday, May 30, 2005 12:13 AM Title Isolation of Some Macromolecules Gist of Experiment Use a variety of techniques to isolate macromolecules from a starting mixture Experiment Procedure and Justification Thereof 1. We start with a yeast-sand mixture a. Yeast cells have: i. Glucan (a polysaccharide) in the cell walls ii. Glycogen, proteins, and nucleic acids in the cytoplasm b. Grind the yeast to rupture the cell walls and release all this stuff 1. Add TCA (trichloroacetic acid) and continue grinding a. Polysaccharides (in this case glucan) are soluble in TCA, so they will go into solution a. But the proteins and nucleic acids will stay suspended! 1. Centrifuge the suspension (so just the non-sand part) a. When you do this on a liquid (remember the polysaccharides are suspended in the liquid) with particles suspended in it (remember these are the nucleic acids and proteins), all the suspended stuff goes to the bottom (it "sediments") and the liquid remains on top i. The sediment is known as the precipitate, also known as "pellet" ii. And the top liquid stuff is the "supernatant" 1. Now we focus just on the pellet (i.e. the nucleic acids/proteins) a. Add NaCl to the pellet i. Nucleic acids are soluble in strong NaCl, so they go into solution! ii. But the proteins remain in suspension 1. Again we centrifuge this, and the proteins become the pellet, and the nucleic acids are the supernatant 1. Now we are going to SPLIT the nucleic acids and the proteins, and do stuff with each Nucleic Acid Portion 1. Alright, first remember that we are dealing with a liquid here, because the nucleic acids were in solution! 1. But the first thing we'll do is to add chilled ethanol, which will cause the acids to precipitate out of solution to form a suspension 1. As before, we centrifuge to isolate the acids (which are in the pellet, of course) 1. But then we take the pellet and we add sulfuric acid, which makes the nucleic acids go into solution again 1. Then we boil the stuff - but only ONE of the test tubes! (We have 2 test tubes' worth of nucleic acid) a. Boiling in acid is a "hydrolyzing process" - it breaks up the nucleic acid into the nucleotide subunits, and then even FURTHER into the base and sugar and phosphoric acid subunits! 1. So now we have one test tube of "hydrolyzed nucleic acid" and another of "unhydrolyzed nucleic acid" Good times! 1. OK, remember we had sulfuric acid in there? Now we have to neutralize the solution! Details below a. We're going to use barium hydroxide, a base, to neutralize this solution a. We're essentially going to perform a titration, where we use litmus paper to figure out when the solution is acidic, when it is basic, and when it is neutral a. The chemical formula is this: H2SO4 + Ba(OH)2 -> BaSO4 + 2H2O i. Note that the precipitate (salt) which forms is barium sulfate - we will filter this out later! Protein Portion 1. OK, so remember that back in the day, we had protein and nucleic acid resulting from a centrifugationWell, now we're dealing with the protein portion, which is solid 1. We take half the protein and add pancreatic enzyme a. This enyzme will hydrolyze the protein into its amino acid subunits a. This simulates how the hydrolytic process is carried out naturally, because in real life it is done with enyzmes! 1. And the other half of the protein we add phosphate buffer, which will not hydrolyze it at all! 1. To both we add thymol crystals, which prevent the growth of bacteria Summary for Quiz Monday, June 06, 2005 10:28 AM Title Characterization of Some Macromolecules Gist of Experiment Use the method of chromatography to separate the proteins and nucleic acids earlier into their individual components Experiment Theory Chromatography is a technique that separates mixtures into their individual components o For example: If we put black washable ink onto a tissue, the ink will spread outwards from the place where we blotted it However, the various components of the ink can't all move at the same speed as it spreads out - so the components will visibly separate The pigment which moves the slowest will "stop" first, followed by the next slowest, and so on The
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