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Biol130L Exam Notes Full.docx

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University of Waterloo
Dragana Miskovic

1.Identification of Some Macromolecules Treatment controls - positive or negative - they are specific samples that are used to check if the experiemental conditions were set properly and/or if the procedure was followed properly so that the changes can be attributed to the actual results - prevent uncertainty Positive control – designed so that it gives a positive result when experiment is done properly, how data should look if experiment works Negative control – designed so that it gives a negative result when experiment is done properly Time points - if time is a variable, must control every time point in experiment - zero time point – samples must be collected and measured immediately after treatment (time zero) Intro Most abundant living material are Carbon, Hydrogen, Oxygen, Nitrogen, Sulfur, Phosphorus Four major biological macromolecules - carbohydrates - lipids - proteins - nucleic acids Grouped based on behaviour due to their functional groups Iodine test for Starch and Glycogen - iodine is pale yellow - Turns black-blue with starch – due to amylose reaction - Turns red-brown with glycogen – due to multibranching Plants and animals store glucose in compounds known as polysaccharides Plants – this polysaccharide is starch – made of glucose units linked by glycosidic bonds - Starch is made of two polymers – amylose and amylopectin - Amylose – unbranched helix, glucose joined by 1-4 linkages - Amylopectin – staright, branched, glucose joined by 1-6 linkages Animals – this polysaccharide is glycogen – same thing but larger Starch and glycogen differ in molecular weight, shape, branching - Glycogen is similar to amylopectin but more branched Benedict’s Test for Reducing Sugars Sugars can exist as straight chains or in ring forms Straight chain form - Terminal aldehyde group making it an aldose sugar – reacts in the benedicts test making glucose a reducing sugar To test presence of reducing sugar, solution is mixed with benedict’s solution (blue), then boiled If precipitate forms coloured yellow, green, red or brown, or combination, there is a reducing sugar ++ Benedict’s solution has cupric ions (Cu ). When mixed with reducing sugar, aldehyde groups reduce cupric ions to cuprous ions (Cu ). So, this combines with oxygen to form precipitate of cuprous oxide (Cu O)2 4Cu + 2OH + 2e  2Cu O + 2H 2 2e + - Amount of cuprous oxide formed is proportional to concentration of aldehyde groups *A sugar with a ketone (Fructose) can also reduce benedict’s solution. The C of ketose isn’t oxidized directly, but the conditions catalyze isomerization of ketose to an aldose through an enediol. This gives a false positive. Biuret Test for Protein Peptide bonds of proteins mixed with Cu ++ ions and alkali give a violet Cu ++- peptide complex. NaOH is added to make it alkaline for the reaction to take place. 2. Isolation of Some Macromolecules Yeast is held together with starchy binding material. Glucose (a cellulose-like polysaccharide) is present in the yeast cell walls, and glycogen, proteins and nucleic acids are present in the cytoplasm. Grinding the cells with sand will rupture the cell walls and cell membranes. TCA (trichloroacetic acid) is added to the sand-cell mixture. Polysaccharides (starch and glycogen) are soluble in TCA and they will go into solution in the TCA. Proteins and nucleic acids are insoluble in TCA and will remain in suspension. Suspension – particles in a liquid Supernatant – liquid Pellet – solid Centrifugation – fractionation technique in which centrifugal force sediments suspended particles at the bottom of the tube (pellet/ precipitate). The liquid remaining above the sediment is supernatant. By controlling the speed and time of centrifugal run, particles of diff sizes or properties can be separated from the same suspension. Pellets containing proteins and nucleic acids are mixed with NaCl. Nucleic acids are soluble in NaCl and will go into solution. Proteins are insoluble in NaCl and will remain in suspension. Proteins and nucleic acids are boiled which coagulates (changes from fluid to solid) the suspended protein molecules. Ethanol is added to nucleic acids because nucleic acids are insoluble in ethanol and will precipitate out of ethanol. It is then mixed with sulphuric acid because nucleic acids are soluble in sulphuric acid. On half is boiled. This is a hydrolyzing process because it will break up the nucleic acid into its nucleotide subunits and eventually into the base and sugar phosphoric acid subunits. Separated into 2 tubes. Mix contents of tube with glass rod. Touch glass rod to red litmus paper. Litmus should remain unchanged to confirm that the solution is acid. Keep adding Ba(OH)2 and test until the red litmus turns blue. This indicates slightly alkaline solution with pH 7. White precipitate forms in tubes. This is the salt (barium sulfate) that forms as a result of the reaction b/w base and acid. H2So4 (acid) + Ba(OH)2 (base) = BaSo4 (salt) + 2H2O (water) BaOH was used as base and sulfuric acid as acid because the salt formed is insoluble and will precipitate out, leaving a salt-free solution of nucleic acids. Pancreatic enzyme hydrolyzes proteins into their amino acid subunits. Protein hydrolysis can also be attained by boiling in a strong acid or base. Using pancreatic enzyme ressembles the naturally occurring hydrolytic process b/c in living cells, hydrolysis is carried out by enzymes. This process is also quicker and equally as efficient as boiling. Works best at pH 7 and buffered solution helps it stay at this pH. Thymol crystals prevent bacterial growth. 3.Characterization of some macromolecules Chromatography - Technique that separates mixtures into their individual components. - Ex. When you touch an ink pen on a tissue, the ink will spread out and separate into 3 distinct bands of different colours. As the water in the ink is absorbed by the cellulose fibres of the tissue, the pigments can’t keep up b/c they are absorbed by the cellulose fibres. - The degree of absorption of each pigment depends on the structure of each individual pigment. The one that sticks the most will be slowed down the most. Thus, each pigment has its own characteristic rate of movement and this difference in the rate results in the separation. Stationary phase in the chromatogram is the matrix - Inert substance like cellulose. 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. The mixture is applied to a spot on the matrix and the matrix is partially immersed in the mobile phase so that the application is above the solvent. As the solvent is absorbed by the matrix, the solvent dissolves the mixture and so allows the components of the mixture to move along the matrix. Solvent is very important. If the substances in the mixture are completely soluble in mobile pahse, they would move as fast as the solvent and no separation would occur. If the they were completely insoluble in the mobile phase, the substances wouldn’t move at all. Solubility, molecular weight, and polarity will influence the mobility of a substance. Chromatography stops when the solvent has almost reached the opposite end of the matrix. R frelative mobility factor) = Distance travelled by substance (cm) / Distance travelled by solvent (cm) R fs always constant for any given substance in a particular solvent system and matrix 4. Spectroscopy A spectrometer consists of a white light source, which is focused on a prism that separates white light into distinct narrow portions of the spectrum. Each distinct portion has a distinct wavelength and can be focused onto a sample specimen which dissolved in a suitable solvent housed in a tube called a cuvette. The beam reaches the specimen and gets absorbed, transmitted or reflected. The transmitted beam then strikes a photoelectric cell which generates an electric current that is proportional to the intensity of the transmitted beam. The electric current is measured by a galvanometer with a graduated scale. The scale is graduated in 2 ways: a) Percent transmittance which is an arithmetic scale b) Absorbance (A) or optical density (O.D.) which is a logarithmic scale To use a spectrometer, a substance is dissolved in a suitable solvent. A cuvette containing only solvent (blank) is put in the machine and it is zeroe
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