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Bio130L Lab Notes

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Heidi Engelhardt

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Biology Lab Study Notes Identification of Some Macromolecules - positive control: should give positive result; negative control: should give negative result - time points: if time is a variable, you have to have a control for every time point in experiment - most abundant elements in living material: C, H, O, N, S, P - biological macromolecules: carbohydrates (mono- and polysaccharides), lipids, proteins, nucleic acids Iodine Test for Starch and Glycogen - normally pale yellow (turns black-blue for starch, reddish-brown for glycogen), change = positive - plants and animals store glucose in the form of high molecular weight compounds (polysaccharides) - in plants, starch is a storage polysaccharide made of a large number of glucose units linked by glycosidic bonds - in animals, glycogen is the same as starch and is a larger polymer in terms of molecular weight - starch and glycogen differ in shape and degree of branching in final polysaccharide structure - starch is mixture of amylose and amylopectin: o amylose: an unbranched, helical molecule where glucose units are joined by ∝(14) linkages o amylopectin: has straight and branched sections resulting from ∝(16) linkages - amylose reacts with iodine for blue colour; reddish-brown colour is due to multibranched components Benedicts Test for Reducing Sugars - carbohydrates can exist as straight chains or ring forms: - in straight chain, aldehyde group ( ) makes it an aldose sugar - aldehyde reacts in Benedicts test to make glucose a reducing sugar - positive: coloured precipitate – yellow, green, red, brown - Benedicts contains blue cupric ions (Cu ), when mixed with reducing sugars the aldehyde group reduces the cupric ions to cuprous ions (Cu ), cuprous ions combine with oxygen, form precipitate of cuprous oxide (Cu O) 2 - ketose and aldose gives a positive test - false positive reaction could happen due to presence of other substances that could be oxidized Buret Test for Protein - used colour reaction of peptides and proteins that is not given by free amino acid - treatment of peptide bonds within proteins with Cu ions and alkali give a violet Cu peptide complex - positive: violet colour Isolation of Some Macromolecules - yeast contains yeast cells held together by a starchy binding material - glucan is in yeast cell walls; glycogen, proteins & nucleic acids are in cytoplasm - grinding cells with sand ruptures the cell walls and membrane - mix TCA (trichloroacetic acid) to sand-cell mixture o polysaccharides (starch and glycogen) are soluble in TCA and will go into solution o proteins and nucleic acids are insoluble and will stay in suspension - decant suspension into tubes - proteins and nucleic acids (suspension), carbohydrates (solution) - centrifugation: a fractionation technique in which force separates suspended particles at bottom of tube o sediment is a precipitate (or pellet) and the liquid is the supernatant - supernatants contain polysaccharides, pellets contain proteins and nucleic acids - add NaCl solution to pellets - nucleic acids are soluble (solution), proteins are insoluble (suspension) - add ethanol to nucleic acids, make a white suspension - nucleic acids are insoluble (precipitate to suspension) - boiling acid is a hydrolyzing process; will break up nucleic acid into component nucleotide subunits, and eventually into the base and sugar and phosphoric acid subunits Biology Lab Study Notes - neutralize with Ba(OH) 2 o touch wet end of rod to red litmus paper, unchanged = acid o add base slowly, when litmus paper turns blue = end of titration (blue indicates slightly alkaline solution) o should see white precipitate (salt – BaSO ),4BaSO is 4nsoluble and will precipitate out  H SO + Ba(OH)  BaSO + 2H O 2 4 2 4 2 - in living cells hydrolysis is carried out by enzymes Characterization of Some Macromolecules - chromatography: a technique that separates mixtures into individual components - ex: separation of pigments of black ink by touching pen to tissue so ink spreads into 3 bands (red, blue, green) - as water is absorbed by cellulose fibers of paper, pigments cant keep up (they‘re absorbed to cellulose fibers) - stationary phase: matrix; an inert substance like cellulose (in the case of paper chromatography) - mobile phase: solvent - separation depends on relative tendencies of molecules to associate more strongly with one of the other phase - mixture is applied to matrix and partially immersed in mobile phase so application is above level of solvent - as solvent is absorbed by matrix, solvent dissolves mixture allowing components to migrate along matrix - if substances are completely soluble in mobile phase, they would move as rapidly as the solvent, and no separation would occur - if completely insoluble in mobile phase, substances would not migrate - chromatography is terminated when solvent has almost reached opposite end of matrix - R fthe relative mobility factor) = Distance travelled by substance Distance travelled by solvent Spectroscopy - light: photons are light particles with no mass that move through space as a wave - human eye is sensitive between 400 nm and 750 nm (violet-red) (visible light spectrum or white light) - spectrophotometer: consists of a white light source which is focused on a prism that separates the white light into bands of radiant energy o each band has distinct wavelengths to be focused through a narrow slit on to a sample specimen o this chosen incident beam hits the sample specimen o the incident beam reaches the sample, and gets absorbed, transmitted or reflected o the transmitted beam then strikes a photoelectric cell which generates an electric current o the generate electric current is measured by a galvanometer with a graduated scale - the scale is graduated two ways: (a) percent transmittance (%T), or (b) absorbance or optical density - to be analyzed in a spectrophotometer, a substance is first dissolved in a suitable solvent - a cuvette containing only solvent (the blank) is inserted into the machine, and the scale is zeroed for that particular solvent - the blank is then replaced with a cuvette that contains solvent plus substance - any reading on the scale that is less than 100% transmittance or more than 0.0 absorbance is because the substance absorbed some of the incident light - the concentration of a light-absorbing solute is directly proportional to the absorbance over a given range of concentrations (Beer’s Law) - there are numerous pigments within chloroplasts which have the ability to absorb part of the visible spectrum - a pigment appears deeply coloured because it is unable to absorb the wavelengths of that particular colour; all other wavelengths are absorbed, but those it does not absorb are reflected or transmitted - pigments can be isolated form chloroplasts to determine their ability to absorb various wavelengths - the major pigments in chloroplasts are the chlorophylls and the carotenoids - Deep Green: spinach leaves are homogenized in anhydrous alcohol, homogenate is filtered through several layers of cheese-cloth and the filtrate was poured into beakers and allowed to evaporate to dryness Enzymes - enzymes are biological catalysts - all enzymes (excluding ribozymes) are proteins, and it has been shown that their specificity is determined by the number and order of component amino acids, and 3-D configuration of the protein molecule Substrate + Enzyme  Substrate-Enzyme Complex  Product + Enzyme Biology Lab Study Notes - temperature, pH, enzyme concentration, substrate concentration, product concentration, energy of activation, etc. affect the rate at which the enzyme converts the substrate into a product - salivary amylase acts on starch by breaking off maltose from starch chains - each rupture in the starch chain consumes a water molecule - hydrolytic reactions: require water to digest substrate (process is called hydrolysis) - phosphorylase acts on starch by “taking off” glucose molecules - phosphorylase ruptures glucose-glucose bonds by consuming phosphoric acid instead of water; hence it is done by phosphorylase and called phosphorolysis, not hydrolysis (Glucose) +nHPO 42-  (Glucose) n-1+ Glucose-1-phosphate - in the above reaction, the phosphoric acid in solution dissociates into hydrogen and phosphate - energy released is trapped in system being used to create a glucose-phosphate bond in glucose-1-phosphate - virtually no energy is released from system during phosphorolysis - water concentration will not affect direction of reaction, as it did in the case of the salivary amylase - direction of reaction mediated by phosphorylase is determined by concentrations of reactants and products - starch synthase is the main enzyme involved in the production of starch in the intact plant cell - when phosphorylase is isolated from plant tissue phosphorylase will convert glucose-1-phosphate into starch - during synthesis, an enzyme can only act by attaching glucose to a pre-existing starch primer molecule which must be made longer to give a positive iodine test (molecules are “short” and do not give a positive result) - phosphorylase will not synthesize starch if a primer molecule is present or act on a “naked” glucose - during synthesis, glucose must be present as glucose-1-phosphate Osmosis - diffusion: molecules moving from an area of high concentration to an area of low concentration - once equilibrium is reached, the molecules are still moving randomly, but at equilibrium - water is the solvent in which many other substance, all collectively called solutes, are dissolved - cell membrane: acts as a semipermeable membrane allowing water to pass through freely in both directions - osmosis: the movement of water through the cell membrane - isotonic: if the fluid that bathes a cell contains the same concentration of solutes as is found in the cytoplasm (the concentration of water on both ides of the cell membrane is identical, no net movement) - hypertonic: if the fluid that bathes a cell contains a higher concentration of solutes that is found in the cytoplasm (the concentration of water is greater in the cytoplasm than the surroundings, net movement of water will be out of the cytoplasm) - hypotonic: if the fluid that bathes a cell contains a lower concentration of solutes than is found in the cytoplasm (the concentration of water is greater in the surroundings than the cytoplasm, net movement of water will be into the cytoplasm) Osmosis in a Model System - the cellophane of dialysis tubing resembles the action of a cell membrane in the sense that it 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” will be monitored by weighing the “cells” Osmosis in Animal Cells - the cytoplasm of an animal cell is surrounded by a thin flexible cell membrane that is slightly elastic so small increases in volume can be contained (if too much water comes into the cell, the cell membrane will rupture) - lysis: the bursting of an animal cell; haemolysis: the bursting of a red blood cell - the hematocrit of whole blood is the percent of the blood that is made up of cells - ex: if a person has a hematocrit of 40, it means that 40% of the blood volume is cells and the rest is plasma - the average hematocrit of a normal human male is 42 and 38 for a normal human female - the degree of bodily activity, anemia, as well as altitude, are among some factors that affect hematocrit Osmosis in Plant Cell - the cytoplasm of a plant cell is surrounded by a cell membrane similar to the one found in animal cells - however, all plant cells have a cell wall outside the cell membrane which is composed of a fairly rigid cellulose network permeable to water - most plant cells have huge vacuole within the cytoplasm so it forms a thin envelope around the vacuole - isotonic environment: the vacuole is filled with sap - hypotonic environm
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