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Bio 130 Lab Final Review.docx

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Dragana Miskovic

Identification of Some Macromolecules - C, H, O, N, S, P are most abundant elements - 4 major biological macromolecules 1. Carbs (mono and polysaccharides) 2. Lipids 3. Proteins 4. Nucleic acids Identification of Some Macromolecules Test Beaker # Composition Iodine Test Benedict’s Test Biuret Test 1 15 mL of 1% glucose - + - 2 15 mL of 0.3% glucose-1- - - - phosphate 3 15 mL 1% maltose - + - 4 15mL honey - + - 5 15 mL 1% sucrose - - - 6 15 mL 1% lactose - + - 7 15 mL 1% glycogen + - - 8 15 mL of 1% starch + - - 9 15 mL protein - - + 10 15 mL beer - + - 11 15 mL water - - - Discussion of results - Glucose-1-phosphate (mono) and sucrose(di) are aromatic structures with no free aldehyde groups, can’t donate electrons to make benedict’s positive - Maltose and lactose are disaccharides - Honey contains reducing sugars: glucose, fructose, maltose - Beer has glucose, fructose and maltose - Iodine test o Normally very pale yellow o Blue-black in presence of starch o Reddish-brown in presence of glycogen - In plants, starch is the storage polysaccharide composed of glucose linked together by glycosidic bonds o Starch is a mixture of polymers, amylose, and amylopectin which reacts with iodine to give the characteristic blue colour o Amylose in starch  Amylose gives off blue colour - Glycogen for animals made of glucose, is larger than starch in terms of molecular weight o Glycogen is similar to amylopectin in overall structure, but is more highly branched - Starch and glycogen differ in their overall shape and degree of branching in the final polysaccharide structure - Cellulose is made of o Amylose  Unbranched, helical molecule where glucose units are joined by alpha (14) linkages o Amylopectin  Straight and branched sections resulting from alpha (16) linkages  Gives off reddish-brown color due to multi branched components - Benedict’s test for reducing sugars o Originally blue o Changes to a range of yellow or green to red or brown or a combination of these for positive results o All sugars can exist as straight chains or as ring forms o In straight chain form, there is a terminal aldehyde group H-C=O  Called aldose sugar  This part is what reacts in the Benedict’s test and makes glucose a reducing sugar o Benedict’s contains blue cupric ions (Cu++) o When mixed in a reducing sugar solution, the free aldehyde groups on the end of the straight-chain sugar molecule will reduce the cupric ions to cuprous ions (Cu+) o Precipitate of cuprous oxide (Cu2O) forms from cuprous ions bonding with oxygen o 4CU+ + 2OH- + 2e-  2CU2O + 2H+ + 2H+ + 2e-  Amount of precipitate formed is proportional to the concentration of free aldehyde groups in the solution - False positives could occur due to the presence of other substances that could be oxidized - Biuret test for proteins o Test for peptide bonds in protein  Solution turns purple from Cu++ ions-peptide complex  Cu++ from the biuret test binds to the nitrogen making it Cu+, (only basic solution because you don’t want nitrogen to bind to hydrogen).  The atoms surround the nitrogen is spread out in a different orientation and causes it to change its wavelengths, therefore, changing the color to a violet-ish color. - Starch has Amylose and Amylopectin o Amylose is the part that reacts with iodine test because it is not branched and allows iodine to enter the molecule. o Amylopectin is not involved in the iodine test - Glycogen has Amylopectin only o Very highly branched but also a very big molecule so allows some iodine to get in. 2. Isolation of Some Macromolecules - yeast + sand will rupture the cell walls and cell membrane - yeast contains yeast cells held together with a starchy binding material o glucan (cellulose-like polysaccharide) is present in the yeast cell walls o glycogen, proteins, and nucleic acids are in cytoplasm - TCA (trichloroacetic acid) o Polysaccharides (i.e. starch and glycogen) are soluble in TCA o Proteins and nucleic acids are insoluble in TCA and remain in suspension (solid particles) - centrifugation o the sediment is known as a precipitate (or pellet) o liquid (above sediment) is known as the supernatant - 10% NaCl o Nucleic acids are soluble in strong NaCl o Proteins are insoluble, remain in suspension - Boiling will coagulate the suspended protein molecules o Coagulate: Change to a solid or semisolid state. - Chilled ethanol o Nucleic acids are insoluble in ethanol and will precipitate out of solution to form a suspension - H2SO4 o Nucleic acids are soluble in sulphuric acid and will go into solution o BOILING  Boiling in acid is a hydrolyzing process; it will break up nucleic acid initially into its component nucleotide subunits, and eventually into the base and sugar and phosphoric acid subunits  Different from denaturation  Denaturation is the separation of a double strange into two single strands by breaking hydrogen bonds - BaOH 2as used as titrating base o Use litmus paper to test until it turns slightly blue. (indicates just past the pH of 7) o The salt formed in the above reaction is insoluble and will precipitate out, thus leaving a salt-free solution of nucleic acids o If the salt didn’t precipitate out but remained in the nucleic acid solution as a contaminant, it would interfere with the chromatographic separation of the nucleic acid - Pancreatic enzyme for protein o Pancreatic enzyme will hydrolyze the proteins into their AA subunits  could also be achieved by other methods such as prolonged boiling in a strong acid or base o PE simulates the naturally-occurring hydrolytic process b/c in living cells hydrolysis is carried out by enzymes o Invitro experiments are better than In vivo o Works best with pH 7  b/c protein is placed in the PE solution which is buffered, the mixture will tend to stay at this optimal pH  thymol (crystal) will prevent/minimize bacterial growth) 3. Characterization of Some Macromolecules - Chromatography is a technique that separates mixtures into their individual components - Degree of absorption depends of each pigment depends on the structure of each individual pigment o The one that sticks the most to the cellulose of the paper will be slowed down the most - Stationary phase in any chromatogram is the matrix o It is generally the inert substance like cellulose paper - Mobile phase is the solvent o Separation depends on the relative tendencies of molecules in a mixture to associate more strongly with one or the other phase o The matrix is partially immersed in the mobile phase in such a way that the application is above the level of the solvent o As the solvent is absorbed by the matrix, the solvent dissolves the mixture  Allows the components of the mixture to migrate along the matrix o Choice of solvent is crucial  If the substance was completely soluble in the mobile phase, they would move as rapidly as the solvent itself, no separation will occur  If substance was insoluble, the substances wouldn’t move at all  Factors such as…influence the mobility of a substance  Molecular weight  Overall polarity of compounds - Chromatography is terminated when the solvent has almost reached the opposite end of the matrix - Rf value (the relative mobility factor/relative frontal mobility) ( ) ( ) o Rf = ( ) ( ) o Rf is always constant - From farthest travelled to least for AA o Methionine alanine  - Formic acid (10 formic acid: 70 isopropanol: 20 water) was as the solvent for separating the substances - Keep applications superimposed on each other, in as small an area as possible o Superimpose to create a high concentration of the application - Refer to Figure 5: Chromatography jar with a chromatograph - Ninhydrin-acetone reagent o Spray entire chromatogram to cause the AA to turn purplish-pink - To separate AA, use acetic acid solvent (15 acetic acid: 60 butanol: 25 water) - View NA under UV lamp o All the nitrogenous bases or compounds containing nitrogenous bases absorb UV light, so that any compound with a base will appear as a dark spot on a pale background - Guanine is insoluble 4. Spectroscopy - Light is a form of energy: photons, light particles with no mass, move through space as waves - Energy content of the certain light depends on the wavelength - Human eye can see wavelength of 400nm (violet)-750nm (red)  visible light spectrum - A spectrophotometer consist of a white light source, which is focused on a prism that separates the white light into its distinct narrow portions of the spectrum-bands of radiant energy - Each distinct energy band has distinct wavelength and it can be selectively focused through a narrow slit and on to a sample specimen - This chosen incident beam this the sample specimen, which is usually dissolved in a suitable solvent housed in an optically selected tube called a cuvette - The incident beam reaches the sample, and gets absorbed, transmitted or reflected - The transmitted remnant, or transmitted beam then strikes a photoelectric cell, which generates an electric current that is proportional to the intensity of the transmitted beam - The generated electric current is measured by a galvanometer with a graduated scale - The scale is graduated in 2 ways: o Percent transmittance (%T)  Arithmetic scale with equidistant units graduated from 0 to 100% o Absorption (A) or Optical density (O.D)  Log scale with unequal divisions graduated from 0.0 to 2.0 o Readings outside of these ranges are not valid or useful measurements - To be analyzed in a spectrophotometer, a substance is first dissolved in a suitable solvent - Next a cuvette containing only solvent (called a blank) is inserted into the machine, and the scale is zeroed for that particular solvent by manually setting the needle at 100% T or 0.0 A. - The blank is then replaced with a cuvette that contains solvent + substance - Any reading that is less than 100% or more than 0.0 A is due to the substance absorbing some of the incident light o Therefore, the energy of the transmitted beam was less than the energy of the incident beam - The spectrophotometer used in lab measured entire visible spectrum, with slight overlaps into the UV range and infrared range o UV range (180-350nm) o Infrared range (780-300000nm) - The concentration of a light-absorbing solute is directly proportional to the absorbance over a given range of concentrations (Beer’s Law) o %T vs concentration (refer to figure 7 pg 33); exponential o Absorbance vs concentration (figure 8, pg 33); linear - First step in analyzing a substance through a spectrophotometer is to plot an absorption spectrum for that substance o Done by reading the absorbance of that substance at many different wavelengths - Once wavelength of max absorption is determined, the next step is to construct a concentration curve for that substance o Done by setting the spectrophotometer at the max abs. wavelength, and measure the absorbance values for several different known concentrations of the substance (min of 2, 3 is preferred) - Ideally, the same cuvette should be used for all samples, including blanks - Cuvette should always be whipped with lint free tissue - The major pigments in chloroplasts are the chlorophylls (a and b) and the carotenoids (carotenes and xanthophylls) - Solvent used in chromatogram of fast green is 90 petroleum ether: 10 acetone - From farthest to closest on chromatogram: o Carotene is shown as orange band o 2 xanthophylls bands of yellow o Chlorophyll a with band of green o Chlorophyll b with band of green - Chlorophyll mostly absorbs blue (~400-460) and red ( ~660-700) Lab 5: Enzymes - Enzymes are biological catalysts o Thousands of enzymes are required to mediate all the reactions necessary for normal cell function o All enzymes (except ribozymes) are proteins o Specificity is determined by # and order of AA and 3D structure o Combine with a substrate (reactant) to form a substrate-enzyme complex  Breaks down into the unaltered enzyme and a product  Substrate + enzyme  substrate-enzyme complex  product + enzyme  Substrate enzyme> product o Temp, pH, enzyme concentration, substrate concentration, product concentration, energy of activation affect reaction rate o All enzyme-mediated reactions are reversible  Direction depends on conditions under which the reaction is taking place - Salivary amylase o Digestive amylase found in saliva o Acts on starch  Breaks off maltose from ends of the starch chains  Each rupture in the starch chain consumes a water molecule  Hydrolysis - McIlvaine’s buffer o Used to maintain an optimal pH for salivary amylase activity (37C) - Phosphorlyase o Enzyme that acts on starch by taking off glucose molecules o Ruptures glycosidic bonds by consuming phosphoric acid instead of water  Phosphorolysis  REFER TO PG 41!~ o Energy released in rupturing the glucose-glucose bond is trapped in the system by being used to create the glucose-phosphate bond in the glucose-1-phosphate  No energy is released from the system during phosphorolysis  Water is neither a product nor a reactant in the reaction o Reaction mediated by phosphorylase is mainly determined by the relative concentrations of the reactions and products - Starch synthase is the main enzyme involved in the production of starch in a plant cell o Phosphorylase can convert glucose-1-phospahte into starch in appropriate concentration gradients - When synthesis takes place, the enzyme can only act by attaching the glucose to
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