Lab 4 summary CC-BY-NC

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Summary of Lab 4 for BIOL 112 (Winter 2011). Week of Jan 31, 2011.

Macromolecules: starch and cellulose
Enzymes: salivary amylase and succinate dehydrogenase

1Introduction to biochemistry

  • Chemical reactions catalysed by specific proteins
    • Catalysts influence rate of reation, recovered unchanged
    • Each enzyme has its own particular substrate (substance upon which it acts)
    • Has an active site - arranged geometrically to bind only to specific types of molecules
    • Active site gives the properties of specificity and catalysis
    • Changing the structure will cause it to lose catalytic properties (e.g. oboiling)

2Benedict's test

  • To check for the presence of a reducing sugar
    • If positive, will change colour from green to yellow to orange to red
    • Tests positive for glucose, but negative for sucrose (not a reducing sugar)
    • Sucrose: glucose and fructose, bonded so that the reducing ends of both are lost
  • Molecules possessing aldehyde functional groups will test positive
  • Maltose and lactose will also test positive

3Iodine test

  • To check for the presence of starch
  • If positive, will turn blue/black/purple
  • Positive for starch solution, negative for cotton (because it's just cellulose)

4Starch and cellulose

  • Macromolecules: carbohydrates, proteins, nucleic acids
  • Each: polymers
  • Carbohydrates: derived during photosynthesis from carbon dioxide and water
    • Examples: sugar, starch (grains, seeds etc), cellulose (wood)
    • Three types: monosaccharides, disaccharides, polysaccharides
    • Classified based on the number of simple sugar molecules linked together
  • Starch and cellulose both polysaccharides, made up of many glucose molecules linked together
    • But in starch, alpha bonds, so the same orientation
    • Cellulose, beta bonds, orientation varies
  • Experiment: learn about the sub-units of the two polysaccharides, as well as specificity of enzyme action
  • Procedure for starch:
    • 8 test tubes
    • 4 water, 2 HCl, 2 amylase
    • For two water, benedict/iodine test immediately
    • The other two water, 30 mins at 100 C, then tests
    • For the amylase, 45 mins at 37 C, then tests
    • For the HCl, 30 mins at 100 C, then tests
  • Results for starch:
    • Water + Benedict negative, because starch is not a reducing sugar by itself
    • Water + iodine test positive, because starch contains starch (...)
    • Amylase + benedict's test positive, because amylase COULD break down the starch into its subunits (glucose)
    • Amylase + iodine test negative, because it was broken down into glucose (i.e. not starch anymore)
    • HCl + benedict's test positive, because the HCl and heat broke it down into its glucose subunits
    • HCl + iodine test negative, for the above reason
    • water + 30 mins at 100 C + benedict = negative, that was not enough to break it down
    • ^ with iodine = positive, see above
    • Basically, Benedict tests if it's broken down, iodine confirms that it's not
    • Note: neutralise the acid with sodium hydroxide first, because Benedict's test is always negative in an acidic solution
  • Procedure for cellulose:
    • 8 test tubes, 4 water, 2 sulfuric acid, 2 amylase
    • Pretty much the same as with starch
  • Results for cellulose:
    • Water + benedict, negative because it was not broken down
    • All the iodine tests were negative because there is no starch ...
    • Amylase + benedict = negative because amylase can't break down the beta bonds in cellulose
    • sulfuric acid + benedict = POSITIVE because acid and high heat can always break the bonds
    • water + heat + benedict = negative, not enough to break the bond
  • What we learned from these experiments etc
    • both composed of glucose subunits
    • Amylase could only break starch and not cellulose into its subunits
      • Glucose monomers joined in same direction; orientations switch in cellulose
    • Specificity of enzyme action - enzymes specific to substrates and reaction
      • In this case, amylase was specific breaking down the alpha bonds in starch

5Enzymes

  • Dehydrogenation in living cells: hydrogen removed from molecules
    • Dehydrogenases - enzymes that influence this
    • Hydrogen transferred from one hydrogen carrier to another
    • Eventually combines with oxygen to form water
    • If an artificial hydrogen carrier is introduced, hydrogen may be diverted to it instead of oxygen
    • For example, the blue dye DCIP ... which incidentally becomes colourless when it accepts hydrogen
  • The enzyme succinate dehydrogenase, from the mitochondria of plant and animal cells, catalyses a step in the Krebs cycle
    • Succinate is dehydrogenated to form fumarate; usually, a derivative of riboflavin accepts the hydrogen that is removed
    • But if we add DCIP, it will take up the hydrogen, and so we can measure the rate of the dehydrogenation by the rate of colourlessness of DCIP
    • Note: this reaction must be done in the absence of oxygen, otherwise DCIP-H2 will spontaneously react with oxygen to form water and blue dye again
      • We used azide to block electron transfer to oxygen
      • Essentially, it binds with oxygen, so it can't react with the hydrogen
  • Competitive inhibitors: chemically resemble the natural substrate
    • In this case, malonate resembles succinate
    • So if we add enough malonate, it will complex with the enzyme, so there is none left to catalyse the succinate
    • Note: succinate deyhydrogenase is unable to dehydrogenate it due to valence difficulties
  • Spectrophotometry: measure light absorption at a given wavelength of light
    • Used in the visible or UV light range
    • As DCIP becomes colourless, light absorption will increase as the reaction proceeds
    • Thus the rate of the dehydrogenase reaction is proportional to the rate of change of absorbance
    • % Transmittance: the proportion of light passing through the sample
      • Not a direct measure of concentration, however
      • Because the molecules shade each other etc
      • However, we can use transmittance to get absorbance, which is equal to <math>\log \left ( \frac{1}{T} \right )</math>
      • Absorbance IS a direct measure of solute concentration
    • We have to use a blank solution (everything but the dye) for each test tube
      • Place the blank solution in, adjust the knobs to 100% transmittance
    • Procedure: 6 tubes + 3 blank tubes
      • Test tube 1: everything but malonate, 0.3 mL mitochondria (so very little enzyme)
      • Test tube 2: same as above but more mitochondria (0.9mL)
      • Test tube 3: Same as above, medium amount of mitochondria (0.6 mL)
      • Test tube 4: Added a bit of malonate
      • Test tube 5: No succinate
      • Test tube 6: boiled mitochondria
      • Note: amount of azide and DCIP and succinate same for all (except when succinate was not used)
      • Amount of assay varied depending on how much mitochondria was there (for a constant total volume I guess)
      • Had to vortex the mitochondria suspension each time, then add parafilm and invert
    • Results:
      • Test tube 1: Slow reaction due to not enough enzyme etc
      • Test tube 2: much faster reaction - more enzyme (although it tapered off near the end, as succinate was running out)
      • Test tube 3: medium rate of reaction
      • Test tube 4: Much less reaction due to the competitive inhibitor (but still some - not enough malonate I guess)
      • Test tube 5: No succinate led to little to no reaction (if there was some succinate in the mitochondria suspension there may have been some reaction)
      • Test tube 6: enzyme was denatured for the most part, so little to no reaction (incomplete denaturation would lead to some reaction)
    • Summary:
      • Initial rate of reaction proportional to enzyme concentration (but levels off over time)
      • Malonate inhibited the reaction, but still some change in absorbance because there was not enough malonate to inhibit it completely
      • Whatever