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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