# Lab 4 summary

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 $\log \left ( \frac{1}{T} \right )$
• 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