Enzyme Lab

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Enzyme Lab
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Enzyme Phenolase

• Reaction catalyzed
• Catechol ½ O2-----------gt 0-benzoquinone
• 0-benzoquinone H2O---gt
• pink/gold melanin ---gt ---gt brown melanin
• Reaction detected by change in Absorbance at 475
  nm due to production of the pink/gold melanin

phenolase
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Standard Reaction Mixture
For Part A begin timing when extract is added
Read A475 at 30 seconds and 5 minutes
For Part B After pre-treating the enzyme, begin
timing when substrate is added
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Experimental Design

• Choose independent variable to study
• Choose a range of values for the independent
  variable
• Hold all other factors constant, including
  reaction volume
• Plan the composition of experimental samples,
  using the standard reaction mixture as a model
• Make sure to have a control sample

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Adjusting the StandardReaction Mixture to
Produce Experimental Samples
Choose different volumes of substrate, add buffer
to reach 4 ml
Choose different volumes of enzyme, add buffer to
reach 4 ml
Must have at least 2 ml of buffered solution,
including extract volume
Varying the Enzyme Concentration
Varying the Substrate Concentration
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Experimental Design

• Use a 5 ml reaction volume
• At least 2 ml of this volume must contain
  solutions that have phosphate buffer at pH 6.0
  (except for pH experiment)
• You can satisfy this requirement with 1 ml of
  potato extract (already buffered) and 1 ml of
  buffer
• You have a choice of potato extract in phosphate
  buffer at pH 6 or potato extract in H2O
• Determine the order in which you will mix the
  reactants and decide whether some components
  should be preincubated together (under certain
  environmental conditions) before the reaction
  starts

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Planning Next Weeks Experiment

• Propose two possible hypotheses to study
• One of these two will be assigned to your group
• Prepare a detailed experimental plan for testing
  the assigned hypothesis
• Obtain your instructors approval for your plan
  BEFORE you begin the experiment