Enzyme Lab
Enzyme: Phenolase
• Reaction catalyzed
phenolase
Catechol + ½ O2-----------> 0-benzoquinone
0-benzoquinone + H2O--->
pink/gold melanin ---> ---> brown melanin
• Reaction detected by change in Absorbance
at 475 nm due to production of the pink/gold
melanin
Standard Reaction Mixture
Potato
Extract
(enzyme)
For Part A:
begin timing when
extract is added
1 ml
Phosphate
Buffer
3 mls
Catechol
(substrate)
1 ml
Read A475 at 30 seconds
and 5 minutes
For Part B: After pretreating the enzyme,
begin timing when
substrate is added
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
Choose
different
volumes of
enzyme, add
buffer to reach
4 ml
Adjusting the Standard
Reaction Mixture to
Produce Experimental Samples
Potato
Extract
(enzyme)
+
Phosphate
Buffer
4 mls
Catechol
(substrate)
1 ml
Varying the
Enzyme Concentration
Potato
Extract
(enzyme)
Phosphate
Buffer
+
Catechol
(substrate)
Choose
different
volumes of
substrate,
add buffer to
reach 4 ml
1 ml
4 mls
Must have at
least 2 ml of
buffered
solution,
including
extract
volume
Varying the
Substrate Concentration
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
Planning Next Week’s 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 instructor’s approval for your
plan BEFORE you begin the experiment