1. Enzymes Biol 101, Lab 8, Section 420 3.18.15
Control + Click for laser pointer

2. Objectives Understand the properties of enzymes
Describe factors that affect enzyme activity
Set up a controlled experiment to test enzyme activity
Make sure you understand catechol and catecholase before you leave
Make sure you can interpret the data you get in the second, cofactors experiment
Make accurate volumetric measurements
Set up timed reactions
Create and interpret graphical data

3. Enzyme Basics
Enzymes are proteins that act as catalysts (lower the activation energy of a reaction/ makes it go faster)
Enzymes are unchanged by the reaction
Enzymes attach to an active site on a substrate
High specificity…Today’s enzyme (O2 and Cat.)
Enzymes are usually named for the substrates on which they work
Lactose, Lactase
Catechol, Catecholase
Sucrose, Sucrase
This is pretty much the first paragraph of the introduction
Enzymes don’t make things happen that wouldn’t normally happen, they just help it happen faster than it naturally would
Substrate is just another name for reactant
Enzymes usually named for the substrate (sucrose, sucrase; catechol, catecholase)

4. Enzymes in This Lab
Catechol O2
Catechol: (Substrate) found naturally in fruits and vegetables, just under skin, colorless
Catecholase: (Enzyme) acts on oxygen and catechol to form benzoquinone and water, reaction would occur anyway but this enzyme speeds it up to happen in a few seconds
Benzoquinone: (Product) antibacterial properties, prevents spoiling of fruits, brown/orange color
Because of the color change from colorless  orange, we can trace the rate of the reaction like we did in photosynthesis using spectrophotometers
Catecholase
Second Paragraph of Report
Talk about apple example. You cut up an apple for lunch, leave it on the counter and come back, it’s brown… why? Benzoquinone. Bruises on apples are squishy, and so is the flesh of an apple after it’s been cut, why? Water
Benzoquinone
H2O

5. We will run two experiments
You will need to address both in your lab report. You should have 2 hypotheses, talk about both methods…
Temperature
Cofactors

6. Procedure: Temperature Controls
Review Spectrophotometer procedure
Why do we measure a blank calibration tube?
%T vs. %A
Absorbance= 2- log10(Transmittance) so %A is on a scale from 0-2
Color change from colorless  Orange/Brown
Spectrophotometer set to 540 (green)
Colors that your eye sees are actually reflected off of the object
All other colors are absorbed, so if something is yellow, purple light has been totally absorbed, and yellow has been reflected
In this experiment, the orange/brown is reflected and the green is absorbed with a higher saturation of orange color. The more orange it appears, the higher the absorbance of green light.
Colorless catechol will absorb no green light
Do some examples with absorbance and reflection

7. Expectations: Temperature Controls
Small
Change, Low ∆A No
Change, ∆A = 0
Large
Change, High ∆A
Nope.
∆A = 0
Tube 1
Substrate Alone
Tube 2
Enzyme Alone
Tube 3
Substrate + Enzyme
Tube 4
Water (Calibration, not control)

8. Enzyme Solution Preparation
Remember to ask why at each step. Include this detailed information in your materials and methods of the lab report.
Why am I using a chilled blender? What do you know about proteins like enzymes and heat?
Why strain the solution? Think back to photosynthesis lab.
Why cut the potato up before blending? Why do we blend instead of cutting as in the apple example we talked about?
Why overflow containers?
Etc…
Note New Pipettes
Reaction is FAST, add enzyme solution last
Recommend calibrating, then adding the enzyme right before your first measurement for each tube

9. Actual: Temperature Controls
There is a some catechol in our potato solution
SO when we thought we had only enzyme solution, there was actually a small amount of substrate too… potential for reaction
In undamaged cells the small amount of catecholase is stored in vesicles and doesn’t interact with catechol
Small
Change, Low ∆A
Little to no change
Tube 1
Substrate Alone No
Change, ∆A = 0
Some change, ∆A higher than expected
Tube 2
Enzyme Alone
Large
Change, High ∆A
Still the largest change, because of intentional substrate addition
Tube 3
Substrate + Enzyme
Nope.
∆A = 0
No, ∆A = 0
Tube 4
Water
How does this function as a control? In the next part of the experiment, temperature experimental, we can use these measurements as a baseline for the change that would already occur… allows us to normalize our data and account for the substrate that may exist in the enzyme solution already.
Report: Talk about this in your introduction. Do not include procedures/materials/methods. Do not include any numerical data. Just talk about why this control was necessary, why what you did works, etc.
Tube 1
Substrate Alone
Tube 2
Enzyme + Little Bit of Substrate
Tube 4
Water (Calibration, not control)
Tube 3
Enzyme +Substrate

10. Experimental: Temperature
9 Test tubes in this round
Even #s will have catechol and enzyme
Odd #s (except 9) will have only enzyme
9 is calibration tube… just H2O
Really, Kimwipe. It will help.
After experiment has run, you will calculate change in Absorbance, and normalize it by pairing an even tube with an odd. Your table in your lab notebook may have headings like below…
Graph Norm. Delta A v. Temperature
Denaturing may take time, freezing too
Temperature:
-you bite two apples, stick one in the fridge, one on the counter. Which one will turn brown faster?
-you bite two apples, leave one on the counter, cook one on the stove. Which one will turn mushier?
What temperature do you think this enzyme works best at? Not Body Temp.
Remember, if Absorbance of the green wavelength increases, that means the solution is growing more orange in color and the reaction is proceeding.
Prepare… measure
First two, in ice bath, take temperature
Second two, 3+ 4, left on lab bench at room temp
Third two, 5+6, 37 degree water bath
Fourth two, 7+8, 60 degree water bath
Wait 10 minutes, remove, shake, take off any parafilm (60 degrees will melt anyway), KIMWIPE, record absorbancy
Tube #
Temp. A0
A10
| A0-A10|
Normalized Delta Absorbance 1
Ice (degrees)
.35
.31
Tube 2 –Tube 1 2
.13
.32

11. Enzyme Solution Preparation 2
Why make a second solution?
Remember to ask why at each step. Include this detailed information in your materials and methods of the lab report.
Why am I using a chilled blender? What do you know about proteins like enzymes and heat?
Why strain the solution? Think back to photosynthesis lab.
Why cut the potato up before blending? Why do we blend instead of cutting as in the apple example we talked about?
Why overflow containers?
Etc…
Note New Pipettes
Reaction is FAST, add enzyme solution last
Recommend calibrating, then adding the enzyme right before your first measurement for each tube

12. Cofactors Cofactors are not proteins
Bond to enzymes and help them do their job in some cases
In this experiment we are working backwards, using guess and check
We know that the reaction works now, so the enzyme has it’s cofactors whatever they may be.
We want to find out what, if anything, is the cofactor, so we are going to remove each potential cofactor
Whatever element causes a plummet in productivity when removed is one that must be necessary for reaction… it’s the cofactor.
Potential cofactors can be removed with a chelating agent that bonds to it more strongly than an enzyme can, higher affinity, stronger attraction
EDTA takes away Calcium and Magnesium
PTU takes away Copper

13. Cofactors (CON’t) Three test tubes plus calibration
What is the control here?
What are the independent and dependent variables?
Why do we have to let it sit for 10 min?
Remember, if Absorbance of the green wavelength increases, that means the solution is growing more orange in color and the reaction is proceeding.
Graph Change in absorbance v. Chelating Agent Used or Cofactor Removed… 4 possibilities
Based on your data, what is the cofactor for catecholase that makes it possible for it to turn catechol into benzoquinone?
Remember EDTA – Calcium and Magnesium, PTU – Copper
Says do it for 10, but if you are running out of time, do it for 5 min.
Make one with EDTA, one with PTU, one with no chelating agent, calibration

14. Review *Not comprehensive*
What were the independent and dependent variables in each experiment? (Mention in intro especially)
Always justify your actions, why did you do what you did?
Why did we do the controls for the temperature lab?
In Results, talk about trends in your data, why do they exist, what do they look like?
In your discussion talk about whether your hypothesis was supported (not proved) and back up your analysis with some qualitative data.
Units on Graphs, Clear tables and graphs in appendix
You will likely not print this in color, your bar graphs may be difficult to understand. It is your responsibility to either use different patterns in Excel OR to distinguish the bars after you print. I drew diagonal lines on one of my bars that was indistinguishable, ex.
PAGE 91 !!
Don’t forget the front of your lab manual too!

15. Due Next Time Enzymes Lab Report Read Anatomy Lab Use Standards
Cite all material (Including Lab Manual)
Read Anatomy Lab