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

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

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)

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

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

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

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)

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

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

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 .35 - .31 Tube 2 –Tube 1 2 .13 .32 .13 - .32

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

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

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

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!

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