In this lab we will be researching the influence of various factors on enzymatic activity. This lab will cover two lab periods and one thing I need to point out right away is we will be wearing goggles and gloves in this lab. A few of our learning goals are to practice collecting and analyzing data.
That data will be based on the enzyme-substrate hypothesis model, so we're going to make some predictions based on the concept that enzymes and substrates interact, lowering the activation energy needed to run a reaction, and how various conditions might affect the rate of that reaction. We're going to investigate those over the next two weeks and we're going to write a lab report over our findings. There we go.
Okay so just a reminder what are enzymes? Enzymes are Proteins that are, if you remember, based on chains of amino acids. So you have a chain of amino acids in a certain order. That's known as the primary structure.
That's based on the DNA that coded for that primary structure. That primary structure can be folded up into a beta pleated sheet or an alpha helix, which is the secondary structure. Those structures themselves can fold around again, binding in various ways forming a tertiary structure. In some cases you have more than one tertiary structure coming together to form a quaternary structure, a very complex globular enzyme. Those enzymes have an active site.
which may bind to a specific substrate. Once that substrate attaches to the enzyme, the enzyme may again change shape slightly. They call that a confirmation change, a change in the shape of the enzyme in relation to the binding of the substrate.
The enzyme we are looking at this week is called catalase and the substrate is hydrogen peroxide. Catalase is found in the peroxisomes of your cell because hydrogen peroxide is produced accidentally, if you will. It's a waste product of certain fatty acid metabolism processes, and so you end up with peroxide being produced.
Peroxide can actually oxidize cells, so we break it down with catalase into water and oxygen gas. One cool thing about enzymes is they bind with substrates, they lower the activation energy needed to run a reaction. They do that in two ways, very important to understand this.
One way is by bringing substrates together like we see here in their proper orientation so they interact and react without needing to add more activation energy. Or they stress existing bonds allowing them to be broken without having to add additional. activation energy. Then once the products are produced and released the enzyme is still usable and goes around the cycle again.
So one thing to think about is that an enzyme is like a hammer. You can build a house with a hammer and when you're done you still have the hammer. Enzymes are the same way.
Enzymes do not get used up in the reaction. So the enzyme we're looking at this week is composed of four polypeptide chains. So four tertiary structures stuck together and it protects cells from oxidative damage as we produce that hydrogen peroxide when we break down fatty acids.
And one catalyzed enzyme can convert millions of peroxide molecules to water and oxygen every second. Here is the reaction for 2H2O2, so that's two peroxides will yield two water molecules. and a molecular oxygen.
This is what the setup will look like. We're going to use a pressure probe. That pressure probe will be attached to a cork.
We're going to mix our peroxide and in this case we're going to use yeast. Yeast is a good model organism in this experiment because yeast is just like you in that it has a nucleus. It has all the major organelles.
It's a eukaryote. And it has catalase just like your cells do. And we're going to put yeast into the test tube and peroxide.
Quickly put that stopper on and we're going to record a rate of change. Change in pressure in that tube over time. And we're going to look at how various things affect that rate. We can look at how different concentrations of peroxide affect that rate. So substrate concentration.
We could look at how temperature affects the rate of that reaction. We could look at how pH. affects the rate of that reaction. And if you can think of something else you'd like to test, we may be able to do that as well. The second week of the experiment, we will likely all test the effects of temperature on the rate of the reaction.
And we'll keep in mind that room temperature will be the control and that we really don't need to go above 60 degrees Celsius because that would not be biologically relevant as 60 is a little bit higher than the hottest temperature ever recorded in Death Valley. So. yeast will not experience that temperature anywhere else on planet Earth unless they're near a thermal vent or something. So what we'll do that week is we'll incubate yeast as well as peroxide at different temperatures for five minutes within three degrees of the target temperature and we're going to use temperature probes and thermometers to keep track of how cool or warm everything is.
It's going to be important to keep in mind we need to get them to temperature before we start the five minute timer. And we can use an incubation table and stagger when things go into the heat blocks and or into the ice baths in order to keep track of how long everything needs to incubate at temperature. Then we're going to use heat blocks like I said.
We can use ice baths to cool things off and heat blocks to heat things up. And the heat blocks are a little bit complicated. I have a series of Written instructions here that we can look at again in lab and I will also attach a video to this pre-lab video on how to use the heat block. We can start by adding water to the heat block to help transfer heat and then power the unit on on the back. You're then going to press the Set Calibrate button on the front of the unit until you see flashing like we see here.
Then use the Shift key to change decimal places. Then up and down arrows will allow you to select the target temperature. In this case I am moving the target temperature to 40 degrees.
Then I wait for the flashing to stop. Now the flashing has stopped and I press Start. The unit now begins heating up to the target temperature.