Enzyme Assays are a laboratory method to show and measure enzyme activity.
It is very important to test what an enzyme can do, test if a potential inhibitor can work on it, or how a co-factor can help.

Goal:

In this article, I will show you two examples of enzyme assays, which can help to show the process of deciding on a specific assay.
The first enzyme assay will be used to show and potentially measure the enzyme activity of bromelain on gelatin.
The second enzyme assay will be used to show and roughly measure the enzyme activity of peroxidase on different fruits and vegetables.

Background Info (Theory):

Bromelain is an enzyme commonly used in cosmetics and medication and is usually extracted from pineapple.
Bromelain has the ability to slowly break down gelatin, which can be used to show bromelain activity.
Peroxidase is an enzyme that various organisms use to break down hydrogen peroxide.
Different organisms have different amounts of peroxidase, and you can use hydrogen peroxidase to figure out which ones contain the highest amounts.
Apples contain relatively low amounts of peroxidase, but potatoes and horseradish contain high amounts of peroxidase, which can be shown when they interact with hydrogen peroxide.

The Procedure:

Step 1 (Extraction time):

For the first enzyme assay, the enzyme needs to be extracted first to get it in its pure form.
It’s quite simple to extract bromelain from pineapple, as it is water-soluble.
So you simply need to cut up some pineapple, to get the highest surface area possible and then put it in a tube filled with water.
Let the tube sit in a place without direct sunlight for about an hour, and the water will have a high enough concentration to be used in the enzyme assay.

Step 2 (Bromelain Assay):

During the extraction process, you can create the gelatin and put half of it in one test tube, and the other half in another test tube.
Label both tubes, so you which one is which, I label my tubes “-C” for control and “+B” for added bromelain.
Add equal volumes of water to the control tube and bromelain extraction to the bromelain tube and wait, until you see a difference.

Bromelain Assay Results and Conclusion:

After an hour or so you should see a stark difference between the two tubes.
The control tube will have no change, but the bromelain tube will have broken down gelatin in the bottom and it will almost look like precipitate.
This shows that pineapple contains bromelain, and this could then potentially be used to test if you added a potential inhibitor, would the breakdown of gelatin stop, or you could add a potential co-factor to see if more gelatin is broken down.

Step 1 (Setting up veggies):

Showing the presence of peroxidase is even easier than the previous enzyme assay, as you simply need to put different samples that you want to try out into different test tubes.
Then when you have your samples you simply need to add enough hydrogen peroxide to cover the samples.
Then depending on the sample the reaction will either happen instantly or will take a couple of seconds to happen.

Peroxidase Assay Results and Conclusion:

I tested six different samples, which are shown below, and it shows that root vegetables like horseradish and potato react the most out of the fruits and vegetables.
Then for the last picture, it shows that small samples of animal liver contain even more peroxidase than the horseradish, which makes sense, as the liver breaks down chemicals to make them nontoxic.

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