1. Soybean Lipoxygenase: Which amino acids matter?
By: Noreen Castellanos, Robert Zuniga, Gerardo Gallardo

2. Soybean Lipoxygenase Enzyme
            - A kind of protein that help a reaction go faster.
Amino acids
            - Are the building blocks of proteins.
            - There are 20 different kinds of amino acids.
           

3. Hypothesis In this experiment, we tested which amino acids are
important to the rate of 
the enzyme soybean 
lipoxygenase.
- We tested a wild type 
and two mutants.
- One enzyme (I553W) was mutated near the active site. The active site is where the reaction occurs. The other mutant (L496F) was mutated far away from the active site.
 - We hypothesize that the amino acid closer to the active site is more important than the one further away.

4. Experiment - To test our hypothesis we will purify each enzyme.
- We will measure the rate of reaction of each enzyme.
- If the reaction rate is slower for a mutant, then it shows that there is a major effect if we change that particular amino acid. 
- So if our hypothesis is correct then the reaction rate of the mutant closer to the active site (I553W) would be slower compared to the reaction rate of the wild type and the other mutant.

5. Centrifuge and Lysis In our experiment, we wanted purified enzymes.
- To obtain purified enzymes we had to break open bacterial cells which is known as lysis.
- To remove all the debris (such as cell walls), we centrifuged our cells.  All the debris went to the bottom and all of the proteins stayed on the top.

6. Affinity Chromatography
At this point we still need to get our enzyme by itself.
 - We poured the mixture of protein into a column
 - The tag of our enzyme sticks to the beads in the column.  
 - Then we poured buffer to clean out all the impurities. (Wash Step)
- Finally, we poured imidazole that breaks the bond between our enzyme and the beads, which gives us our enzyme by itself. (Elution Step)
                                                

7. Gel Electrophoresis
Gel electrophoresis is a technique we used to determine if our purification worked.
- We load our samples on the top. 
 - Light proteins will move down quickly, while giant proteins will not be as fast.

8. Data: what does our gel tell us?
Our gel tells us that our purification worked!
In the wash lanes, we see all of our impurities.
In the elution lanes, we see a single band that represents our pure enzyme! (soybean lipoxygenase) 
Wild Type
L496F
I553W
elution
wash

9. How do we measure a reaction rate?
- Linoleic Acid Hydroperoxide is the product that absorbs the light and what allows us to see the rate of the reaction.
- Because the product absorbs light at 234nm, we could use a spectrophotometer to measure the rate of reaction for each enzyme.
- A spectrophotometer is a machine that measures the absorbance properties of a sample.
Linoleic Acid Hydroperoxide

10. Data: What rates did we actually see?
- The wild-type reaction went the fastest, enzyme L496F was 5 times slower, and enzyme I553W was really slow (50x!). 
- The main reason that enzyme I553W was the slowest is because the mutation was closest to the active site and therefore it was more important for the reaction.
-The mutation in enzyme L496F was farther from the active site  and it was not as necessary to carry out the chemical reaction.
Wild-type Enzyme
L496F Enzyme 
I553W Enzyme

11. Summary: Was our hypothesis correct?
Original Hypothesis:  The closer a mutation was to the active site the more necessary it was for the reaction.
Conclusion: Our hypothesis was proven right by our experiment because we saw that the enzyme with the mutation closest to the active site was the slowest.