Soybean Lipoxygenase
For the past two weeks we have been working with Lara on our project.
We are analyzing Soybean Lipoxygenase. Soybean Lipoxygenase is an enzyme
found in soybeans. Enzymes are catalyze that increase the rate of a reaction,
they are made up of amino acids which means enzymes are proteins. The job of
an enzyme is to speed up the rate of a chemical reaction. Our goal in this
experiment was to answer if all the amino acids in the enzyme are important.
Amino acids are basically the building blocks of proteins in which case we would
suspect that all are relativeley important however, to actually see which amino
acids are important to the soy bean lipoxygenase we had to purify the enzyme.
To do this, we needed a WILD type and two mutations, the WILD type is the
controlled variable with no mutation in the enzyme. The mutations in our two
enzymes were in different locations, one mutation was near the active site and it
was titled as I553W. The enzyme with the mutation farther from the active site is
known as L496F. This brings to our Hypothesis, we hypothesize enzyme I553W
will slow down the rate of reaction because we think the mutation will slow down
the reaction since its close to the active site. Enzyme L496F will slow down as
well since it has mutation however the mutation is farther from the active sight
which suggests that the reaction will not be slow as Enzyme I553W. The wild
type will be the normal reaction that will go faster than the enzymes with
mutations.
In order to get our DNA from the bacteria we must first break the cell
open. the process of opening the cell is called lysis. During lysis you shake the
bacteria inside a chemical that break the cell apart. then we put it in a centrifuge
and all the heavy parts of the cell are pulled down to the bottom and the lighter
stuff like the proteins stay at the top.
Affinity chromatography is the process in which we get our enzyme by its
self. Our enzyme has a tag on it that allows us to tell it apart from the other
enzymes. That tag also hooks on to the chemichal on the water. Everything else
flows through the column. After that we do three washes to make sure that we
only have the enzyme inside of the column. Then we do three elutions to make
sure that we take off the enzyme completely.
Gel Electrophoresis is a process that allows us to check that we had the
right kind of enzyme after the affinity chromatography. We can look at the results
of the gel electrophoresis and if by elution three we have mostly our enzyme then
we know that we did the affinity chromatography right. Also it allows us to see
what was came out of the washes and the first two elutions.
At the end of the the experiment we tested our enzymes in Linoleic Acid
Hydroperoxide. This acid helps us so we can see when the reaction occurs. Our
enzyme goes through the reaction of 234 nm. We tested our reaction by using
the spectrophotometer. The spectrophotometer measures the absorbence of our
product. We put all three of the enzymes in a container at seperate times with
the acid and used the machine to see the absorbence rate of each individual.
Once we put our enzymes in the spectrophotometer, the rates came out in
a graph. The wild type went the fastest. Then the second L496F was the second
slowest it was 5 times slower. The slowest one was I553W this was 50 times
slower. This mutation was the slowest because it was closer to the active site.
The closer to the active site the more of an effect it will have on the reaction rate.
The second slowest was further from the active site so it had less on the impact
on the reaction rate. This enzyme was not need to conduct the chemical reaction.
At the end of this experiment we need to see if our hypothesis was
correct. Our hypothesis was the closer our mutation is to the active site the more
necessary it is for the chemical reaction. With the results we go from the
experiment and testing we saw that our hypothesis was correct. Since the
mutation closer to the active site went slower then the wild type which is normal
speed. This shows the mutation made could not conduct the chemical reaction
need and it will be a great disadvantage if this amino acid went missing. The
purpose of this experiment was to which amino acids however, this practice can
be applied in other experiments relating to proteins or enzymes. This is helpful to
us because some mutations in our body do no damage and others can do
damage such as defects.
We had a wonderful experience working with Lara on our projects and
experiments because we were able to acquire an insight on how do people work
in there environment. In this case it was in a lab and we learned many methods
and practices on how to use certain tools when working in a lab. One tool we
used for example, was the micropipettes. This tool was used to extract microliters which what we needed in order to get the proteins out and moving on to the
next step. While working in the lab, an instrument i found interesting was the gel
electropherises, it was surprising when we observed the results on a gel that was
violet. Overall, this experiment has changed our eyes in how look at science
because we worked together efficiently on the experiment and project. We like to
give thanks to our project leader Lara for teaching us patiently when sometimes
we did not understand however, she pushed us and we got through it.