Enzymes are used to catalyze reactions, so they can metabolize more quickly, without
expending any additional energy.
The majority of enzymes are proteins, and are specific to
But because these “biological systems are very complex and difficult to study
,” (Farrell, 2006), enzymes are isolated from their systems usually through a combination
of different methods and examined individually.
In this experiment, Lactate dehydrogenase
(from now on referred to as
enzyme found in the cell cytosol, which makes it easy to isolate” (Farrell, 2006) will be purified
through a series of centrifuging, salting out and affinity chromatography.
Its subunits will be
analyzed by electrophoresis and the remaining LDH will be assayed to examine its kinetics.
“Enzyme kinetics is the study of enzyme rates and how these rates are affected by enzyme
concentration substrates, and any inhibitors or activators,” (Farrell, 2006).
Centrifuging seperates substances in a solution based on their densities.
The denser a
substance is the further down the test tube it falls.
The more force that is applied in the
centrifuge, the more substances will break apart and separate.
For example, at “10,000 g
mitochondria, peroxisomes, and lysosomes will spin down” (Farrell, 2006) but at 100,000
can begin to break apart the endoplasmic reticulum.
Centrifuging will also spin down any
precipitates that may form.
During the “salting out” process, a salt is added to the solution containing the enzyme to
The salt, in this experiment (NH
, is added slowly to prevent the protein from
forming hydrogen bonds with the water molecules.
This causes the proteins to react with each
other instead of the water and precipitate out of solution.
This sample can then be centrifuged
again and decanted to separate the protein.
Affinity chromatography uses specific resins that are chosen based on its ability to bind
to the protein of interest, in this instance, LDH.
The LDH binds to the resin, while the remaining
proteins are eluted out.
Once the remaining proteins are washed out of the column, the LDH can
be eluted out by using a concentrated salt solution to “disrupt the [electrostatic] interactions and
cause the bound molecule to be released,” (Farrell, 2006).
Electrophoresis involves the use of an electric current running through a gel medium and
moving charged particles across the medium.
The velocity at which a molecule moves across the
gel depends on its charge, the more negative the charge, the faster it will be.
Likewise, the larger
the molecule the faster it will be, this is because larger proteins are more negative
Materials and Methods:
Purifying LDH from crude protein:
Begin the experiment by cutting up the beef heart in to small pieces, and trim off any fatty or