Testing the activity of enzymes.
The vast majority of enzymes are proteins. Their function is directly related to their very
precise 3-D shape (configuration). If that 3-D shape is altered, the enzyme will not
function. The enzyme and its substrate must fit together like a lock and key. In some
cases, cells control the activity of enzymes by temporarily altering their shape, but these
enzymes might be re-activated by returning to their original shape. On the other hand,
some enzymes (and other proteins) can be permanently altered and their function
irreversibly destroyed. This irreversible damage to enzymes/proteins is called denatured.
Once an enzyme/protein is denatured, its 3-D shape cannot be restored. A variety of
factors can denature enzymes. Excessive heat, extremes in pH, and the presence of
cellular toxins are a few examples. Heating to 100 degrees C is perhaps the easiest.
Altering pH is not so difficult, but to know the beginning and ending pH requires the use
of a pH meter. If we use heat, we can safely assume that we start at room temperature
(20 C) and take the enzyme to 100 C (boiling point of water). Give or take a few degrees
for fluctuations in atmospheric pressure (pure water boils at 100 C at one atmosphere of
pressure, but atmospheric/barometric pressure is in constant flux).
Candidate enzymes:
There is an enzyme in pineapples that digests (catalyzes the hydrolysis of) animal protein.
There is an enzyme in potatoes (and lots of other things) that catalyzes the breakdown of
hydrogen peroxide (H2O2) to water and oxygen gas. There is an enzyme in saliva that
digests starch (starch is officially "amylose" and the enzyme of which I speak is called
amylase). I could go on for the rest of my corporeal existence with this, but I will stop
for now with these 3, because they are all readily available and the reactions they catalyze
are easy to observe.
Pineapple fruits look a bit like pine cones, but they are in the plant family Bromeliaceae.
Bromelain is the common name given to the protein-digesting enzyme found in the
plant. Fresh pineapple juice is often used as a marinade to tenderize beef (it partially
breaks down the protein of muscle tissue). How can we observe bromelain activity
(protein digestion)? Gelatin is an animal protein (from cows) used in Jell-O. Gelatin is
what makes Jell-O gel. If the gelatin is digested, the Jell-O won't gel. Pretty easy.
Hydrogen peroxide (H2O2) is a harmful (toxic) byproduct of many metabolic processes in
many organisms. It must be chemically decomposed to water and oxygen. The reaction
is simple enough: 2H2O2 - 2H2O + O2. The enzyme that catalyzes this reaction is
called catalase. It must be an important enzyme if it is named for what every enzyme
does, and this is in fact the case. Almost all organisms produce catalase. Mammals make
lots of it, especially in liver cells. I don't like dealing with raw meat in the lab, so the
simple potato tuber (the modified, underground stem that we usually call "potato") makes
a lot of catalase too. I'm more comfortable with raw vegetables than I am with raw meat.
So how can we observe catalase activity? How about looking for and testing for the
release of oxygen gas? How can you tell if it's really oxygen?
Amylase is an enzyme that digests (hydrolyzes) starch to sugar. The digestion of starch
begins in the mouth as a result of the activity of salivary amylase. Saliva is always
available. How could you test for amylase activity? Well, if you know how to detect
starch and if you know how to detect glucose, you could determine if starch is being
digested. You know how to do this.
So have 3 readily available enzymes and we can easily detect their activity.
Designing an experiment:
You mission is to design a controlled experiment to answer the question: Does heating
_______ (whatever enzyme you are testing) to the boiling point of water (100 C) affect
its activity, and if so, how?
Use your template for controlled experiments as a guide. A controlled experiment must
have an experimental variable (the independent variable), and all other variables such as
reaction time, temperatures, amounts/proportions, etc. must be kept constant or
controlled. An experimental control is sometimes used. In the case of a liquid that
contains an enzyme (pineapple juice, potato juice, or saliva, for example) the
experimental control could be a liquid without said enzyme… water is the candidate of
choice.
Once you and your colleagues have designed such an experiment, you must present it to
your instructor who is a highly skilled biologist who can comment on your procedural
outline and determine if you have designed an experiment that will answer your question
and which adequately controls unwanted variables.
Once your experimental design has been approved, the necessary materials will be
provided. At this point you and your colleagues can conduct the experiment to answer
your question.
Note: Saliva is a body fluid, and in addition to amylase, it contains numerous bacteria
and viruses that we might not want to mess with.
Note #2: It is not necessary to work with large quantities of enzymes and substrates.
Keep your volumes as low as possible to make the necessary determination of enzyme
activity.
Note #3: If you look for experimental designs online, you might be cheating yourself out
of the opportunity to design your own experiment. However, once you come up with a
general outline and cannot avoid the temptation to compare your idea with those of
others, you absolutely MUST give credit to the source on information. Science demands
integrity. The sooner you learn this the better.