Name: _____________________

LAB #3: ENZYMES (based on a lab by Melanie O'Brien)
I. Introduction.

        Enzymes are proteins that play an essential role in metabolism: they catalyze
biochemical reactions, making the reactions occur much faster than they would occur
without the enzymes. Enzymes work by binding briefly with reactant molecules, holding
them in an orientation that accelerates the reaction, and then releasing the product
molecules. The location on the enzyme that binds to the reactants is called the active
site. The shape of the active site is critical: it must match precisely the shapes of the
reactants for the reactants to bind successfully. If an enzyme looses its shape it is said
to be denatured. A denatured enzyme will not work as effectively as one that is intact,
or it may not work at all. Various physical and chemical factors may cause enzymes to
denature. Today we will examine two such factors: changes in temperature and pH. We
will examine the affect of temperature on the enzyme rennin and the affect of pH on the
enzyme catalase.

II. Rennin and Milk.

       Rennin (or chymosin) is an enzyme that causes milk to coagulate. It occurs
naturally in the stomachs of calves, where it causes milk to curdle; the curdled milk
passes through the stomach more slowly, allowing for better digestion. (Our rennin
comes from biotechnology - no calves were killed to do this experiment.) Rennin has
been used for centuries in making cheese and other dairy products. The reaction it
catalyzes breaks casein, the main protein in milk, into two polypeptides. One of these
then joins together to form a network that solidifies the milk; useful to cheese makers as
well as calves.

       A. Rennin Experiment

Measure 15 ml of distilled water in a graduated cylinder, and then transfer it to a small
beaker. Using a mortar and pestle, grind-up a rennin tablet. Add the powder to the 15 ml
of water, and swirl it to mix - it will remain cloudy.

Use a wax pencil to label three test tubes with your initials and "C" (for cold), "W" (for
warm), and "B" (for boiled). Pour the rennin mixture into the three test tubes, so that
there is about the same amount in each. Put the C tube in the ice bath, the W tube in
the warm water bath, and the B tube in the boiling water bath.

Label three more test tubes with your initials and "C", "W", and "B". Put three squirts of
milk into each of these. (A squirt is as much fluid as a transfer pipette will hold if you
squeeze the bulb completely and fill it with as much as it will hold - about 1 ml.) Put the
C tube in the ice bath, and both the W tube and the B tube in the warm water bath.

                                          Lab #2 -1
Leave all the test tubes in their baths for about five minutes, and then transfer 10 drops
of the C rennin mixture to the C milk tube, 10 drops of the W rennin mixture to the W
milk tube, and 10 drops of the B rennin mixture to the B milk tube. Leave all of the milk
tubes in their water baths: C in the cold, W and B in the warm. Write down the time that
you finished adding the rennin to the milk tubes.

¿ Time: __________

You can discard the remainder of the rennin in the sink. Rinse out the rennin test tubes,
wipe-off the labels, and leave them in the wash tub.

Wait 30 minutes (you can begin the next experiment during this time), and then record
your observations on the three milk tubes below.

¿ Cold Milk: ____________________________________________________________


¿ Warm Milk ___________________________________________________________


¿ Milk with boiled Rennin _________________________________________________


¿Did your results come out as expected? If not, what might have gone wrong? ______



¿ Explain why the milk did not solidify under the cold conditions. __________________



¿ Explain why the milk did not solidify with the boiled rennin. _____________________



¿Which temperature was most like that found inside a calf's stomach? ______________

                                         Lab #2 -2
III. Acids and Bases.

        In pure water, at a low frequency, some water molecules split apart to form
hydrogen ions and hydroxyl ions: H2O  H+ + OH-. Thus, even pure water has some
ions of each type in it. Some materials, when dissolved in water, release more hydrogen
ions and increase their concentration in the solution. Such solutions are known as acids.
Other materials release hydroxyl (OH-) ions in water, making bases. In basic solutions,
hydroxyl ions combine with hydrogen ions to make water, reducing the concentration of
hydrogen ions. The acidity of a solution is measured with the pH scale, which measures
the concentration of hydrogen ions. The pH scale has the following properties:
       1. Neutral solutions, with equal numbers of H+ and OH- have a pH of 7.
       2. Acid solutions have a pH < 7; the lower the pH, the lower the OH-
       concentration, the higher the H+ concentration, and the stronger the acid.
       3. Base solutions have a pH > 7; the higher the pH, the higher the OH -
       concentration, the lower the H+ concentration, and the stronger the base.
       4. The pH scale is based on powers of 10, so, for example, a solution with a pH
       of 3 has 10 times the concentration of hydrogen ions as a solution with a pH of 4,
       and 100 times the concentration of hydrogen ions as a solution with a pH of 5.

        The chemical environment is very important to the survival of living organisms.
Changes in the chemical environment, especially changes in pH, can drastically affect
living cells. We humans, like most creatures, carefully regulate our internal chemical
environment to maintain optimal conditions for our cells. An example of this is our ability
to maintain a blood pH of close to 7.4 (slightly basic), regardless of external conditions
or what we have been eating.

       A. pH Test. The pH of a solution can be fairly accurately measured using test
paper that changes color in response to the pH of a solution.

 Samples of each fluid can be found in small beakers on the side of the room along with
boxes of pH paper. To measure pH, dip the colored pads on a piece of pH paper into
the solution and compare the color of the pads to the color chart on the pH paper

¿ Record the pH of the solution on Table 2, below.

Table 2. pH readings for test solutions.

Solution:     DI Water      Bottled       7-up          Grape         Milk          Rice
                            Water                       Juice                       Drink

pH:        _____      _____     _____      _____      _____      _____

                                         Lab #2 -3
¿Which solution is the most acidic? ___________________

¿Which solution is the most basic? ____________________

¿Which solution is nearest to neutral? ___________________

¿Do any of the results surprise you? Why? ___________________________________


IV. Catalase

      Catalase is a very useful enzyme. Hydrogen peroxide is a toxic chemical that is
produced as a byproduct of ordinary chemical reactions within cells. Catalase takes two
hydrogen peroxide molecules and converts them into two water molecules and a
molecule of oxygen: 2 H2O2  2 H2O + O2

Thus catalase takes molecules that are toxic and makes from them molecules that are
useful to the cell. Catalase is probably found in all living cells; for our experiments we
will use potatoes as a source of catalase.

       A. Catalase Experiment - Comparing Catalysts.

When working on this experiment you must wear protective gloves and goggles!

For this and the next experiment we will use the amount of bubbling produced in test
tubes as a measure of the reaction. Rate the amount of bubbling from 0 (no bubbles) to
3 (very strong bubbling). It may be hard to judge at first, but after you have done a few
tests you will be able to make reasonable ratings.

Cut two small cubes of potato - small enough to fit into the test tubes - from the inside of
a potato. Chop one of the cubes into tiny pieces.

With a wax pencil, label four test tubes A,B,C, and D. Put two squirts of hydrogen
peroxide solution in each tube.

To test tube A, add a small amount of sand - about the same amount as your chopped
To test tube B, add a similar amount of manganese oxide.
To test tube C, add the cube of potato.
To test tube D add the chopped potato.

                                         Lab #2 -4
¿ Rate the amount of bubbling in each tube. (0-3 scale)

      Tube          Bubbling

        A           _______
        B           _______
        C           _______
        D           _______

¿ Which substances showed some (any) ability to catalyze the reaction? ___________


¿ What was the purpose of testing sand? ____________________________________


¿ Why was there more bubbling when the potato was chopped than when it was left in a


¿What is in the bubbles? _________________________________________________

      B: Catalase Experiment: The Effect of pH.

Cut three fresh cubes of potato, the same size as before, and chop them into tiny
pieces, keeping the piles separate.

Using a wax pencil, label three clean test tubes "Acid", "Neutral", and "Base". Put one
chopped potato cube into each tube.

Add one squirt of 0.01M HCl (hydrochloric acid) to the Acid tube.
Add one squirt of Deionized Water to the Neutral tube.
Add one squirt of 0.01M NaOH (sodium hydroxide) to the Base tube.

Let the potato pieces soak in the solutions for 2 minutes.

Add two squirts of hydrogen peroxide solution to each tube, and

                                        Lab #2 -5
¿ Rate the bubbling (0-3 scale).

      _Tube_        Bubbling

      Acid          _______
      Neutral       _______
      Base          _______

¿ Did the pH treatment affect the catalase reaction? (yes or no) __________________

¿ Which treatment produced the most bubbling? _______________________________

¿ What do the rennin and catalase experiments tell you about the necessity for living
things to maintain temperature and pH homeostasis?






                                       Lab #2 -6

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