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EXPERIMENT 4

TITLE

: Enzymes

AIM

: 1. To study the effect of temperature on enzyme activity.


2. To study the effect of pH on enzyme activity.
3. To study the effect of enzyme concentration on enzyme activity.
4. To study the catalase activities in different tissues.

MATERIALS

: Test tubes, test tube rack, pipettes, thermometer, liver puree, deionized

& APPARATUS
water, ice bath, hydrogen peroxide, sodium citrate buffer with different pH,
chicken, apple, potato and carrot.

PROCEDURE

: Refer to Lab Manual page 25-28.

RESULTS

A. Control

Enzyme Activity (1-5)

Description

Positive Control

No bubbles produced

Negative Control

Bubbles produced overflow

1. Did the reaction give off heat?

Yes.

B. Effect Of Temperature On Enzyme Activity

Temperature (oC)

Enzyme Activity (1-5)

Tube 1 (ice bath)

1.0

Tube 2 ( room temperature)

27.0

Tube 3 (37oC)

37.0

Tube 4 (100oC)

100.0

1. Is there a temperature at which the catalase activity was the most effective? Was it the
temperature you were expecting? If it was not, how can you explain the results?

The catalase activity was the most effective at 37oC. Yes, it was the temperature I was
expecting. However, if it was not the results can be explained by stating that mistakes took
place when the experiment was conducted.

2. Why put the tubes of H2O2 (and not just the liver tubes) into the water baths?

The tubes of H2O2 are being left in the water baths to obtain the temperature similar to the
liver puree for a better and more accurate results.

C. Effect Of pH On Enzyme Activity

Enzyme Activity (1-5)


pH 3

pH 7

pH 11

1. At what pH does catalase exhibit the greatest activity?

At pH 7 does catalase exhibit the greatest activity.

D. Effect of Enzyme Concentration On Enzyme Activity

[Enzyme]

Enzyme Activity (1-5)

Description

100%

Bubbles produced overflow

10%

More bubbles produced

1%

Less bubbles produced

1. Were the results of this experiment as you expected? If they were not, can you explain why they
were not?

Yes. The results obtained from this experiment is similar from what I expected.

2. How effective is catalase? In this experimental setup, do you think you would detect a difference
in reaction rates between a 100% and a 50% liver solution?

50% enzyme concentration of liver solution produces less bubbles and reacts slower with
hydrogen peroxide compared to 100% enzyme concentration of liver puree. In other words,
the higher the enzyme concentration, a higher rate of reaction would be displayed.

E. Catalase Activity In Different Tissues

Tissue

Enzyme Activity (1-5)

Description

Liver

Bubbles produced overflow

Chicken

Less bubbles produced

Apple

Less bubbles produced

Potato

More bubbles produced

Carrot

Less bubbles produced

1. What do your results tell you about the functions of the different types of tissues?

The results show that different types of tissues have different types of catalase.

2. Why was it important that the puree of each substance is obtained by blending the same amount
of substance in a similar volume of water?

In order to achieve the same concentration for each substances.

QUESTIONS

1. List the conditions you tested to determine which catalase exhibited the greatest activity. How do
these conditions compare to those of a cell in the body?

The catalase exhibited the greatest activity at 37oC and in a pH 7 (neutral) condition. These

conditions are some what similar to the cell in the body. However, in the human body, the
pH value differs from one place to another.

2. H2O2 is commonly used as a disinfectant for scrapes and cuts. What exactly are you trying to do
when you apply H2O2 to your scraped knee? What on your knee causes the H2O2 to bubble up
when you apply it?

Hydrogen peroxide is used as an antiseptic to reduce the possibility of infection, sepsis or


putrefaction. The reason why it foams is because blood and cells contain an enzyme
called catalase. Since a cut or scrape contains both blood and damaged cells, there is lots of
catalase floating around. When the catalase comes in contact with hydrogen peroxide, it
turns the hydrogen peroxide (H2O2) into water (H2O) and oxygen gas (O2). Thus, causing
bubbles.

3. The browning that occurs when fresh potatoes and apples are cut is a result of this reaction:

catacholase
Catechol + O2 -----------> Benzoquinone
(colorless)

(reddish brown)

Why do mashed potatoes stay white?

This is because mashed potatoes are cooked. The heat from cooking them denatures the
enzyme polyphenol oxidase, which causes raw potatoes to turn brown through oxidation when
they are exposed to air. Since, mashed potatoes are cooked, polyphenol oxidase is absent
causing it to remain white.

4. Some people put fresh lemon juice on fruit salad to keep it from browning what might the
chemical explanation be for this practice?

Lemon juice is used on fruit salad to keep it from browning because it has a low pH value
which denatures the enzyme polyphenol oxidase that catalyzes oxidation. When the enzyme
denatures, it loses it structure and function. Without it being a catalyst, oxidation process
occurs more slowly.

5. a) The U.S Food & Drug Administration recommends that cooked beef be refrigerated for no
more than 3-5 days before it's eaten; for cooked fish they recommend only 1-2 days. Why do
you think fish might not keep as long in the refrigerator?

Probably because fish spend their entire lives in a fairly constrained low temperature due
to the chemical and bulk thermal proprties of water. "Meat" (land mammals) must be
able to endure much larger environmental swings. The structural proteins and enzymes
of
the mammals are designed to survive the extremes they might encounter without getting
out of control or denaturing (losing their operational structure). Most spoilage is due to
the action of cellular enzymes and/or bacteria. Cow meat in a standard US fridge (42F,
7oC) is well below the design temp of its enzymes, which may therefore have 1/8th the
activity they would have in a living cow. For an arctic ocean fish, that same temperature
is
above its normal ambient temperature, and the fish will steadily go bad, even if
scrupulously cleaned and prepared before storage.

b) It is recommended that uncooked ground beef is refrigerated for only 1-2 days as well. Why
might this be?

This is because ground meat has a larger surface area for bacteria to infect and the
temperature in the refrigerator can still support bacteria activity causing the beef to go
bad even when regfrigerated.

6. Fever is a common symptom of a viral or bacterial infection. What are two different functions of
a rise in body temperature in this case? What would the danger be if the temperature got too high
(above about 42oC, in humans)?

The two different functions of a rise in body temperature are first, to fight the infection and
second, to slow down the body's function. If the temperature got too high, most enzymes
would denature causing the body system to slow down and malfunction leading to death.

7. Lizards and snakes may often be found sitting in sunny spots (on exposed rocks, in the middle of
the road) in the morning. They do not used the heat generated by their bodies to heat themselves:
they obtain heat from the environment. After a cold night, they are sluggish and must heat up
before they can be active.

a) What do they need heat for?

They need heat to regulate their body temperature. They also need heat from the sun to
retain energy that they will need for locomotion besides helping them to metabolize and
function as a bodily organism.

b) From where does the body heat from mammals and birds come? (The Second Law of
Thermodynamics might help you answer this.)

Sugar is a complex molecule with lots of energy stored in its bonds. When we break down
sugar, and other molecules, that bond energy is released, creating a blip of heat. Mammals
and birds have to break down much more sugar than the rest of the animal kingdom to
maintain their constant temperature.

DISCUSSIONS

A. Effect Of Temperature On Enzyme Activity

Like most chemical reactions, the rate of an enzyme-catalyzed reaction increases as the temperature
is raised. A ten degree Centigrade rise in temperature will increase the activity of most enzymes by
50 to 100%. Variations in reaction temperature as small as 1 or 2 degrees may introduce changes of
10 to 20% in the results. In the case of enzymatic reactions, this is complicated by the fact that
many enzymes are adversely affected by high temperatures. As shown in Figure 13, the reaction
rate increases with temperature to a maximum level, then abruptly declines with further increase of
temperature. Because most animal enzymes rapidly become denatured at temperatures above 40C,
most enzyme determinations are carried out somewhat below that temperature. Over a period of
time, enzymes will be deactivated at even moderate temperatures. Storage of enzymes at 5C or
below is generally the most suitable. Some enzymes lose their activity when frozen.
B. Effect of pH On Enzyme Activity.

Enzymes are affected by changes in pH. The most favorable pH value - the point where the enzyme
is most active - is known as the optimum pH. This is graphically illustrated in Figure 14. Extremely
high or low pH values generally result in complete loss of activity for most enzymes. pH is also a
factor in the stability of enzymes. As with activity, for each enzyme there is also a region of pH
optimal stability. The optimum pH value will vary greatly from one enzyme to another, as Table II
shows:
Table II: pH for Optimum Activity

Enzyme

pH Optimum

Lipase (pancreas)

8.0

Lipase (stomach)

4.0 - 5.0

Lipase (castor oil)

4.7

Pepsin

1.5 - 1.6

Trypsin

7.8 - 8.7

Urease

7.0

Invertase

4.5

Maltase

6.1 - 6.8

Amylase (pancreas)

6.7 - 7.0

Amylase (malt)

4.6 - 5.2

Catalase

7.0

In addition to temperature and pH there are other factors, such as ionic strength, which can affect
the enzymatic reaction. Each of these physical and chemical parameters must be considered and
optimized in order for an enzymatic reaction to be accurate and reproducible.

C. Effect of Enzyme Concentration On Enzyme Activity

In order to study the effect of increasing the enzyme concentration upon the reaction rate, the
substrate must be present in an excess amount; i.e., the reaction must be independent of the
substrate concentration. Any change in the amount of product formed over a specified period of
time will be dependent upon the level of enzyme present. Graphically this can be represented as:

These reactions are said to be "zero order" because the rates are independent of substrate
concentration, and are equal to some constant k. The formation of product proceeds at a rate which
is linear with time. The addition of more substrate does not serve to increase the rate. In zero order
kinetics, allowing the assay to run for double time results in double the amount of product.

Table I: Reaction Orders with Respect to Substrate Concentration


Order Rate Equation
Comments
zero

rate = k

rate is independent of substrate concentration

first

rate = k[S]

rate is proportional to the first power of substrate concentration

second rate = k[S][S]=k[S]2 rate is proportional to the square of the substrate concentration
second rate = k[S1][S2]

rate is proportional to the first power of each of two reactants

The amount of enzyme present in a reaction is measured by the activity it catalyzes. The
relationship between activity and concentration is affected by many factors such as temperature, pH,
etc. An enzyme assay must be designed so that the observed activity is proportional to the amount
of enzyme present in order that the enzyme concentration is the only limiting factor. It is satisfied
only when the reaction is zero order.
In Figure 5, activity is directly proportional to concentration in the area AB, but not in BC. Enzyme
activity is generally greatest when substrate concentration is unlimiting.

When the concentration of the product of an enzymatic reaction is plotted against time, a similar
curve results, Figure 6.
Between A and B, the curve represents a zero order reaction; that is, one in which the rate is
constant with time. As substrate is used up, the enzyme's active sites are no longer saturated,
substrate concentration becomes rate limiting, and the reaction becomes first order between B and C.
To measure enzyme activity ideally, the measurements must be made in that portion of the curve
where the reaction is zero order. A reaction is most likely to be zero order initially since substrate
concentration is then highest. To be certain that a reaction is zero order, multiple measurements of
product (or substrate) concentration must be made.
Figure 7 illustrates three types of reactions which might be encountered in enzyme assays and
shows the problems which might be enountered if only single measurements are made.

B is a straight line representing a zero order reaction which permits accurate determination of
enzyme activity for part or all of the reaction time. A represents the type of reaction that was shown
in Figure 6. This reaction is zero order initially and then slows, presumably due to substrate
exhaustion or product inhibition. This type of reaction is sometimes referred to as a "leading"
reaction. True "potential" activity is represented by the dotted line. Curve C represents a reaction
with an initial "lag" phase. Again the dotted line represents the potentially measurable activity.
Multiple determinations of product concentration enable each curve to be plotted and true activity
determined. A single end point determination at E would lead to the false conclusion that all three
samples had identical enzyme concentration.

CONCLUSION

: Enzymes work best at the optimum temperature that is 37oC and at the
optimum pH of pH7. Different tissues have different catalase.