Enzymes

ACTIVITY # 5
Biomolecules Lab

Prepared by Justin Rachelle Dimaguiba

I. Catalase activity in Pineapple
Measurement of the rate of catalytic activity with varying amounts of the substrate (H 2O2)

Improvised eudiometer
(inverted 10mL graduated cylinder)

Glass tubing

To be replaced with
Erlenmayer flask
(completely sealed)
Contents:
• 50mL pineapple extract
• 8 mL 0.1M NaCl buffer
• 2mL of H2O2 (30% v/v)
 Factors affecting enzyme activity
Substrate concentration
• An increased substrate concentration will
increase the rate of enzyme activity
• Velocity- improvised eudiometer (mL/min)
 Principle of Catalase Test
• The enzyme catalase mediates the breakdown
of hydrogen peroxide into oxygen and water.
• Hydrogen peroxide was introduced into the
presence of the bromelain enzyme in
pineapple extract
• rapid elaboration of oxygen bubbles occurs
 Factors affecting enzyme action
1. Enzyme concentration
• Increased enzyme
concentration=
velocity of the
reaction increase
• for determining the
activities of serum
enzymes during the
diagnosis of diseases.
 Factors affecting enzyme action
2. Substrate concentration
• the rate of enzymatic reaction increases as the substrate
concentration increases until a limiting rate is reached
• after which further increase in the substrate concentration
produces no significant change in the reaction rate.
 Factors affecting enzyme action
3. Effect of
temperature
• Enzyme activity occurs
within a narrow range of
temperatures
• Optimal temperature – a
point at which an enzyme is
more active
• 37 to 40C°
 Factors affecting enzyme action
4. Effect of temperature

• Enzyme activity gradually lowers as the temperature rises more

than the optimal temperature .
• Enzyme activity stops completely due to the change of its
natural composition.
• temperature below the optimal temp.,enzyme activity lowers
• enzyme activity stops completely at 0 C°
• if the temperature rises again, the enzyme gets reactivated
 Factors affecting enzyme action
5. Effect of pH
• Enzymes are protein substances that contain acidic carboxylic
groups (COOH–) and basic amino groups (NH2).
• Enzymes are affected by changing the pH value.
• Optimum pH: maximum efficiency of the enzyme
• If the pH is lower or higher than the optimal pH, the enzyme
activity decreases until it stops working
Example:
• pepsin works at a low pH, i.e, it is highly acidic
• trypsin works at a high pH, i.e, it is basic
• Most enzymes work at neutral pH 7.4
 Factors affecting enzyme action
6. Effect of activators
• Some enzymes may require certain inorganic
metallic cations (Mg2+, Mn2+, Zn2+, Ca2+, Co2+,
Cu2+, Na+, K+ etc.) for their optimum activity.
• Rarely, anions are also needed for enzyme
activity. e.g. a chloride ion (CI–) for amylase
 II. Effect of pH on salivary amylase
activity
pH Distilled 0.05 N HCl 0.05 N NaOH
water pH 2 pH 13
pH 7

Enzyme activity activated inactivated inactivated

Reaction with Yellow- blue solution blue color

Lugol’s iodine orange
 III. Effect of temperature on
salivary amylase activity
TEMPERATURE Room Water bath Boiling water
Temp 370C (human bath
(250C) body temp) (95-970C)

Enzyme activity inactivated activated Enzyme

denaturation

Reaction with blue Yellow-orange blue

Lugol’s iodine
 Answers on Enrichment Activity
1. Effects on the function of proteins as affected
by pH, temperature, and other factors
(answered on slides 6-11)
 Answers on Enrichment Activity
2. Molecular explanation of functionally
inactivated protein subjected to denaturing
factors ( pH, temperature, etc)
• disruption of the alpha-helix and beta sheets
in a protein
• uncoiling it into a random shape
• Denaturation occurs because the bonding
interactions responsible for the secondary
structure (hydrogen bonds to amides) and
tertiary structure are disrupted.
 Answers on Enrichment Activity
4. Repair mechanisms of misfolded or denatured
proteins
•Gentle denaturing, removal of
denaturing agent may restore the original
attractions between the amino acids reshape
the protein to resume its function
Ex: Proteins in a slightly beaten egg can refold.
•Extreme denaturation cannot be reversed.
 Answers on Enrichment Activity
5. Is it enough to observe color changes in
determining enzyme activity? Why?
Photometric protein determination
• based on the relationship between
concentration and light absorbency
• Beer-Lambert Law, which states that the
concentration of a light-absorbing species is
proportional to its absorbance
 Answers on Enrichment Activity
6. Are enzymes in fruits and vegetables still
functional upon exposure to air?
•Enzymatic browning is one of the most
important reactions that occur in fruits and
vegetables, usually resulting in negative effects
on color, taste, flavor, and nutritional value.
•polyphenol oxidase (PPO)- triggers the
generation of dark pigments upon exposure to
air.
 Answers on Enrichment Activity
7. If 2 proteins have the same amino acid
sequence, can they have different functions?

Isomers
each of two or more compounds with the same
formula but a different arrangement of atoms in
the molecule and different properties.
 Answers on Enrichment Activity
8. Will the enzymes in fruits and vegetables
helpful in physiological activities in our bodies?

Enzymes from fruits and vegetables can also

help to prevent diseases
 Enzymes from fruits and
vegetables
Papain
• Found in unripened papaya
• helps break down proteins into smaller units,
called amino acids
• useful for treating bedsores, skin ulcers and
wounds from surgery
• may also promote wound healing from burns
 Enzymes from fruits and
vegetables
Asparagus
• contains glutathione peroxidase, an enzyme
the body uses to create antioxidants, which
are used to combat cancer-causing agents
• best eaten raw, as the cooking process
destroys much of the glutathione peroxidase
• Asparagus is also high in folic acid and other
vitamins and minerals.
 Answers on Enrichment Activity
9. Is it better to eat fruits/ vegetables raw or
cooked?
•Many enzymes are destroyed when heated
above 115 degrees F, it's better to eat raw fruits
and vegetables for the maximum benefit.
•the highest amounts in raw form include
apples, avocados, carrots, grapefruit, spinach
and tomatoes
Thank you!