Biology Department
Physiology Lab manual
Enzymes
Objectives
The student will be able to:
1- Notice the chromic point of amylase enzyme.
2- Examine the enzymatic activity and specificity of
salivary amylase depending on changes in temperature
and pH.
3- Determine the narrow range of temperature and pH
values at which salivary amylase exhibits its optimum
activity.
Proteins act as catalysts without chemical changes but accelerate
the rate of reaction.
1-Specfic
2-Inhbitors
3-Activator
4-pH
5-Tempreature.
LAB (1)
Enzymes Estimation
Effect of salivary amylase on starch digestion
Principle
Nutrients are absorbed when broken down into their monomers
(simplest form). In human and animal.
Carbohydrate is macromolecule, consisting of carbon(C),
hydrogen (H), and oxygen (O) atoms.
Carbohydrate function mainly as a source of chemical energy
for generating ATP, or their primary function in the body is to
supply energy. The major carbohydrate in human diet is starch
Saliva contains two types of secretions; the serous and the
mucous. The serous secretion contains the enzyme salivary
amylase, which also called salivary alpha-amylase.
This enzyme is responsible for the initial digestion of
carbohydrates, particularly the starch which secreted by the parotid
gland.
A chromic point is that stage at which the last trace of erythro
dextrin converted to achro-dextrin. The interval taken to reach the
chromic point called the chromic period.
Salivary amylase converts starch soluble starch
Higher dextrin erythrodextrine achrodextrine
maltose. (colorless)
• Preparation of Saliva
o Rinse the mouth with little warm water, circulate it by the
tongue for 2 min. and collect 10-15 mL. of this water in clean
beaker.
• Procedure
1. Put exactly 1mL.of unfiltered saliva into 100 mL. Cylinder dilute to
100 ml with water, mix well.
2. In test tube, put 5ml of dilute solution.
3. Add 5 ml soluble starch.
4. Add 2ml sodium chloride followed by 2 ml carbonate buffer pH 6.6.
5. Put the tube in water bath at 37 С .
6. Using a series of test tubes (5-10) containing iodine soul. Test
enzyme action.
7. Record the time at which no change in iodine observed (achromic
point).
Observations:
Conclusion:
Supervisor
LAB (2)
Effect of Temperature on salivary amylase enzyme
Principle
At 4°C, enzymatic reaction of salivary amylase occurs slowly or not at all due
to lack of energy and heat. The optimum temperature of salivary amylase
ranges from 32°C to 37°C.This applies to the human body since salivary
amylase is suitable to function within these temperatures. After 37°C, the
graph then steeply declines because of loss of activity. At 50°C and 70°C,
salivary amylase is denatured. The molecular conformation of the enzyme
altered as the hydrogen bonds responsible for its structures are broken.
• Preparation of Saliva
o Rinse the mouth with little warm water, circulate it by the tongue
for 2 min. and collect 10-15 mL. of this water in clean beaker.
• Procedure
1. Put exactly 1mL. of unfiltered saliva into 100 ml. Cylinder dilute to 100
ml with water, mix well.
2. In test tube (A), put 5ml of dilute solution.
3. Add 5 ml soluble starch.
4. Add 2ml sodium chloride followed by 2 ml carbonate buffer pH 6.6.
5. Put the tube in water bath at 37°C fo 5 min then add 2-3 drops of
iodine solution.
6. In test tube (B), put 5ml of dilute solution.
7. Add 5 ml soluble starch.
8. Add 2ml sodium chloride followed by 2 ml carbonate buffer pH 6.6.
9. Put the tube in ice bath at 4°C For 5 min. then add2-3 drops of iodine
solution.
10. In test tube (C), put 5ml of dilute solution.
11. Add 5 ml soluble starch.
12. Add 2ml sodium chloride followed by 2 ml carbonate buffer pH 6.6.
13. Put the tube in water bath at 45 °C For 5 min 2-3 drops of iodine solution.
14. In test tube (E), put 5ml of dilute solution.
15. Add 5 ml soluble starch.
16. Add 2ml sodium chloride followed by 2 ml carbonate buffer Ph 6.6 .
17. Put the tube in water bath at 55 °C For 5 min.
18-Add 2-3 drops of iodine solution in tubes. (A-B-C-E).
Observations:
Conclusion:
Supervisor
