NORTHWESTERN UNIVERSITY

COLLEGE OF ALLIED HEALTH SCIENCES

Laoag City

LABORATORY MANUAL
IN
BIOCHEMISTRY
(For Allied Health Science Courses)

____________________________________________
Name of Student

____________________________________________
Course and Year

Semester___________ School Year__________

_________________________________________________________________________

Name of Instructor
1
INTRODUCTION 2

SAFETY INSTRUCTIONS TO STUDENTS 3

Title Page

EXPERIMENT 1 Determination of Physiological Compounds 6

EXPERIMENT 2 Osmotic Behavior of RBC 11

EXPERIMENT 3 Carbohydrates 15

EXPERIMENT 4 Determination of Sugar in Body Fluids 22

EXPERIMENT 5 Proteins 26

EXPERIMENT 6 Determination of Protein in Body Fluids 33

EXPERIMENT 7 Lipids 37

EXPERIMENT 8 Determination of Cholesterol Level in the Blood 42

EXPERIMENT 9 Enzymes 45

EXPERIMENT 10 Factors Influencing Enzyme Reaction 48

EXPERIMENT 11 Digestion 53

Bibliography 58

2
 INTRODUCTION

This laboratory manual was developed with the objective of inculcating to

the students the gift of the laboratory that is discipline in scientific method.
Laboratory experiments also develop students’ logical reasoning and the ability to
write scientific reports. In that sense, every student could be developed to be
research-oriented individuals.

It has been said that “biochemistry is the mortar which builds together the
bricks (facts of other science) into a structure of life which is essentially a
chemical foundation.” These foundations are primarily the known facts of organic
chemistry and physical chemistry.

This Laboratory Manual in Biochemistry has been designed to understand

better basic concepts of biochemistry particularly in the field of Health Sciences
like nursing and medical technology. The laboratory activities were constructed in
line with the course content in Biochemistry for Allied Health courses centered on
the actual needs of the students. The manual includes the characterization of the
three essential organic compounds to sustain life- carbohydrates, proteins and
lipids. It also includes the actual determination of these compounds in body fluids
like blood and urine, thus, activities on basic instrumentation and blood specimen
collection and processing are also incorporated. The incorporation of actual
laboratory testing of body fluids will make students in the Allied Health courses
realize and appreciate the importance of Biochemistry in the field.

Prior to each laboratory activity, each student need to spend some time
reading the Laboratory Manual and concepts associated with the activity. This
reading will provide background information and an outline of the procedures to
be performed, thus, preventing laboratory time to be wasted. This will also help
students answer the pre- laboratory questions and gives the student an idea of the
expected results in every activity.

The biochemistry laboratory course, like all laboratory courses, is an

exploration of procedures. This means that, in order to get full benefit from the
course, the student will read the manual, and should participate as much as
possible in the actual laboratory work. The more effort student put into the course
work, the more he will learn. The class is an opportunity to learn valuable skills;
take full advantage of it!

3
 SAFETY INSTRUCTIONS TO STUDENTS

I. General

1. Smoking, eating and drinking are not permitted in the laboratory.

2. Bags must be placed in lockers provided for the purpose and not allowed
to obstruct gangways or bench tops.
3. Suitable laboratory coats must be worn in the laboratory and removed
after leaving. Safety spectacles must be worn when carrying out any
procedure where may be slightest risk of eye injury; gloves of the
appropriated type must be worn when necessary. Long hair must be
restrained e.g.by means of cap, ribbon or headband.
4. Procedure of each activity should be read before coming to the laboratory
and take note of any safety precautions. Students are NOT permitted to do
any experimental work unless the instructor or the laboratory assistant is
present.
5. The use of unfamiliar equipment and the handling of potentially hazardous
materials will be explained to students. If a student, for some reason,
misses the appropriate explanation, then it is the student’s responsibility to
bring the lack of knowledge to the attention of the instructor or the
laboratory assistant.
6. Glassware and plastic ware that is being used should always be labeled to
avoid confusion.

II. Substances and procedures hazardous to health

1. Where a potential hazard exists in a particular activity, this will be
discussed before the activity and the details of safe working methods
will be highlighted in the procedure notes.
2. Students must NOT use unfamiliar equipment or procedures without
them having been given instruction. All safety instructions given in the
preliminary laboratory discussion and procedure notes must be
adhered to.
3. Mouth pipetting is forbidden for any reagent.
4. All hazardous materials are deposited in fumehood.
5. Never use toxic substances without taking the proper precautions and
making arrangements for safe working. Use volatile solvents in
fumehood.
6. Never use broken glasswares and must be disposed properly.
7. Used plastic pipette tips must be placed in the appropriate labeled
container.

III. Waste
1. Bench should be left waste-free and tidy at the end of each activity-
this reduces potential for accidents and spillages and is considerable
help to the laboratory staff.
4
 2. Laboratory equipment must be left clean and dry after each activity.
Return all equipments’ borrowed to the laboratory assistant.

IV. Accidents
1. All accidents and spillages, including personal injuries and damage
caused to equipment, must be reported as soon as practicable to the
instructor or to the laboratory assistant.
2. Concentrated acid or alkali on the skin: a). flood the splashed surface
thoroughly with water and continue until satisfied that no chemical
remains in contact with the skin. Soap will help to remove chemical
which are insoluble in water; b). remove all contaminated clothing,
take care not to contaminate yourself in the process.
3. Splashes in the eye. Eye protection should be worn for any work
where there is a potential hazard but if accident occurs; a).flood the
eye thoroughly but gently with water; b). seek medical advice for all
eye injuries from chemicals.
4. Burns and scalds. Cool affected area by immersing in cold water.
Continue for at least 5 minutes or until pain is relieved.
5. Spillages must be cleared up immediately and the area
decontaminated; they must NOT be left as a hazard to others.
6. A first aid box is available in the preparatory room. Ask the instructor
or the laboratory assistant if you need items from the first aid box.
7. Bring any injured person for proper treatment in the school clinic.

TECHNIQUES AND PROCEDURES TO MINIMIZE

LABORATORY INFECTIONS WHEN HANDLING BIOLOGICAL
SPECIMEN
1. Never perform direct mouth pipetting in the laboratory.
2. Do not blow out pipettes that contain infectious material.
3. Do not mix infectious materials by bubbling air through the liquid.
4. Use only needle- locking hypodermic syringes. Avoid using whenever
possible. Dispose of needle in special containers designed for that purpose.
5. Sterilize reusable glassware immediately after use by placing it into
a pan containing phenolic compound.
6. Never leave a discarded tube or infected material unattended or
unlabeled.
7. Handle with disposable gloves serum specimens.
8. As a part of the disposable process, sterilize all biological specimens
(except urine) by autoclaving or incineration. Urine may be poured
into a drain.
9. Do not allow smoking or consumption of food or beverages in the
laboratory.

5
10. Encourage frequent hand washing; this practice is mandatory before
leaving the laboratory.
11. To reduce the possibility of self- inoculation, develop the habit of
keeping your hands away from your mouth, nose, eyes and any other
mucous membranes when handling biological specimen.

6
Name:__________________________Date: _____________________________

Course&Year:___________________Rating: ___________________________

Experiment No. 1

Determination of Physiological Compounds

Inrtoduction:

Organic compounds were believed to be obtained from vegetable and

animal sources. Until about 1850, many chemists believed that organic
compounds must have their origin in living organisms and consequently could
never be synthesized from inorganic material.

Objective:
To identify sources of the 5 major elements found in the different
physiological compounds both in plants and animals.

Materials:

3 Test tubes Bunsen burner Rubber cork

Bent glass tube Crucible Mortar & pestle
Test tube holder Test tube brush Test tube rack
Crucible tong Iron stand with ring Clay triangle
Glass rod Spatula

Reagents/Chemicals

Muscle tissue Horn Ammonium molybdate sol’n

Feather Dry albumin ½ inch potassium nitrate
Hair Sucrose ½ inch of stick potassium hydrate
Lecithin Soda lime Lead acetate solution
Cupric oxide Ammonia Litmus paper
Lime water conc. HNO3 Filter paper
HCl

Procedure:
7
A. Test for Carbon
1. Place a small amount of cane sugar in a test tube.
2. Heat over a Bunsen flame. Note the changes undergone by the
heated sucrose and determine whether gas is produced. Explain the
changes observed.

Result:
______________________________________________________

______________________________________________________

______________________________________________________

B. Test for Hydrogen

1. Mix a small amount of powdered sugar in a dry mortar with about
10x the quantity of cupric oxide.
2. Place the mixture in a dry test tube provided with a rubber cork
perforated by a bent glass tube which dips into Ca(OH)2 solution.
3. Heat the test tube over a Bunsen flame and note the changes in the
lime water. Explain the phenomenon observed and state the
chemical reaction involved.
Result:
______________________________________________________

______________________________________________________

______________________________________________________

C. Test for Nitrogen

1. Burn muscle tissue, horn, hair or feather. Note the characteristic
odor produced.
2. Mix a small piece of dry albumin and about 20x the amount of
soda lime in a mortar.
3. Introduce the mixture into a test tube and heat the material over a
Bunsen flame. Determine if gas is present. If it is ammonia,
confirm it by the following tests:
a) by its odor
b) using red litmus paper held over the mouth of the tube
c) it gives white fumes with a glass rod held over the mouth of the
tube originally dipped in HCl

8
 ODOR BLUE/RED LITMUS PRESENCE/ABSENCE
PAPER OF FUMES

D. Test for Sulfur

1. Add 2 drops of a neutral lead acetate solution to a few ml of NaOH

solution. Note that the lead hydroxide which is precipitated is
dissolved.
2. Heat a small portion of the albumin with this alkaline solution.
Note the change. State the chemical reactions involved in the test.
Result:
______________________________________________________

______________________________________________________

______________________________________________________

E. Test for Phosphorous

1. Place a small amount of lecithin in a porcelain crucible.

2. Add ½ inch of stick potassium hydrate and potassium nitrate. Heat
strongly.
3. Cool. Treat with water and filter.
4. Place 3 ml of the filtrate in a test tube and add to it 2ml of conc.
HNO3 and 4 or 5 ml of ammonium molybdate solution.
5. Warm gently for sometime. Note the color of the precipitate
formed. What is the precipitate formed?
Result:
______________________________________________________

______________________________________________________
_______________________________________________
_______

______________________________________________________

Questions:

9
 1. What is the role of bone black or the activated carbon in
decolorization?

_________________________________________________________

_________________________________________________________

_________________________________________________________

2. Differentiate dialysis, chromatograph and electrophoresis.

_________________________________________________________

_________________________________________________________

_________________________________________________________

_________________________________________________________

_________________________________________________________

Give the principle involved in each method.

_________________________________________________________

_________________________________________________________

_________________________________________________________

_________________________________________________________

_________________________________________________________

3. Give other methods of purification and separation of organic

compounds.
_________________________________________________________

_________________________________________________________

_________________________________________________________

_________________________________________________________

Conclusion/s:

10
 ______________________________________________________
___

_________________________________________________________

_________________________________________________________

_________________________________________________________

Name:____________________________ Date:___________________________
Course & Year: ____________________Rating: ________________________
11
 Experiment No. 2

Osmotic Behavior of Red Blood Cells (RBC)

Introduction:

Whenever two solutions of unequal concentrations are separated by a

semi-permeable membrane, the fluid tends to flow from the side of low osmotic
pressure to that of higher osmotic pressure until equilibrium is reached. The
process is called osmosis.
The osmotic pressure of a solution depends upon the number of particles
dissolved, regardless of size.
Cells behave differently in different osmotic solutions. When cell is
brought in contact with a solution with the same osmotic pressure, the water
passing in and out of the cell into the solution is balanced provided that the
membrane is impermeable to solutes. The solution is called isotonic. The volume
of the cell remains constant and the tone is maintained. The cell when placed in a
solution with higher concentration (hyperosmotic or hypertonic) will shrink
while it swells when placed in a hypotonic solution, a solution with lower
concentration.

Objective:

To demonstrate and describe the characteristic reactions of RBC (red

blood cells) in the different solutions.

Materials:

Glass slides (4) Dropper Microscope

Applicator sticks Cover slip

Reagents/Chemicals:

0.3% NaCl Solution

0.9% NaCl Solution
5.0% NaCl Solution
Blood sample

Procedure:
1. Prepare four (4) clean properly labeled slides.
2. Perform skin puncture using the middle finger. Wipe the first two drops of
blood.
3. Put one drop of blood in each of the four slides. Do not add any solution to
the first slide. This serves as a control.

12
4. To the second slide, add one drop of 0.3% NaCl ; to the third slide, add
0.9% NaCl and to the fourth slide, add one drop of 5% NaCl.
5. Mix the drop of blood and the solution with an applicator stick.
6. Observe the preparations under the microscope.

Observations:

a. Control
____________________________________________________________

____________________________________________________________

____________________________________________________________

____________________________________________________________

b. 0.3% NaCl
____________________________________________________________

____________________________________________________________

____________________________________________________________

____________________________________________________________

c. 0.9% NaCl
____________________________________________________________

____________________________________________________________

____________________________________________________________

d. 5% NaCl
____________________________________________________________

____________________________________________________________

____________________________________________________________

____________________________________________________________

7. Make a drawing of each observation describing what happened in each or

the appearance of each.

13
 Illustrations:

Control 0.3% NaCl

0.9% NaCl 5% NaCl

Conclusion:

____________________________________________________________

____________________________________________________________

____________________________________________________________

____________________________________________________________

14
Questions:

1. Define the following:

a. Osmotic Pressure:
____________________________________________________________

____________________________________________________________

____________________________________________________________

b. Osmolarity:

____________________________________________________________

____________________________________________________________

____________________________________________________________

c. Physiological Salt Solution:

____________________________________________________________

____________________________________________________________

____________________________________________________________

2. What caused the RBC to react differently in different solutions? Explain

the principle involved.
____________________________________________________________

____________________________________________________________

____________________________________________________________

___________________________________________________________

Name: __________________________Date:__________________________
Course & Year: __________________Rating: ________________________

Experiment No. 3

15
 Carbohydrates

Carbohydrates are the most abundant compounds found in nature, which

may occur as simple sugars and as complex sugars. The carbohydrates are
polyhydroxy alcohols having potentially active aldehyde or ketone groups and
compounds that can be hydrolyzed. A carbohydrate that can be hydrolyzed to 2
monosaccharide molecules is called a disaccharide. A carbohydrate that can be
hydrolyzed to many monosaccharide molecules is called polysaccharide.

A monosaccharide maybe further classified into an aldose, if it contains an

aldehyde; ketone if it contains a keto group. They can also classified according to
the number of carbon atoms they contain like triose, tetrose, pentose, hexose, etc.
A keto pentose is a five-carbon compound containing a ketone group or an aldo
hexose is a six- carbon compound containing an aldehyde group.

Carbohydrates that reduce Fehling’s, Benedict’s or Tollen’s reagent are

known as reducing sugars. All monosaccharides, whether notable exception for it,
is a non- reducing sugar.

Objectives:
1. To differentiate the types of carbohydrates as to their physical and
chemical properties.
2. To test the hydrolysis of sugar and starch.

Materials: Reagents:
Watch glass(7) Glucose Alcohol Seliwanoff’s Rgt.
Test tubes (30) Fructose 10%NaCl Benedict’s
Rgt.
Bunsen burner Galactose 5%Na2CO3 Iodine solution
Test tube rack Maltose 12%HCl 10%NaOH
Test tube holder Lactose Conc. NaOH
Water bath Sucrose Conc. HCl
Beaker (250 ml) Starch Molisch Rgt.
Spatula Water H2SO4

Procedure :

A. Macroscopic Appearance:
1. Take a small amount of glucose, sucrose, maltose, fructose,
galactose and starch and put each one in a cover glass.
2. Examine each one as to form, color, odor and taste.

16
 Glucose Fructos Galactose Maltose Lactose Sucros Starch
e e

Form

Color

Odor

Taste

B. Solubility

1. Prepare 7 test tubes in the rack and introduce into each one 5 ml of
the ordinary solvents.

2. Add a small amount of glucose to the above solvents and

determine the solubility.

3. Repeat the same procedure using sucrose, maltose, fructose,

galactose, starch.

Alcohol Water 10% 5% 12% Conc Conc.

NaCl Na2CO3 HCl NaOH HCl
Glucose

Fructose

Galactose

Maltose

Sucrose

Starch

C. Color Reactions:

1. Molisch’s Tests:
a. Place about 5ml of 1% glucose, fructose, maltose, galactose, sucrose,
lactose in separate test tubes.

17
 b. Add 2 drops of Molisch’s reagent in each tube.
c. Shake and mix thoroughly.
d. Incline the test tube and pour slowly and carefully down the sides of
the test tube H2SO4 to keep the acid below and sugar solution above in
separate layers.
e. Note and observe for the characteristic violet ring produced between
the two liquids.

RESULT:

Glucose Fructose Maltose Galactose Sucrose Lactose

Result

2. Seliwanoff’s test :
a. Place 5ml of Seliwanoff’s reagent in a test tube.
b. Add few drops of glucose solution.
c. Heat the contents of the tube to boiling. Note the production of a color
and the separation of a colored precipitate. What is the color of the
precipitate?
d. Add alcohol and note down the changes.
e. Repeat the same procedure with the other sugar.

RESULT:
Glucose Fructose Maltose Galactose Sucrose Lactose
After
boiling

After
addition
of
alcohol

D. Reducing property of Sugars:

1. Fehling’s Test:
a. Mix 1 ml of Fehling’s A and Fehling’s B in a test tube.
b. Add 2 ml of glucose solution.
c. Note the color of the solution before and after boiling the mixture in
water bath for 2-5 minutes.
d. Repeat the procedure using fructose, galactose, maltose, lactose and
sucrose. Which are reducing sugars? Which are non- reducing sugars?

18
 POSITIVE RESULT: Yellow precipitate

RESULT:
Glucose Fructose Maltose Galactose Sucrose Lactose
Before
boiling
After
boiling
Reducing
sugar

2. Benedicts’ Test :
a. Mix 2ml of glucose and 2ml of Benedict’s reagent.
b. Boil in water bath for 2 minutes. Observe the color
c. Repeat the procedure using fructose, sucrose, maltose, lactose and
galactose.
d. What are the colors developed?

POSITIVE RESULT: Green to orange red solution with yellow to

brick red precipitate

RESULT:
Glucose Fructose Maltose Galactose Sucrose Lactose
Result

Reducing
sugar

E. Other Tests:

1. Iodine Test
a. Prepare a starch solution. Dissolve a small amount of starch in 1
ml. water. Shake well to dissolve the starch.
b. Put 2 ml water in a test tube. Add 5 drops of starch solution and
shake well.
c. Add a drop of iodine solution. What is the color produced?
d. Repeat the test using glucose, fructose, maltose, galactose, sucrose
and lactose in place of starch. Compare the results.

POSITIVE RESULT: Violet or purple color

RESULT:
Glucose Fructos Maltose Galactose Sucros Lactose Starch

19
 e e
Resulting
color
Interpretation
of result

2. Hydrolysis of Sugar
a. Prepare sucrose solution.
b. To 5 ml sucrose solution, add 2 drops of concentrated HCl and heat in
a water bath for 30 minutes.
c. Test the solution with Benedict’s test.
d. Repeat the same procedure with glucose, fructose, maltose, galactose
and lactose.

RESULT:
Glucose Fructose Maltose Galactose Sucrose Lactose
Resulting
Color

Interpretation
of Benedict’s
test result

3. Hydrolysis of Starch

a. Preparation of Starch paste

Thoroughly dissolve about 2 grams of starch powder with a small
amount of cold water in a beaker. Add about 200ml of boiling
water with continuous stirring until a paste like structure is formed.

b. Transfer 3 ml each of the starch paste into test tubes and perform:
b.1 Fehling’s test
b.2 Benedict’s test
b.3 Iodine’s test

c. Observe and record the results.

RESULT:
Fehling’s Test Benedict’s Test Iodine’s Test
Resulting
Color

20
 Interpretation of
result

d. To 10ml of the paste solution add drops of concentrated HCl and place
the tube in boiling H2O.

e. At interval of 1 minute, transfer a drop of the solution into an

evaporating dish and perform the regular iodine test.

f. Note the color change and time of change.

RESULT:
Time Interval Color Change

g. When the blue color with iodine has completely disappeared, heat the
tube in boiling water for 5 minutes more, cool and neutralize with solid
KOH or better 10% NaOH drop by drop. Observe and note the result.

RESULT:
_______________________________________________________

Conclusion:
__________________________________________________________________

__________________________________________________________________

__________________________________________________________________

Questions :

1. What is the general test for carbohydrates?

21
 _______________________________________________________________

_______________________________________________________________

_______________________________________________________________

2. What is the chief function of carbohydrates to the body?

_______________________________________________________________

_______________________________________________________________

_______________________________________________________________

3. What do you call the linkage that binds one monosaccharide unit to another?

_______________________________________________________________

_______________________________________________________________

_______________________________________________________________

Name: _________________________Date:_______________________
Course & Year: _________________Rating: _____________________

22
 Experiment No. 4

Determination of Sugar in the Body Fluids

Introduction:
Carbohydrate is the major energy source for the human body. Glucose is
the commonly determined sugar in body fluids. The glucose level in body fluids
particularly blood is kept within a narrow range through a variety of influences.
Although there is some variation in glucose level in body fluids as circumstances
change (feeding, prolonged fasting), levels above or below the normal range
usually indicate disease. Determinations of glucose in body fluids are usually
ordered by clinicians for the diagnosis and follow-up of abnormalities of
carbohydrate metabolism.

Objective:
1. To determine qualitatively the presence of sugar in urine.
2. To determine the level of sugar in the blood.

Materials: Reagents:
Beaker (250 ml) Test tube (4) Benedict’s Rgt.
Pasteur pipet Graduated cylinder (10 ml) Glucose standard
Bunsen burner Spectrophotometer
Water bath
Test tube rack
Test tube holder

Procedure:

I. Determination of Sugar in Urine using Benedict’s Test

1. Measure 5 ml Benedict’s solution in a test tube.

2. Add 8 drops of freshly collected urine sample.
3. Mix the preparation.
4. Place in a boiling water bath for 5 minutes.
5. Observe for the result immediately after the heating process.

RESULTS AND INTERPRETATION:

(-) no change from the original blue color
(trace) green solution without any precipitate
(+) greenish yellow solution with yellow precipitate
(++) yellowish green solution with yellow precipitate
(+++) yellowish orange solution with orange precipitate
(++++) orange red precipitate with brick red precipitate

23
 RESULT:
______________________________________________

II. Determination Of Glucose Level in the Blood using Glucose

oxidase Reagent

A. Collection and Preparation of Blood sample

I. Venipuncture (Syringe method)
a. Collect blood sample by venipuncture using syringe method.
b. Use on the antecubitsl fossa in collecting blood.
c. Tie the tourniquet about 3 inches above the flex of the elbow.
Locate for the prominent vein on the antecubital fossa.
Remove the tourniquet.
d. Sterilize the area with 70% isopropyl alcohol using circular
motion from center going outside. Using the same
technique, wipe the area with dry sterile cotton.
e. Re-tie the tourniquet.
f. Open a syringe with needle. Make sure the needle is tightly
attached to the syringe. Remove any air in the syringe.
g. With needle bevel up, puncture the chose vein.
h. Once blood enters the syringe, slowly pull the plunger until the
desired amount of blood enters.
i. Remove the tourniquet, put dry sterile cotton on top of the
needle then slowly withdraw the needle from the vein.
j. Apply slight pressure on the site of puncture then put plaster or
let the patient flex his elbow until bleeding stops.
k. Remove the needle from the syringe, then dispense blood on
the tube slowly by passing on the sides of the tube.
l. Allow blood to clot.

NOTE: Be careful in covering he needle to avoid needle

puncture. Dispose needle and syringe in the proper receptacle.

II. Preparation of Serum

1. Once the blood has been clotted, rim the sides of the tube
with applicator stick.
2. Centrifuge the tube for 5-10 minutes at 2500 RPM.
3. Remove the tube from the centrifuge.
4. Using a clean Pasteur pipette, slowly separate the clear
yellow fluid known as SERUM from the packed red cells.

B. Determination of Glucose Level in the Blood

24
 1. Switch on the TC 84 spectrophotometer to stabilize the
temperature at 37C.
2. Label the three test tubes as blank, standard and sample.
3. Put 1.5 ml of glucose buffer in each of the tubes.
4. Add 10 microliter (using micropipette) of serum to sample test
tube, 10 microliter of standard solution to standard test tube and 10
microliter of distilled water to the blank test tube.
5. Incubate the test tubes in the built-in incubator of the
spectrophotometer for 3 minutes.
6. Add 50 microliter of glucose enzyme reagent to all test tubes. Mix
gently by swirling.
7. Re- incubate at 37ºC for 10 minutes.
8. Read the absorbance of the preparations in spectrophotometer at
505-550 nm wavelength.
9. Compute the level of sugar in the blood using the following
formula.

Absorbance of sample
Concentration = ------------------------------------ x concentration
of
of sugar Absorbance of standard standard

NORMAL LEVEL OF SUGAR IN BLOOD: 70 – 105 mg/dl

RESULT AND INTERPRETATION:

______________________________________________________

______________________________________________________

Conclusion:

__________________________________________________________________

__________________________________________________________________

__________________________________________________________________

__________________________________________________________________

Questions:

25
 1. Give the significance of determining the presence of sugar in urine. What
is renal threshold? Give the renal threshold for glucose.

____________________________________________________________

___________________________________________________________

____________________________________________________________

____________________________________________________________

____________________________________________________________

____________________________________________________________

____________________________________________________________

____________________________________________________________

2. What is the clinical significance of increase and decrease sugar level in the
blood?

_________________________________________________________________

_________________________________________________________________

_________________________________________________________________

_________________________________________________________________

_________________________________________________________________

_________________________________________________________________

_________________________________________________________________

_________________________________________________________________

________________________________________________________________

Name: _________________________ Date:_______________________

26
Course & Year: _________________ Rating: _____________________

Experiment No.5

Proteins
Introduction:

Proteins are highly complex nitrogen formative substances in all living

things including man himself and of high molecular weight that yield 20-25
different amino acids on hydrolysis. These amino acids are connected to one
another through a peptide bond. Protein molecule is often built up of hundreds
and even thousands of the amino acids, and the varied proteins found in nature are
largely due to the differences in the number, the kind and the arrangement of such
amino acids in the protein molecule.

Objective:
To describe and identify proteins according to the differences in their
color and precipitation reactions.

Materials:

10 Test tubes Bunsen burner Test tube holder

Test tube rack Test tube brush pH meter
pH paper Dropper Pipette

Reagents/Chemicals:

Dilute egg albumin solution

2% solution- egg white, egg albumin, casein or milk
Amino Acids:
Glycine, alanine, arginine, histidine, lysine

Millon’s reagent dil. KOH/ NaOH 3% CuSO4 solution

Albumin solution conc. HNO3 NH4OH solution
Glacial acetic acid protein solution conc. H2SO4
Glyoxilic acid Molish reagent Picric acid
dil. HgCl solution dil. Lead acetate dil. Silver nitrate
dil. Copper sulfate dil. Ferric chloride dil. Barium
chloride
conc. HCl conc. KOH conc. HC2H3O2
10% Sodium tungstate 1% HAc 95% alcohol

Procedure:

27
A. Colored Reactions:

1. Millon’s Reaction:
a. Add a few drops of Millon’s reagent to 5 ml of dilute solution of egg
albumin in a test tube. Note the change in the mixture. Note the further
change in the contents of the tube by heating it to almost boiling.
b. To what group of the protein molecule does you, attribute the changes
observed?
Result:
_________________________________________________________
________________________________________________
________

2. Biuret test:
a. Fill the test tube to almost ½ its height with dilute KOH/ NaOH and
add to it 8-10 drops of 3% CuSO4 solution until a slight though distinct
blue color is produced.
b. Get another test tube and place 5ml of albumin solution. Add slowly
the NaOH and CuSO4 solution, slow enough to have the reagent
stratified over the albumin solution.
c. Note the production of a colored ring at the point of contact. What
causes the production of this color?
Result:
_________________________________________________________
________________________________________________
_________

3. Xanthoproteic Test:
a. Add a small amount of concentrated HNO3 to 3ml of egg albumin
solution in a test tube and note the character of the precipitate formed.
b. Heat the contents of the tube until dissolution takes place.
c. Cool and render the solution alkaline with NH4OH, NaOH or KOH.
Note further color changes.
d. To what group of the protein molecule do you attribute these color
changes?
Result:
_________________________________________________________
________________________________________________
_________

4. Tryptophane Reaction:
a. Place 5ml of glacial acetic acid in a test tube.
b. Add 3-10 drops of the protein solution.

28
 c. Mix well and in an inclined position, pour concentrated H2SO4 down
the side of the tube so that it forms a layer underneath the acetic acid.
d. Note the development of a colored ring at the point of contact for a
positive test.
e. Agitate the tube so as to mix the sulfuric acid and acetic acid. Note
that the whole solution may become violet. What causes the
production of the violet ring at the zone of contact?
Result:
_________________________________________________________
________________________________________________
_________

5. Hopkin’s Cole/ Glyoxilic Acid Reaction :

a. To 1 ml of egg albumin add 5 drops of glyoxilic acid.
Carefully run down 5 drops of concentrated sulfuric acid on the side of
the test tube.
b. Note the color formed at the point of contact of the 2 liquids. What
group of protein is responsible for the reaction?
Result:
_________________________________________________________
________________________________________________
_________

6. Molisch Test:
a. To 2 ml of egg albumin solution, add 1 ml of Molish reagent.
b. Shake the mixture carefully.
c. Incline the test tube and carefully add 1 ml of concentrated sulfuric
acid down the side of the tube. What color is formed at the point of
contact of the 2 liquids?
d. To what group of protein do you attribute the change in color?
Result:
_________________________________________________________

_________________________________________________________

B. Precipitation of Proteins:

Use 2% solution- egg white, egg albumin, casein or milk

1. By Heavy metal Salts:
a. Pour 5 ml dilute albumin solution into each 6 test tubes.
b. Add dilute solution of HgCl, drop by drop and note the appearance of
a precipitate.
c. Add more mercuric chloride solution until excess of it is present and
note further change on the precipitate.

29
 d. Repeat the above procedure using lead acetate, silver nitrate, and
copper sulfate, ferric chloride and barium chloride (dilute). Tabulate
your results.

PRECIPITANTS CHARACTER & FURTHER CHANGES ON

DILUTE SOLUTION PRECITATE

DROP BY DROP EXCESS

1. HgCl2

2. Pb ( C2H3O2)2

3. AgNO3

4. CuSO4

5. FeCl3

6. BaCl3

1. By using mineral acids/ alkalines/ organic acid

a. Prepare 4 test tubes with 2 ml each of concentrated H 2SO4 concentrated
HCl, concentrated KOH and HC2H3 O2.
b. Add 5 ml of egg albumin solution into each test tube.
c. Observe and note down changes, which may occur.
d. Heat the tubes and note if there are any further changes.

PRECIPITANTS CHARACTER AND FURTHER CHANGES

ONPRECIPITATE
ON IMMEDIATE UPON HEATING
ADDITION AFTER 24 HRS.

1. conc. H2SO4

2. conc. HCl

3. conc. KOH

4. conc. HC2H3O2

30
2. By Heat
a. Heat 2 ml egg albumin solution to boiling.
b. Observe if coagulation takes place.
c. Add 2 drops pH acetic acid and note the effect.
Result:
____________________________________________________________
_______________________________________________________
_
_
3. By alkaloidal Reagents
a. Picric Acid
To 5 ml of albumin solution, add an aqueous solution of picric acid
drop by drop until an excess has been added. Note the formation of
a yellow precipitate. What causes the production of a yellow
precipitate?
Result:
______________________________________________________
__________________________________________
____________

b. Tannic Acid
Slightly acidify 5 ml of albumin solution with H 2SO4 in a test tube
and acid a few drops of dilute alcoholic solution of tannic acid.
Note the formation of brownish precipitate. What causes this?
Result:
______________________________________________________
_______________________________________________
_______

c. Tungstic Acid
Slightly acidify 5 ml albumin solution with H2SO4 in a test tube
and then add a few drops f 10% solution of sodium tungstate. Note
the formation of white precipitate. What causes this?
Result:
______________________________________________________
_______________________________________________
_______

4. By Alcohol:
a. Place 2 ml of egg albumin solution in a test tube.
b. Add 1 or 2 drops of 1% HAc and 2 ml of 95% alcohol.
c. Shake well. Note the color of the precipitate formed.

31
 Result:
____________________________________________________________

______________________________________________________

C. Acidity and Basicity:

By using the pH meter or pH paper, determine the pH of the following
solutions containing amino acids:

Amino Acids pH
1) Glycine
2) Alanine
3) Arginine
4) Histidine
5) Lysine
6) Aspartic Acid

Conclusion:
_______________________________________________________________

_______________________________________________________________

_______________________________________________________________

_______________________________________________________________

_______________________________________________________________

_______________________________________________________________

_______________________________________________________________

_______________________________________________________________

Questions:

1. What are peptides? Do all proteins possess peptide bonds?

_______________________________________________________________

_______________________________________________________________

_______________________________________________________________

_______________________________________________________________

32
2. What is meant by denaturation of proteins? Give examples of protein
denaturants.
_______________________________________________________________

_______________________________________________________________

_______________________________________________________________

_______________________________________________________________

3. Why is silver nitrate used in cauterization of wounds?

_______________________________________________________________

_______________________________________________________________

_______________________________________________________________

4. Why is egg white used as an antidote for lead and mercurial poisoning?
_______________________________________________________________

_______________________________________________________________

_______________________________________________________________

5. Give the rationale for the following:

a. Use of picric acid in burns.
_______________________________________________________________

_______________________________________________________________

_______________________________________________________________

_______________________________________________________________

b. Use of Tannic acid in diarrhea.

_______________________________________________________________

_______________________________________________________________

_______________________________________________________________

_______________________________________________________________

33
Name: _________________________ Date: _______________________
Course & Year: __________________ Rating: _____________________

Experiment No. 6

Determination of Protein in Body Fluids

Introduction:

The human body contains countless different proteins, some soluble in

fluids within or outside the cells, some insoluble and structural. The only ones
available for investigation are the soluble proteins of the extracellular fluids. In
plasma, more than 300 proteins have been identified, but they differ greatly in the
concentration and only the most abundant will be determined. The plasma
proteins move between the blood and other extracellular fluids by active transport
as well as passive diffusion. When cells are damaged, soluble proteins normally in
cells or on their surfaces may also be present in extracellular fluid. Most of the
proteins in urine and spinal fluid are derived from plasma.
The total concentration of all proteins in plasma may vary because of
changes in the volume of plasma water or changes in the amounts of individual
proteins. Decrease in the volume of plasma water causes the concentration of all
proteins to be increased known as hyperproteinemia as seen in dehydration due to
excessive water loss as in vomiting. Increase in plasma water causes a decrease in
the concentration of all proteins known as hypoproteinemia that occurs in water
intoxication or salt retention syndrome.

Objective:
1. To determine qualitatively the presence of protein in urine.
2. To determine the level of total protein in the blood.

Materials:
Beaker (250 ml) Test tubes (4)
Pasteur pipette Graduated cylinder (10 ml)
Bunsen burner Spectrophotometer
Water bath micropipette
Test tube rack syringe
Test tube holder torniquet

Reagents/Chemicals:
Biuret Rgt. Isopropyl alcohol
Protein standard
Distilled water
10% Acetic acid
Conc. Nitric acid
Procedure:
34
I. Determination of Protein in the Urine
A. Heat and Acetic Acid Test
1. Fill a test tube about 2/3 full with freshly voided urine sample.
2. Clamp the mid-portion of the tube, and then heat evenly the upper
1/3 of the preparation by turning the bottom of the tube with one
hand while the other holds the test tube holder. Allow the
preparation to boil for a while. Observe for the presence of
cloudiness at the heated portion against a black background.
3. Add 3 drops of 10% acetic acid then repeat the preparation as in
procedure 2, and observe for cloudiness.

NOTE: If cloudiness initially formed disappears after the addition

of acetic acid and re-heating process, the substance present is
amorphous urates/ phosphates. However, if the cloudiness
initially formed does not disappear, the substance possibly
present is protein.

RESULTS AND INTERPRETATION

(-) absence of cloudiness
(trace) cloudiness is barely visible
(+) cloudiness is distinct but not granular
(++) cloudiness is granular and granular
(+++) cloudiness is heavy with distinct flocculi
(++++) cloudiness is dense with large flocculi

RESULT:
_______________________________________________________________

_______________________________________________________________

_______________________________________________________________

_______________________________________________________________

B. Heller’s Nitric Acid Test

1. Place 2 ml of concentrated nitric acid in a test tube.
2. Stratify 2 ml of freshly voided urine sample on the nitric acid
3. Observe for the result
POSITIVE RESULT: white ring at the point of contact of urine
and nitric acid.

RESULT:

35
 _______________________________________________________________

_______________________________________________________________

_______________________________________________________________

_______________________________________________________________

II. Determination of Total Protein Level in the Blood using Biuret

Test
1. Prepare serum.
2. Label three test tubes as blank, standard and sample.
3. Put 1 ml of Biuret reagent in each reagent of the 3 test tubes.
4. Add 10 microliter distilled water in the test tube labeled blank.
5. Add 10 microliter standard in the tube labeled standard.
6. Add 10 microliter of serum sample in the tube labeled sample.
7. Mix the preparations in each tube.
8. Stand the preparations at room temperature (15-30ºC) for 5
minutes.
9. Read the absorbance of the preparations in spectrophotometer at
505-550 nm wavelength against the blank.
10. Compute the level of total protein in the blood using the following
formula.

Absorbance of sample
Conc. of total protein = ×conc.of standard
Absorbance of standard

NORMAL LEVEL OF TOTAL PROTEIN IN BLOOD: 5.8-8.0 g/dl

RESULT AND INTERPRETATION:

_______________________________________________________________

_______________________________________________________________

_______________________________________________________________

_______________________________________________________________

_______________________________________________________________

_______________________________________________________________

Conclusion:
36
 _______________________________________________________________

_______________________________________________________________

_______________________________________________________________

_______________________________________________________________

Questions:

1. Give the significance of determining the presence of protein in the urine.

_______________________________________________________________

_______________________________________________________________

_______________________________________________________________

_______________________________________________________________

2. Give conditions associated with increase and decrease total protein in the
blood.
_______________________________________________________________

_______________________________________________________________

_______________________________________________________________

_______________________________________________________________

Name: _________________________ Date:_______________________

37
Course & Year: _________________ Rating: _____________________

Experiment No.7

Lipids

Introduction:

Lipids are like carbohydrates from which they are derived these fatty
substances are composed only of Carbon Hydrogen & Oxygen; unlike
carbohydrates, they contain far less oxygen and more hydrogen. They are
separated from the carbohydrates by the chemical process of reduction, which has
removed oxygen or OH and substituted H for it. This chemical change involves a
marked loss of solubility to water and the acquisition of fatty peculiarities.

The lipids are a large and diverse group of naturally occurring organic
compounds that are related by their solubility in non-polar organic solvents (e.g.
ether, chloroform, acetone & benzene) and general insolubility in water

Fats (lipids) are familiar to all biologists as:

a. The chief form in which excess foodstuffs are stored;
b. They help to give the protoplasm its properties of containing large
amount of water but not dissolving;
c. Therapeutic agents; and
d. They give rise to a group of active substances called biocatalyst-
catalytic agents, vitamins and hormones.

Objectives:
1. To describe the solubility of lipids in the different solvents
2. To determine the characteristic chemical reactions of lipids when
subjected to the different tests.

Materials:
Test tubes Litmus paper Pork fat
Microscope Filter paper Dropper
Evaporating dish Bunsen Burner Iron stand with ring
Spatula Clay triangle Glass slides
Pipette

Reagents/Chemicals:

38
 Coconut oil (Fresh & Rancid) Distilled water
Linseed oil 0.5% Sodium carbonate solution
Potassium bisulfate Hubl’s solution dilute HCl
Cottonseed oil Ether
dilute NaOH Alcohol
Carbon tetrachloride Chloroform

Procedure:

A. Solubility Test
Put two drops of coconut oil in 1 ml of each of the following
solvents contained in separate test tubes: Water, dilute HCl, dilute
NaOH, cold alcohol, hot alcohol, chloroform, ether and carbon
tetrachloride.

In what solvents is the oil soluble?

What are these solvents called?

_____________________________________

B. Spot test
Place 1 drop of coconut oil on a piece of paper. Note the
formation of semi-translucent spot. Allow to evaporate spontaneously.
Does it appear?

_________________________________________________________

C. Reaction with Litmus Paper

Test the reaction of fresh and rancid coconut oil with litmus
paper previously moistened with water.
Results:
______________________________________________________

______________________________________________________

______________________________________________________

How do you account for the difference in reaction between the

fresh and rancid oil?

39
 ______________________________________________________

______________________________________________________

______________________________________________________

D. Emulsification

Place a few drops of fresh coconut oil in 1 ml. water. Shake and
observe the result.

Results:
______________________________________________________

______________________________________________________

In another test tube, place 1 ml of water and 1 ml of 0.5% Sodium

carbonate solution. Add few drops of coconut oil and shake.

What is the effect of Sodium carbonate?

Results:
______________________________________________________

______________________________________________________

______________________________________________________

E. Acrolein Test

To 1 ml. of coconut oil in a test tube, add a pinch of potassium

bisulfate. Heat gradually and note the odor produced. To what is due?
Equation:
______________________________________________________

F. Test for Unsaturation

1. Dissolve 5 drops of linseed oil in 3 ml of chloroform.

40
 2. Add Hubl’s solution drop by drop, shaking between additions, until a
permanent brownish tinge is obtained
3. Record the no. of drops needed to obtain this color.
No. of drops used _____________________
3. Repeat steps 1 and 3 using cottonseed oil instead of linseed oil.
No of drops used: __________________________________

What conclusion can you make based on the number of drops of

Hubl’s solution used for each oil?

______________________________________________________

______________________________________________________

______________________________________________________

What is meant by iodine number? Give its significance?

___________________________________________________

___________________________________________________

G. Fat Crystals
1. Heat a small piece of pork fat in an evaporating dish. (do not add water or
any other substance).
2. Get 1 ml of the extract and dissolve it in 2 ml of ether.
3. Stopper loosely with filter paper and allow to evaporate spontaneously until
crystals begin to separate.
4. Examine the crystals under the microscope and draw them.
5. Repeat steps 1 to 4 using beef fat.

Illustrate:

Crystals of Pork Fat Crystals of Beef Fat

Conclusion:
_______________________________________________________________

_______________________________________________________________
41
 _______________________________________________________________

_______________________________________________________________

_______________________________________________________________

_______________________________________________________________

Questions:

1. What is emulsification?
_______________________________________________________________

_______________________________________________________________

_______________________________________________________________

2. What are the types of emulsion? Differentiate each.

_______________________________________________________________

_______________________________________________________________

_______________________________________________________________

3. What is an emulsifying agent? Give an example.

_______________________________________________________________

______________________________________________________________

_______________________________________________________________

4. What is saponification number? Iodine number?

_______________________________________________________________

_______________________________________________________________

_______________________________________________________________

Name:___________________________ Date:________________________
Course & Year: __________________ Rating: ______________________

42
 Experiment No.8

Determination of Cholesterol Level in the Blood

Introduction:
The most abundant steroid in the human body and the most important is
cholesterol. Cholesterol serves as plasma membrane component in all animal
cells such as red blood cells. It also serves as a raw material for the synthesis of
other steroids such as sex and adrenocorticoid hormones and bile salts.

Cholesterol; exists both in the free form and esterified with fatty acids.

Because of the correlation between high serum cholesterol levels and

diseases as atherosclerosis, many people are afraid of cholesterol. Many regard it
as a poison. Unknowingly, the liver manufactures cholesterol that satisfies our
needs even without dietary intake. When the cholesterol level exceeds 150
mg/100 ml, cholesterol synthesis in the liver is rescued to half the normal rate of
production.

Objective:
To determine cholesterol level in the blood.

Materials:
Dropper Test tubes (3) Test tube rack
Test Tube brush Micropipette Spectrophotometer
Syringe Tourniquet Cotton
Centrifuge Pasteur pipette

Reagents and Chemicals:

Isopropyl Alcohol Cholesterol reagent
Distilled water

Procedures

Determination of Cholesterol Level in the blood using Cholesterol

Oxidase

1. Prepared serum.
2. Switch on the TC 84 spectrophotometer to stabilize the temperature at
37ºC.
3. Label three test tubes as blank, standard and sample.
4. Put 1.25 ml of cholesterol buffer in each test tube.
5. Add 10 microliter (using micropipette) of serum to sample tube, 10
microliter of cholesterol standard solution to standard tube and 10
microliter of distilled water to blank tube.

43
 6. Incubate the tubes in the built-in incubator of the spectrophotometer for 3
minutes.
7. Add 50 microliter of cholesterol enzyme reagent to all test tubes. Mix.
8. Re- incubate at 37ºC for 10 minutes.
9. Read the absorbance of the preparations in spectrophotometer at 505-550
nm against the blank.
10. Compute the level of cholesterol in the blood using the following formula.

Absorbance of sample
Conc.of glucose= ------------------------------ × conc.of standard
Absorbance of standard

NORMAL LEVEL OF CHOLESTEROL IN THE BLOOD: 70-105 mg/dl

RESULT AND INTERPRETATION:

_______________________________________________________________

_______________________________________________________________

_______________________________________________________________

_______________________________________________________________

Questions:

1. Write the formula of cholesterol.

______________________________________________________________
____

__________________________________________________________________
`

2. In what parts of the body is cholesterol found?

__________________________________________________________________

__________________________________________________________________

3. What foods are rich in cholesterol?

__________________________________________________________________

__________________________________________________________________
44
4. What is the clinical importance of the quantitative determination of cholesterol
in the blood?

__________________________________________________________________

__________________________________________________________________

__________________________________________________________________

__________________________________________________________________

5. What are gallstones?

__________________________________________________________________

__________________________________________________________________

Name: __________________________ Date: ____________________________

Course&Year: __________________ Rating: ___________________________

Experiment No. 9

45
 Enzymes

Introduction:

Enzymes are catalysts produced as a result of cellular activity. They are of

an organic nature. They act on biochemically important substances.

The chemical properties of enzymes, as well as their stability and

characteristics, are closely related to those of proteins. As more and more
enzymes have been isolated in a crystalline form and have proved to be single
components, it has become certain that enzymes are indeed proteins. The
enzyme (protein) without its prosthetic group is referred to as the apo
enzymes.

Objective:
To determine the different reactions and functions of the different
enzymes.

Materials:
Potato Cheesecloth pipette
Beaker 150 mL 10 test tubes Test tube rack
Test tube brush Watch glass Dropper
Aspirator Thermometer Water bath

Reagents/Chemicals:

Guiac solution 1% phenol

1% catechol solution Pyrogallol solution
Hydrogen peroxide 0.1 M NaF solution

Procedures:
A. Potato oxidase/ peroxidase
Preparation of Potato Extract:
1. Wash and peel a small sized potato and grate rapidly to a fine pulp.
2. Grind this pulp with 50 ml of 0.1 NaF solution and strain through
several layers of cheesecloth. The filtrate obtained is the enzyme
extract to be used for all subsequent tests.
3. Place 1 ml of the enzyme extract in each of the 4 clean test tubes.
To the first test tube, add 3 drops of 1% phenol; to the second test
tube, add 3 drops of 1% cathecol; to the third test tube, add 3 drops
of guiac solution; and to the fourth, add 3 drops of pyrogallol.
4. Mix the contents of the tubes by shaking gently and place in a
water bath at 37ºC.
5. Examine the tubes after 5 minutes by holding up to light.

46
 B. Potato perioxidase
1. Boil 5 ml of the enzyme extract for 1 minute.
2. Place 1 ml of the boiled extract in each of the 4 clean test
tubes. To the first test tube, add 3 drops of 1% phenol; to the
second test tube, add 3 drops of 1% cathecol; to the third test tube,
add 3 drops of guiac solution; and to the fourth, add 3 drops of
pyrogallol.
3. Add to each tube 3 drops of hydrogen peroxide.

Result:
______________________________________________________

______________________________________________________

C. Catalase
1. Prepare 2 clean test tubes . Put 1 ml of the unboiled potato extract in
one tube, and one ml of boiled potato extract in the other tube.
2. Add to each test tubes 3 drops Hydrogen peroxide.

Result:
_________________________________________________________

_________________________________________________________

_________________________________________________________

Conclusion:

_________________________________________________________

_________________________________________________________

_________________________________________________________

_________________________________________________________

_________________________________________________________

Questions:

1. What are the different classifications of enzymes? Give the functions

of each.
____________________________________________________________

47
 ____________________________________________________________

____________________________________________________________

____________________________________________________________

____________________________________________________________

____________________________________________________________

____________________________________________________________

____________________________________________________________

Name: ___________________________Date:___________________________
Course & Year: ___________________ Rating:_________________________

Experiment No.10

Factors Influencing Enzyme Reaction

48
Introduction:

Knowledge of basic enzyme kinetic theory is important in enzyme

analysis in order to understand the basic enzymatic mechanism and to select a
method for enzyme analysis. The conditions selected to measure the activity of
an enzyme would not be the same as those selected to measure the concentration
of its substrate.
Several factors affect the rate at which enzymatic reactions proceed-
temperature, pH, enzyme concentration, substrate concentration and the
presence of any inhibitors or activators.

Objective:
To determine the factors affecting the rate of enzymatic reactions.

Materials:

Potato Cheesecloth pipette

Beaker 150 mL 10 test tubes Test tube rack
Test tube brush Watch glass Dropper
Aspirator Thermometer Water bath

Reagents:
0.01 m cathecol 0.4% HCl 1% lactic acid
Na2 Co3 Distilled water

Procedures:

A. Substrate Concentration
1. Label 3 test tubes A, B and C and proceed as follows:
Test tube A: 2 ml of 0.01 M Cathecol
4 ml water

Test tube B: 4 ml of 0.01 M Cathecol

2 ml water

Test tube C: 6 ml of 0.01 M Cathecol

2. Add 2 ml of enzyme extract (prepared in experiment 8) to each
tube.
3. Place all test tubes in water bath at 37ºC, shake each test tube
every 3 minutes.
4. Examine each tube after every 5 minutes interval (three times).

RESULT:

49
 _________________________________________________________

_________________________________________________________

_________________________________________________________

_________________________________________________________

CONCLUSION:
_________________________________________________________

_________________________________________________________

_________________________________________________________

_________________________________________________________

B. Enzyme Concentration

1. Label 3 test tubes A, B and C and proceed as follows:

Test tube A: 1 ml of 0.01 M Cathecol
1ml enzyme extract

Test tube B: 1 ml of 0.01 M Cathecol

5 drops of enzyme extract
10 drops of water

2. Place all test tubes in water bath at 37ºC, shake each test tube every 3
minutes.
3. Examine each tube after every 5 minutes interval (three times).

RESULT:
_________________________________________________________

_________________________________________________________

_________________________________________________________

CONCLUSION:
_________________________________________________________

_________________________________________________________

_________________________________________________________

50
 _________________________________________________________

C. Temperature
1. Label 3 test tubes A, B and C.
2. Add 1 ml of enzyme extract to each test tube.
3. Place each test tube in water bath for 10 minutes at the following
temperatures:
Test tube A: 0ºC
Test tube B: 37ºC
Test tube C: 70ºC
4. Add 1 ml of 0.01 M cathecol to each test tube and shake gently.
5. Wait for 15 minutes.
6. Examine each test tube for any color changes.

RESULT:
_________________________________________________________

_________________________________________________________

_________________________________________________________

_________________________________________________________

CONCLUSION:
_________________________________________________________

_________________________________________________________

_________________________________________________________

_________________________________________________________

D. pH

1. Prepare 4 clean test tubes A, B C and D.

Test tube A: 2 ml of 0.04 % HCl (pH=1)
Test tube B: 2ml 0.1% lactic acid (pH=5)
Test tube C: 2 ml distilled water
Test tube D: 2 ml of Na2CO3
2. Add 1 ml of 0.01 M cathecol solution to each tube.
3. Add 1 ml of enzyme extract to each tube.
4. Wait for 15 minutes.
5. Examine each tube for color changes.

51
 RESULT:
_________________________________________________________

_________________________________________________________

_________________________________________________________

_________________________________________________________

CONCLUSION:
_________________________________________________________

_________________________________________________________

_________________________________________________________

_________________________________________________________

Questions:
1. Why should peeled potatoes not be exposed to air longer than necessary?

_________________________________________________________

_________________________________________________________

_________________________________________________________
2. Compare the stability of oxidase and peroxidase to heat.

_________________________________________________________

_________________________________________________________

_________________________________________________________

_________________________________________________________

_________________________________________________________

_________________________________________________________

3. Differentiate the action of oxidase, peroxidase and catalase.

_________________________________________________________

_________________________________________________________
52
 _________________________________________________________

_________________________________________________________

_________________________________________________________

_________________________________________________________

4. What are the optimum conditions for enzyme activity in human body?

_________________________________________________________

_________________________________________________________

_________________________________________________________

_________________________________________________________

_________________________________________________________

_________________________________________________________

5. Give other factors which influence enzyme activity and state the effect of
each factor.

_________________________________________________________

_________________________________________________________

_________________________________________________________

_________________________________________________________

_________________________________________________________
Name: ___________________________Date:___________________________
Course & Year: ___________________ Rating:_________________________

Experiment No.11

Digestion

Theory:

53
 The digestion of carbohydrates, fats and proteins is accomplished by a
series of hydrolytic change brought about by enzymes. These changes are carried
on in the digestive tract, which includes the mouth, esophagus, the stomach and
the small and large intestines. Secretions from the pancreas and the bile find their
way into the small intestine and as we shall see, play important roles in the
digestive process.

The initial digestion of carbohydrates is in the mouth by the action of

saliva that contains salivary amylase which is secreted by the salivary glands.
Mastication is the process of chewing that initially breaks food into small pieces.
The food initially digested in the mouth with the saliva is called “bolus”.

Protein is digested in the stomach by the action of gastric juice. Gastric

juice contains the enzyme pepsinogen and trypsinogen which are activated by
hydrochloric acid to pepsin and trypsin.

Carbohydrate is completely digested in the small intestine by the action of

pancreatic amylase in pancreatic juice secreted by the pancreas. Lipids are
emulsified in the small intestines by bile secreted by the liver and digested by the
action of lipase, enzyme secreted by the pancreas. Complete digestion of food
occurs in the small intestine with the help of intestinal juice.

Objective:
To determine the effects of enzymes in digestion.

Materials:
Saliva sample Test tube Test tube rack
Test tube brush Stirring rod Spot plate
Dropper Water bath Thermometer
Pipette Test tubes Test tube rack
Test tube brush

Reagents/Chemicals:
Paraffin wax Distilled water Litmus paper
Starch Iodine Benedict’s reagent
5% pancreatic solutions 0.5% Na2CO3 solution conc. HCl
Egg albumin solution Pepsin solution 10% NaOH

Procedure:

I. Digestions of carbohydrates:
A. Procedure for Collection of Saliva:
1. Rinse the mouth thoroughly with water and chew a piece of
paraffin wax to stimulate secretion of the saliva.

54
 2. Collect 10 ml of saliva in a test tube.
3. Filter the saliva and test the filtrate with red and blue litmus
paper, is it acidic? basic? or neutral?

Result:
______________________________________________________

______________________________________________________

______________________________________________________

B. Action of Saliva on Carbohydrates:

1. Make a thin paste by mixing half teaspoonful starch with little
cold water Add 100 ml boiling water. Stir while boiling for 2-3
minutes. Cool.
2. Put 50 ml of starch solution in a test tube. Add 1 drop of iodine
solution. This is the standard test for starch. What is the color
change?
Result:
___________________________________________________

________________________________________________

__________________________________________________

___________________________________________________
3. Put 10 ml of starch solution in a test tube. Add 2 or more drops
of saliva. Set in water bath at 37ºC for about half an hour to
become colorless.
4. Add 4 drops of this colorless solution in 5 ml Benedict’s
solution. Boil for 2 minutes and cool slowly. Red, yellow or
green precipitate indicates the presence of glucose.

Result:
______________________________________________________

______________________________________________________

______________________________________________________

C. Action of Pancreatic Juice on Starch:

1. Place 2 ml each of starch solution into 3 test tubes.
2. To one test tube, add 1 ml of 5% pancreatic solutions; to the second
test tube, add 1 ml of 5% pancreatic solution and 1ml of 0.5%

55
 Na2CO3 solution and to the third test tube, add 1ml of 0.5% Na2CO3
solution. Label each test tube.
3. Place the test tubes in a water bath maintained at 40 0 C and keep at
this temperature for about an hour.
4. Pipette sample from each tube and test with iodine solution. In
which tube was the color change faster?
5. Test the content of each tube with 2 ml of Benedict’s reagent.
Describe the color change in each test tube.
Result:
______________________________________________________

______________________________________________________

______________________________________________________

______________________________________________________

II. Digestion of Fats

Effect of pancreatic Enzymes on fats:
1. To three clean test tubes, place 1 ml each of milk or olive oil.
2. To test tube 1, add 1 ml of 5% pancreatic solution; test tube No.2,
add 1 ml pancreatic solution and 2 ml of 5% Na 2CO3 solution; test
tube No.3 add 2 ml of 5% Na2CO3.
3. Place all test tubes in a beaker containing hot water maintained at
30-400C for about an hour.
4. Test each tube with blue litmus paper. Observe which of the test
tubes changes the color of the litmus paper? What substances do
fats hydrolyze before they can be utilized by the body?

Result:
______________________________________________________

______________________________________________________

______________________________________________________

______________________________________________________

III. Digestion of Proteins

Action of Gastric Proteoses:
1. To 3 clean test tubes, place 2 ml one each of case-in or boiled
egg albumin solution. To test tube 1, add 1 ml of pepsin
solution; to test tube 2, add 1 ml of pepsin and 2 drops of conc.
HCl; and to the test tube 3 add nothing.
2. Keep all the test tubes in a beaker with hot water maintained at
380 – 400 for one hour or longer.

56
 3. Filter the content of each test tube and perform the Biuret’s test.
4. To each filtrate on the 3 test tubes, add 2 ml or 10% NaOH and
2 ml of dilute CuSO4 solution. In which of the test tubes would
you say hydrolysis has taken place? Why?

Result:
_________________________________________________

__________________________________________________

___________________________________________________

___________________________________________________

Conclusion:
________________________________________________________________

______________________________________________________________

_____________________________________________________________

_____________________________________________________________

Questions:

1. What are the factors that aid digestion?

_________________________________________________________

_________________________________________________________

_________________________________________________________

_________________________________________________________

_________________________________________________________

57
 _________________________________________________________

2. What are the 3 phases of digestion? Explain each.

_________________________________________________________

_________________________________________________________

_________________________________________________________

_________________________________________________________

_________________________________________________________

_________________________________________________________

3. What is secretion? absorption?

_________________________________________________________

_________________________________________________________

_________________________________________________________

_________________________________________________________

_________________________________________________________

_________________________________________________________

BIBLIOGRAPHY

Kaplan, Alex et.al Clinical Chemistry: Interpretation and Techniques, 4th

Edition. Educational Publishing House, Manila.1995

Murray, R.K. et al Harper’s Biochemistry, 24th Edition. Lange Medical

Publication, Connecticut, U.S.A. 1996````

58
Seelay, R.R. et al Essentials of Anatomy and Physiology, 5th Edition.
Mcgrawhill Educational Co. New York, U.S.A. 2005

Strasinger, Susan K. Urinalysis and Body Fluids, 4th Edition. A.A Davis Co.
Philadelphia U.S.A, 2001

Tietz, Norbert W. Fundamentals of Clinical Chemistry, 3rd Edition. W.B

Saunders, Co. Philadelphia, U.S.A

Brandt, Mark. Biochemistry Laboratory Manual, 3rd Edition. Retrieved at

http://www.rosehulman.edu/~brandt/publications/422_Manual_3
rd_Ed.pdf on June 2, 2010

L. Ivanoviene, R. Laboratory Manual on Biochemistry. Retrieved at

http://www.kmu.lt/nsc/biochemija/Laboratory_manual_PART
%20I.pdf retrieved on June 2, 2010

Lemg, Fenfei. Biochemistry Laboratory Manual, Revised 2009.Florida

International University, Department of Chemistry. Retrieved
from http://www2.fiu.edu/~lengf/biochemistry-lab-manual-
spring%202009.pdf on June 2, 2010

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