UMM AL-QURA UNIVERSITY

Approach to Medical Sciences Biochemistry

FACULTY OF MEDICINE
DEPARTMENT OF BIOCHEMISTRY

Approach to Medical Sciences

Biochemistry

Laboratory Manual
(Part II)

1431 1430

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Approach to Medical Sciences Biochemistry

Table of contents:
page

1Qualitative Analysis of Lipids.

.. 02
2Qualitative Identification of Carbohydrates (I)..
.. . 07
3Qualitative Identification of Carbohydrates (II)
. 11
4Tutorial to assess the student understanding of lessons 1,2,3.
.
5Ionic Properties of Amino Acids.
14
6Protein size and Separation.
18
7Enzyme Activity. ...
.. 22
8Tutorial to assess the student understanding of lessons
5,6,7..

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Approach to Medical Sciences Biochemistry

QUALITATIVE ANALYSIS OF LIPIDS

Introduction :
Lipids are naturally occurring compounds that are esters of long chain fatty acids.
They are insoluble in water but soluble in fat solvents such as acetone, alcohol,
chloroform or benzene.
Alkaline hydrolysis (known as saponification) gives rise to the alcohol and the Na
or K salts of the constituent fatty acids.
Chemically, lipids can be divided into two main groups:
1- Simple lipids.
2- Compound lipids.
Steroids and the fat soluble vitamins are also considered as lipids because of their
similar solubility characteristics and are known as derived lipids.
Major Roles of Biological Lipids:
Lipids of physiological importance for humans have four major functions:
1. They serve as structural components of biological membranes.
2. They provide energy reserves, predominantly in the form of
triacylglycerols.
3. Both lipids and lipid derivatives serve as vitamins and hormones.
4. Lipophilic bile acids aid in lipid solubilization.

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Approach to Medical Sciences Biochemistry

1- Simple lipids:
Esters of glycerol and fatty acids are known as acylglycerols or glycerides.

Triglyceride
The acylglycerols are known as neutral lipids, they are called fats or oils,
depending on whether they are solid or liquid at room temperature.
Fatty acids:
Fatty acids are long-chain hydrocarbon molecules containing a carboxylic acid
moiety at one end.
Fatty acids that contain no carbon-carbon double bonds are termed saturated fatty
acids; those that contain double bonds are unsaturated fatty acids.
Saturated fatty acids of less than eight carbon atoms are liquid at physiological
temperature, whereas those containing more than ten are solid. The presence of
double bonds in fatty acids significantly lowers the melting point relative to a
saturated fatty acid.
2- Compound lipids:
Complete hydrolysis of a compound lipid yields at least one other component as
well as the usual alcohol and fatty acids. These compounds are essential structural
components of cell membranes.
3- Derived lipids:

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Approach to Medical Sciences Biochemistry

Include Steroids and fat soluble vitamins.

Cholesterol:
Cholesterol is an extremely important biological molecule that has roles in
membrane structure as well as being a precursor for the synthesis of the steroid
hormones and bile acids.
The synthesis and utilization of cholesterol must be tightly regulated in order to
prevent over-accumulation and abnormal deposition within the body. Of particular
importance clinically is the abnormal deposition of cholesterol and cholesterolrich lipoproteins in the coronary arteries. Such deposition, eventually leading to
atherosclerosis, is the leading contributory factor in diseases of the coronary
arteries.

Cholesterol

PRINCIPLES:
The main groups of lipids have different solubility characteristics. When fats and
oils are heated with alkali, free fatty acids and glycerol are liberated and this
process is known as saponification. The excess alkali present reacts with the
liberated fatty acids to form the Na or K salts which give the solution a
characteristic soap appearance. Soaps are soluble in water but precipitated on the
addition of excess NaCl. The Mg and Cl salts on the other hand are insoluble and
give rise to the cum formed when soap is lathered in hard water.
The fatty acids in animal fats are usually fully saturated whereas those found in
vegetable oils contain one or more double bonds. Hydrogenation of the double

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Approach to Medical Sciences Biochemistry

bonds converts the oils into solid fats and this is carried out commercially for the
production of margarine.
Halogens also readily add across the double bonds and the decolorization of a
solution of bromine or iodine by a lipid indicates the presence of double bonds.
MATERIALS:
Reagents

HCl. Conc.
NaOH (1N).
NaCl (solid).
Bromine solution (1 ml Br2/20
ml Chloroform).
H2SO4 Conc.
KI (10% w/v).

Ethanol (absolute).
Diethyl ether.
Petroleum ether.
Chloroform.
Benzene.
Copper acetate (1% w/v).
Alco. KOH (100 g/l in
ethanol).

Standard liquids
Phospholipids: Lecithin.
Sterols: cholesterol (solid and
0.5% in ethanol).

Fatty acids: palmitic acid,

Stearic acid.
Fats: Butter.
Oils: Olive oil.

1SOLUBILITY:
Note the physical state of:
f) Water.
a) Palmetic acid.
g) Ethanol.
b) Stearic acid.
h) Diethyl ether.
c) Oleic acid.
i) Petroleum ether.
d) Olive oil.
j) Chloroform.
e) Butter.
Carefully observe the differences between the above groups of lipids, write your
remarks.
2GREASE STAIN TEST:
Place one drop of the above lipids on a filter and leave to dry, observe the formation
of a clear grease spot and give your remarks.

3-

COPPER ACETATE TEST:

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Approach to Medical Sciences Biochemistry

Dissolve a few drops of the oil in 3 ml. of petroleum ether and add equal volumes of
copper acetate (1%), mix once by inversion (Dont shake). Leave the tube until the
emulsion will separate into two layers.
A. Saturated fatty acid: upper layer is clear and precipitate in the lower layer.
B. Unsaturated FA: upper layer is greenish-Blue color and lower layer is
colorless.
C. If the two layers are clear then the test.
4TEST FOR UNSATURATION:
Add one drop of Olive oil, one spatula-point of Butter and one spatula-point of
Lecithin to separate dry test tubes, and dissolve the lipids in about 1 ml. of
chloroform.
Add 1 ml. of chloroform to other test tube to act as Blank.
By means of Pasteur pipette add drop wise a solution of bromine in chloroform until
define yellow color is produced, Note the number of drops in each case, and
comment on the results.
CH3 (CH2)7 CH=CH (CH2)7 COOH

Oleic acid

Br2
Bromine

H
CH3 (CH2)7 C
Br

H
C (CH2)7 COOH
Br

Dibromo-Stearic acid
5TEST FOR CHOLESTEROL (Liebermans test):
Add 10 drops of acetic anhydride and 2 drops of conc. H2SO4 to 2 ml of each of the
following:
a) Cholesterol solution in chloroform (0.5 %).
b) Egg yolk solution in chloroform (0.5%).
c) Butter solution in chloroform (0.5%).
d) Chloroform.
Give possible interpretation of the reaction in each case.

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Approach to Medical Sciences Biochemistry

CARBOHYDRATES
Introduction:
Sugars can be defined as polyhydroxy aldehydes or ketones. Hence the simplest
sugars contain at least three carbons. The most common are the aldo- and ketotrioses, tetroses, pentoses, and hexoses. The simplest 3C sugars are glyceraldehye
and dihydroxyacetone
Carbohydrates can be classified as either monosaccharides, oligosaccharides or
polysaccharides. Anywhere from two to ten monosaccharide units, linked by
glycosidic bonds, make up an oligosaccharide. Polysaccharides are much larger,
containing hundreds of monosaccharide units. The presence of the hydroxyl
groups allows carbohydrates to interact with the aqueous environment and to
participate in hydrogen bonding, both within and between chains. Derivatives of
the carbohydrates may contain nitrogens, phosphates and sulfur compounds.
Carbohydrates also can combine with lipid to form glycolipids or with protein to
form glycoproteins.
Monosaccharides:
The monosaccharides commonly found in humans are classified according to the
number of carbons they contain in their backbone structures. The major
monosaccharides contain four to six carbon atoms.

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Approach to Medical Sciences Biochemistry

Cyclic Fischer Projection of

Haworth Projection of

-D-Glucose

-D-Glucose

Disaccharides:
Covalent bonds between the anomeric hydroxyl of a cyclic sugar and the
hydroxyl of a second sugar are termed glycosidic bonds, and the resultant
molecules are glycosides. The linkage of two monosaccharides to form
disaccharides involves a glycosidic bond. Physiogically important disaccharides
are sucrose, lactose and maltose.
Sucrose: prevalent in sugar cane and sugar beets, is composed of glucose and
fructose
through
an
-(1,2)-glycosidic
bond.

Sucrose
Lactose: is found exclusively in the milk of mammals and consists of
galactose and glucose in a -(1,4) glycosidic bond.

Lactose

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Approach to Medical Sciences Biochemistry

Maltose: the major degradation product of starch, is composed of 2 glucose

monomers in an -(1,4) glycosidic bond.

Maltose
Polysaccharides:
Most of the carbohydrates found in nature occur in the form of high molecular
weight polymers called polysaccharides. The monomeric building blocks used to
generate polysaccharides can be varied; in all cases, however, the predominant
monosaccharide found in polysaccharides is D-glucose. When polysaccharides
are composed of a single monosaccharide building block, they are termed
homopolysaccharides. While the Polysaccharides composed of more than one
type of monosaccharide are termed heteropolysaccharides.
PREPARATION OF REAGENTS
1.

2.

3.

4.

5.

6.

7.

8.

Molischs reagent
5% naphthal in alcohol, i.e., 5g of naphthal dissolved in 100ml of
ethanol.
Anthrone test:
Anthrone (2g/L in conc H2SO4).
Iodine solution
0.005% in 3% KI, i.e., 3g of KI dissolved in 100ml water and then 5mg of
iodine is dissolved.
Benedicts solution
17.3g of sodium citrate and 10g of sodium carbonate are dissolved in 75ml
of water. 1.73g of CuSO4.5H2O is dissolved in 20ml of water. Mix the
CuSO4 solution with alkaline citrate with constant stirring, finally the whole
volume is made up to 100ml with water.
Barfoeds reagent
13.3g of copper acetate in 200ml of water and add 2ml of glacial acetic acid.
Fearons test:
(methylamine Hydrochloride (MH) 5% in H2O + NaOH (20%).
Bials reagent
Dissolve 300mg of orcinol in 100ml of concentrated HCl.
Seliwanoffs reagent
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Approach to Medical Sciences Biochemistry

9.
10.
11.

Dissolve 50g of resorcinol in 100ml of con.HCl in the ratio of 1:2.

Concentrated HCl
Concentrated H2SO4
Osazone Reagent
Phenyl hydrazine hydrochloride
Sodium acetate
Acetic acid

Mechanistic principles of qualitative identification of Carbohydrates

PRINCIPLE OF REACTIONS:
1.Molischs test:
Con. H2SO4 dehydrates carbohydrates to form furfural and its derivatives.
This product combines with sulphonated naphthal to give purple colour.
2.Anthrone test:
The same principle outlined above, except that the furfural reacts with
anthrone (10-keto-9,10-dihydroanthracene) to give a blue-green complex.
3.Iodine test:
Iodine
forms
a
coloured
absorption
complex
with
polysaccharides due to the formation of micellae aggregate.
Iodine will form a polysaccharide inclusion complex.
4.Benedicts test
Carbohydrates with a potential aldehyde or ketone group
have reducing property when placed in an alkaline solution.
Cupric ions present in the solution will be reduced to cuprous
ion. This will give a red coloured precipitate. Moreover, this
test is more specific for reducing sugars.
5.Barfoed test:
Barfoeds reagent is weakly acidic and it is only reduced by
monosaccharides.
Prolonged boiling may hydrolyze the
disaccharide to give false positive test.
6.Bials test:
When pentose is heated with con.HCl, furfural, which
condenses with orcinol in the prescence of ferric ion to give
a blue green colour.

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Approach to Medical Sciences Biochemistry

7.Seliwanofs test:
Ketoses are dehydrated more rapidly than aldose to give a
furfural derivatives, which then condenses with resorcinol to
form a red colour complex.
8.Osazone test:
Compounds containing aldehyde and keto groups form crystalline osazone
with phenyl hydrazine hydrochloride. Osazone crystals have characteristic
shape and melting point which helps in the identification of reducing sugar.

GENERAL PROCEDURE FOR QUALITATIVE ANALYSIS OF

CARBOHYDRATES
No.
1.

2.

3.

EXPERIMENT

OBSERVATION

Molischs test
To 1ml of test solution, add Violet coloured ring is
2 drops of Molischs reagent. formed at the junction
Then add con. H2SO4 carefully of the 2 layers.
along the sides of the test tube.
Anthrone test
To 5 drops of sugar solution
add 2 ml. Anthrone reagent.

Blue green color

complex is formed

Iodine test
To 1ml of the test solution, (i) Deep blue colour is
2 drops of iodine is added and obtained.
observe the colour change.
(ii)Dark brown colour
is obtained.

(ii)No characteristic

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ONCLUSION
Presence of
carbohydrate.

Presence of
carbohydrate.

Presence of
polysaccharide.
(Starch)
Presence of
polysaccharide.
(Glycogen)
Absence of
polysaccharide.

Approach to Medical Sciences Biochemistry

colour change.
4.

Benedicts test
5ml of Benedicts reagent
is mixed with 1ml of test
solution and the contents are
boiled for a few minutes.

(i)Orange red
precipitate is obtained.

Presence of
reducing sugar.

(ii)No characteristic

Absence of
reducing sugar.

colour change.

5.

Barfoeds test
To 2ml of test solution,
2ml of Barfoeds reagent is
added and boiled for 3
minutes and the colour change
is noted.

(i)Brick red precipitate Presence of

is obtained at the
reducing
bottom of test tube.
monosaccharide
(ii)No characteristic
colour change.

6.

FEARON'S TEST
To 1ml of the test solution,
2ml of Fearons reagent is
added and the content is
heated. Then NaOH was
added to the cold mixture.

7.

Absence of
reducing
monosaccharide.

i)Red coloration appear. Presence of

reducing
disaccharide.
ii)no color change.
Absence of
reducing
disaccharide.

Bials test
To 1ml of the test solution, (i)Blue green colour
5ml of Bials reagent is added.
is obtained.
The contents are boiled and
cooled.
(ii)No characteristic
colour change.

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Presence of
pentose sugar.
Absence of
pentose sugar.

Approach to Medical Sciences Biochemistry

8.

Seliwanoffs test
To 1ml of the test solution, (i)Cherry red colour
3ml of Seliwanoffs reagent is
is obtained.
added and the contents are
boiled
(ii)No characteristic

Presence of
fructose.
Absence of
fructose.

colour change.

9.

Osazone test
To 1ml of
the test solution,
, add 2-3 drops
of glacial acetic
acid, followed
by the addition
of a pinch of
phenyl
hydrazine
hydrochloride
and twice the
amount of
sodium acetate
and the contents
are boiled

(i)Yellow colour crystals are formed in 5

minutes, as observed through a
microscope.

Presence of
fructose is
confirmed

Haystack structure form of fructosazone.

(ii) Yellow colour crystals are formed in
5-10 minutes, as observed through a
microscope.

Presence of
glucose is
confirmed.

Haystack structure form of glucosazone

(iii) Yellow colour crystals are formed in
10 minutes.
Broad fan structure crystals of
galactosazone are observed through
microscope.

Presence of
galactose is
confirmed.

(iv) Yellow colour crystals are formed in

20-25 minutes,

Presence of
Maltose is
Sunflower shaped crystals of maltosazone confirmed.
are observed through the microscope.
(v)Yellow colour crystals are formed in 510 minutes,
scattered needles shaped crystals of

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Presence of
Xylose is

Approach to Medical Sciences Biochemistry

Xylosazone are observed through the

microscope.
(vi) Yellow colour crystals are formed in
20-30 minutes,
Powderpuff shaped crystals of
Lactosazone are observed through the
microscope.

confirmed.

Presence of
Lactose is
confirmed.

IONIC PROPERTIES OF AMINO ACIDS

OBJECTIVES:
1- To investigate the ionization of compounds with two or more dissociable
groups.
2- To demonstrate the particular importance of histidine as a potential buffer at
physiologic pH.
Introduction:
Amino acids serve as basic building blocks for proteins. There are a large number of
chemically possible amino acids, but only a few of these occur naturally.
In case of the 22 or so amino acids found in proteins, nearly all of them are -amino
acids, where the amino group is present on the -carbon atom.
The strong positive charge on the -NH3+ group induces a tendency for the COOH
group to lose a proton, so that amino acids are strong acids. The pKa for (the COOH
of) Glycine, for example (2.3), is much lower than acetic acid (4.75), and other
amino acids containing an aliphatic side chain have similar pK values to Glycine.
Ionic properties of amino acids:
Amino acids are polyprotic, i.e. they all contain at least two dissociable
protons, when dissolved in water, The proton from the -COOH group transfers to the
-NH2 end of the molecule, because the NH2 group is a stronger base than -COOforming the so called (zwitter ion) which contain both positive and negative
charges ,while the net charge on the ion will be zero.

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Approach to Medical Sciences Biochemistry

If the zwitter ion is treated with acid, H+ will be added to the COO- to form
COOH, the resulting shape is the cation bearing a net positive charge. Similarly,
treating the zwitter ion with base will result in the loss of the removable proton
attached to the NH3+ group to form NH2, and the result anion bears a net negative
charge. The following pH dependent equilibrium can be drawn.
K1
H3N+-CHR-COOH

K2
H3N+-CHR-COO-

H2N-CHR-COO-

(K1 , K2: are the ionization constants of the COOH and NH 3 respectively.)

e.g. : Glycine :
pKa=

2.34

H3N+---CH2---COOH
Gly+

9.60
H++H3N+---CH2---COO-

H++H2N---CH2---COO-

Glyo

Gly-

Buffering:
According to Henderson-Hasselbalch equation:
pH = pKa + log[A-]/[HA]
At the point of the dissociation where the concentration of the conjugate base [A-] is
equal to that of the acid [HA]: pH = pKa + log[1]
The log of 1 = 0. Thus, at the mid-point of titration of a weak acid:
pH = pKa
At this point, when the pH = pKa, the slope of the curve (i.e. the change in
pH with addition of base or acid) is at a minimum, so the buffer solution best resists
addition of either acid or base, and hence has its greatest buffering ability. As a
general rule, buffer solution can be made for a weak acid/base in the range of 1 pH
unit from the pKa of the weak acids.

addition of strong base produces weak conjugate base:

CH3CO2H + OH- ------------> CH3CO2- + H2O

addition of strong acid produces weak acid:

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Approach to Medical Sciences Biochemistry

H3O+ + CH3CO2------------> CH3CO2H + H2O

Blood Buffering:
The pH of blood is maintained in a narrow range around 7.4. Even relatively
small changes in this value of blood pH can lead to severe metabolic
consequences. Therefore, blood buffering is extremely important in order to
maintain homeostasis. The primary buffers in blood are hemoglobin in
erythrocytes and bicarbonate ion (HCO3-) in the plasma. Buffering by hemoglobin
is accomplished by ionization of the imidazole ring of histidines in the protein.

EXPERIMENT-1
Preparation of Normal Titration Curve for Glutamic Acid
The dissociation of glutamic acid can be represented as:

We are going to use the pH meter to explore the acid-base behavior of Glutamic
acid .
Materials:
-

Buret
Beaker.
pH standards.
0.05M NaOH.

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0.05M HCl.
0.05M Glu.
0.05M Hi.
Ph meter.

Approach to Medical Sciences Biochemistry

Procedure:
1- Titrate 10 ml. of 0.05M Glutamic acid against 0.05M NaOH.
Repeat the titration with 0.05M HCl.
2- Record your data in the given table.
3- Sketch a curve from your data on a graph paper.
(Plot pH (Yaxis) versus volume of NaOH expended (Xaxis).
Volume (ml.)
NaOH 0.05M
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0

pH

Volume (ml.)
HCl. 0.05M
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0

pH

EXPERIMENT-2
Preparation of Normal Titration Curve for Histidine
Repeat the above procedure using 0.05M Histidine.
The dissociation of Histidine can be represented as:

Here , is the ionizable groups of Histidine:

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Approach to Medical Sciences Biochemistry

PROTEIN SIZE AND SEPARATION

PROTEINS:
Proteins are polymers of the bifunctional monomers, amino acids. The twenty
common naturally-occurring amino acids each contain an -carbon, an -amino
group, an -carboxylic acid group, and an -side chain or side group. These side
chains (or R groups) may be either nonpolar, polar and uncharged, or charged,
depending on the pH and pKa of the ionizable group.
Amino acids form polymers, the amino group from one amino acid attached to
the carboxylic group of the adjacent amino acid, the resulting link between them
is an amide link which biochemists call a peptide bond. In this reaction, water is
released. In a reverse reaction, the peptide bond can be cleaved by water
(hydrolysis).
O

C ------ HN

H2N

R1

R2

OH

Peptide bond
When two amino acids link together to form an amide link, the resulting structure
is called a dipeptide. Likewise, we can have tripeptides, tetrapeptides, and other
polypeptides. At some point, when the structure is long enough, it is called a
protein. There are many different ways to represent the structure of a polypeptide
or protein.
Separation of proteins
The molecular weights of proteins are very high. Due to their wide variety of
amino acid configurations, proteins behave very differently. These differences
constitute the bases of the biochemical function of proteins. And these basic
differences are also the parameters which are used to separate proteins:
1- Physical Size:
Which reflects molecular weight of the protein, Gel filtration uses this
parameter, also referred to as Exclusion chromatography.

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Approach to Medical Sciences Biochemistry

2- Electric Charge:
Some amino acid residues are positively charged while others are negatively
charged. Variations in the pH of an amino acid system cause variations in the
charge of amino acid residues. Consequently, the net surface charge of a
protein (comprised of amino acid residues) also varies with its environment.
It is these variations in the net charge of proteins which allow them to be
separated by such techniques as ion exchange chromatography or by
electrophoretic techniques.
3- Hydrophobic character:
Hydrophobic regions available to interact with a hydrophobic stationary
phase provide the important characteristic of proteins which is used in
adsorption chromatography.
4- 3-dimentional substructure:
Provide the basis for very specific separation methods. Bio-specific affinity
chromatography is used to separate proteins according to their biological
functions. Proteins, such as enzymes, antibodies and glycoproteins are
particularly appropriate for separation by affinity chromatography.
Experiment:

EXCLUSION CHROMATOGRAPHY

Objectives:
a- To demonstrate the principle of molecular exclusion (gel permeation)
chromatography using a bead of G-100 Sephadex gel to separate a mixture
of:
Fluorescin
: Yellow M.wt. = 332 (size of a dipeptide).
Hemoglobin

: Red

M.wt. = 68,000 (protein).

Blue Dextran

: Blue

M.wt. = 200,000 (large protein).

b- To show that the technique can be used to determine the Molecular Weight
of a newly discovered protein.

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Approach to Medical Sciences Biochemistry

Principle:
In gel filtration the gel acts as a molecular sieve separating molecules with
differences in molecular size and weight. The gel matrix contains numerous
porous beads (stationary phase) with (mobile phase) in between. If the ample of
mixture is applied at the top of the column, the large molecules in the sample will
not be able to enter the pores in the bead but will pass between them and so be
eluted first, smaller molecules that have access to the pores are retarded in the gel
to a certain extent and will therefore be eluted after the large molecules in order of
decreasing M.wt. and size.
Materials:
Sephadex G-100: 0.5 g of (S.G-100) /100 ml. of 0.3% (w/v) NaCl; leave to
swell for 1 hr. prior to experiment.
Eluant: 0.3% (w/v) NaCl.
Mixture of : Flurescin, hemoglobin and blue dextran (each 0.1 g/10 ml.
H2O). To prepare Hemoglobin dilute 1 ml. of blood to 10 ml. with H2O.
Chromatography column (0.1 to 1.5 cm. diameters).
Microperpox peristaltic pump: Flow rate = 0.5 ml/min.
Disposable syringes (5ml, needle size 20 G).
Method:
1. Set up the column replacing the waist flask by a fraction collector.
2. Pour slurry of Sephadex G-100, into the column and allow the gel beads to
settle. Once about 10 cm. have settled, allow the solvent to run out of the
button of the column. Do not allow the liquid level to fall below the top of
the Sephadex. Continue to add Sephadex until you have a column whose
settled height is at least 15 cm.
3. Carefully place a small filter paper disc on top of the gel bed. This will
prevent disturbance of the surface when sample or solvent are added.
4. Let the liquid level fall to the filter paper disc. Carefully add 0.2 ml. of the
colored mixture with a pipette to the top of the column and let this run into
the gel. Then add 0.5 ml. of the eluant and run this into the gel.
5. Fill the column with the eluant by connecting it to the reservoir and elute
the sample, collecting fractions of ml. in numbered test tubes (Flow rate =
0.5 ml/min.)
6. Stop collecting fractions when the last visible band has been eluted and
record the appearance of your fractions.
7. Repeat the procedure after adding 1% of protein to the mixture.

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Approach to Medical Sciences Biochemistry

Treatment of data:
1. Measure the absorbance of the known fractions:
Fluroscin
: 490 nm.
Hemoglobin : 420 nm.
Blue Dextran : 630 nm.
2. Plot a graph of absorbance versus fraction numbers.
3. Measure the absorbance of the fractions containing the protein of unknown
M.wt.
Plot M.wt. versus fraction numbers and read the M.wt. of the unknown
protein.
Fig.: Exclusion Chromatography.

Separation of the sample

Theory of separation

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Approach to Medical Sciences Biochemistry

ENZYME ACTIVITY
Enzymes are biological catalysts that increase the rate of chemical reactions in the
living organisms. Unlike most inorganic catalysts, enzymes have a very narrow
specificity, i.e., they will only catalyze a comparatively small range of reactions
or, in some cases, only one reaction.
The overall reaction can be represented as follows:

The activity of enzymes can be measured by measuring the concentration of their

products and this approach is widely used in clinical biochemistry and medical
laboratories.
Enzymes function only under certain conditions of pH, temperature, substrate
concentration, cofactors, etc. and some of these properties are illustrated in the
following experiments.
ALP Reaction:
The enzyme catalyze the following reaction:
P

p-Nitrophenyl phosphate

p-Nitrophenol

(Substrate)

(Yellow colour)

Materials:
1.
2.
3.
4.
5.

Carbonate buffer with different pH.

p-Nitrophenyl phosphate (0.01 M)
NaOH 1M.
Alkaline phosphatase Enzyme (0.1 mg/ml.)
p-Nitrophenol (50 mol/L.)

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Approach to Medical Sciences Biochemistry

Experiment -1-

Effect of Temperature

This experiment demonstrates the apparent existence of an optimum, above and

below which the overall rate of the reaction decreases. The effect is the resultant
of tow opposing processes:
An increase in temperature increases the velocity of the a chemical
reaction.
An increase in temperature causes an increased rate of thermal inactivation
of the enzyme, with the result that the velocity of the enzymatic reaction is
diminished.
Set the following tubes and incubate them at the stated temperature:
Contents (ml.)
Buffer (pH9.2)
Substrate0.01M
Temp. ()
Absorbance

1
2
2

2
2
2
18

3
2
2

4
2
2
37

Tube No.
5
6
2
2
2
2
45

7
2
2

8
2
2
55

9
2
2

10
2
2
80

Conc. of the
product
1. When the tubes have been given time to reach the desired temperature, add
2 ml. of enzyme to tubes: 1,3,5,7 and 9. mix thoroughly and incubate for
your chosen time (Experiment Time course).
2. At the end of the incubation period stop the reaction by adding 2 ml. of 0.1
M NaOH to all tubes, and then 2 ml. of enzyme to tubes: 2,4,6,8, and 10.
Mix well.
3. Read the absorbance starting with the faint tube.
4. After substracting the values of the Blank (i.e. the even numbered tubes);
use the calibration curve to calculate the amount of p-Nitrophenol liberated,
and plot these values (which give reaction velocity v) against temperature.
5. Calibration curve:
6. Prepare a standard curve. Stock of p-Nitrophenol (50 umol/L.). dissolve
278.4 gm/dl. In the buffer and dilute 1 ml. of the solution to 100 ml. with
the buffer. Working solutions: 5,10,20,30,40 and 50 umol. (final
concentrations).
7. Add 3 ml. of NaOH (1M) to 1 ml.of each of the above solutions into a test
tube, mix well and read the absorbance at 405 nm. Plot a graph of
absorbance against concentration.
8. Use the standard curve to calculate the amount of product (p-Nitrophenol)
liberated. Plot this against reaction temperature. Measure the initial rate and
choose a suitable incubation temperature for experiment 2.
26

Approach to Medical Sciences Biochemistry

Experiment -2-

Optimum pH

Set the following tubes in a batch at 37 using buffers of different pH.

Contents
(ml.)
Buffer pH
Buffer ml.
Substrate
0.01M
Enzyme

1
8.5
2
2

2
9.0
2
2

3
9.2
2
2

Tube No.
4
5
6
9.6
10
10.5
2
2
2
2
2
2
2

7
11
2
2

8
11.5
2
2

9(BL.)
9.2
2
2

Absorbance
Conc. of
the product
Incubate the tubes at 37 for your chosen period (time course), then add 2 ml. of
1 M NaOH and mix. After adding 2 ml. of enzyme to the blank tube 9, read the
absorbance of all tubes.
Experiment -3-

The time course of the reaction

Set the tubes at 37 as follows:

Contents
(ml.)
Buffer pH
=(9.2)
Substrate
0.01M
Enzyme

Tube No.
5
6
2
2

1
2

2
2

3
2

4
2

7
2

8
2

9
2

10
2

Time
(min.)
Absorbance
Conc. of
the product
1. Pre-incubate the tubes for 5 mts.
2. Stop the reaction by adding 2 ml. of 1 M NaOH at the following times:
3. Tubes: (1,2:4 mts.);(3,4:8 mts.);(5,6:12 mts.);(7,8:16 mts.);(9,10:20 mts.).

26

Approach to Medical Sciences Biochemistry

4. Blank: add 2 ml. of enzyme to tubes: 2,4,6,8 and 10. mix well.
5. Read the absorbance of the solutions (all tubes) in order of increasing color
density at 405 nm.
6. prepare a standard curve (calibration curve). Stock of p-Nitrophenol
(50umol/L.). dissolve 278.4 mg/dl. In the buffer and dilute 1 ml. of the
solution to 100 ml. with the buffer. Working solutions: 5,10,20,30,40 and 50
umol. (Final concentration.).
7. add 3 ml. of NaOH (1M) to 1 ml. of each of the abovesolutions into a test
tube, mix well and read the absorbance at 405 nm. Plot a graph of
absorbance against concentration.
8. use the standard curve to calculate the amount of product (p-Nitrophenol)
liberated, and plot this against reaction time (point-c). measure the initial
rate and choose a suitable incubation period for experiment 2.
Experiment -4-

Effect of enzyme concentration:

Set the following tubes and add the enzyme last:

Contents (ml)
Buffer pH 9.2
Subst. 0.01M
Water
Enzyme
Absorbance

0
2
2
2
0.0

1
2
2
1.8
0.2

Tube No.
2
3
4
2
2
2
2
2
2
1.5
1
0.7
0.5
1
1.3

5
2
2
0.3
1.7

6
2
2
0.0
2

Conc. of the
product
1. Incubate the tubes for a suitable period.
2. Stop the reaction by adding 2 ml. of 1M NaOH.
3. Read the absorbance of the solution.(405 nm)
4. Use the standard curve to calculate the amount of p-Nitrophenol liberated in
each tube and plot this against the volume of enzyme added. Choose the
enzyme concentration at which the activity is linear ( data based on linear
activity or rate gives precise and accurate estimates of the enzyme
parameters such as Michael's constant (Km) of the enzyme.

26

Approach to Medical Sciences Biochemistry

Further readings:
1. Skoog, D. A.; Principles of Instrumental Analysis, 6th ed.; Thompson
Brooks/Cole: Belmont, CA, 2006, Chapter 28.
2. Smith AL (Ed) et al. (1997). Oxford dictionary of biochemistry and
molecular biology. Oxford [Oxfordshire]: Oxford University Press. ISBN
0-19-854768-4.
3. Grisham, Charles M.; Reginald H. Garrett (1999). Biochemistry.
Philadelphia: Saunders College Pub. pp. 4267. ISBN 0-03-022318-0.
4. Plummer D. T.: An Introduction to Principle Biochemistry, 2nd. ed. 1978.

26