UNIVERSITY OF ZIMBABWE

DEPARTMENT OF BIOMEDICAL SCIENCES

BMS 1: PRACTICALS

Experiment 1 Acid-Base Titration

Experiment 2 Use of the Spectrophotometer
Experiment 3 Properties of Proteins
Experiment 4 Enzymology: Determination of the effect of pH on Enzymatic
Activity
Experiment 5 Enzymology: Demonstrate the effect of Enzyme Concentration
on Reaction Velocity
Experiment 11 Determination of Sugar in Blood- Method 1 (Folin and WU)

1
 EXPERIMENT 1

ACID-BASE TITRATION

Biochemical Significance of acid-base balance

Mammalian cells are actively involved in various metabolic processes which

maintain the viability of that organism. The enzymes (bio-catalysts) which enhance
these biochemical processes require optimal pH ranges for efficient catalysis.

Sometimes these metabolic processes can cause an undesirable increase in either

H+ concentration (a condition called acidosis) or a decrease in H + concentration (a
condition called alkalosis). Such unfavorable conditions will tend to depress the
activities of the biological catalysts and hence the metabolic efficiency of the cells.
There is therefore a need to maintain acid-base balance in mammalian cells. The
body/cell has many buffer systems in order to achieve this balance.

PURPOSE: Volumetric determination of a mixture of sodium hydroxide and sodium

carbonate

NOTE: Bicarbonate, an intermediate in this reaction, is an important buffer

salt in the cell and body.

GENERAL THEORY AND PRINCIPLE

Carbonate and hydroxide are bases, which can be determined volumetrically with a
single standard acid like HCL. A complete quantification of these two bases can be
achieved by using two indicators in the same titration.

Use of indicators

Several methods can be used to establish the end point of an acid – base titration.
Use of indicators is one of the methods. An indicator is usually a weak acid which
dissociates in solution.

AH A - + H+
(indicator (indicator
colour 1) colour 2)

It exhibits one colour in the free, unionized form (colour1) and another colour in the
ionized form (colour 2). Therefore the colour of any given indicator depends on its
degree of ionization which in turn is dependant on the pH of the solution. This is the
basis on which indicators are utilized to estimate end-points during an acid-base
titration.

2
 1. Neutralization of Na2CO3 by HCl:

Complete neutralization of Na2CO3 by HCL occurs in 2 stages (Fig 1)

i) Na2CO3 + HCl NaHCO3 + NaCl pH 8.3

ii) NaHCO 3 + HCl NaCl + CO2 + H2O pH 3.8

pH 8.3 (i) Partial neutralization

3.8 (ii) complete neutralization

Acid Added (ml)

Fig. 1. Titration curve of Na2CO3

2. Neutralization of NaOH by HCL:

The neutralization of NaOH by HCl occurs in one stage only (Fig 2):
1) NaOH + HCl NaCl + H2O

9
(i) complete neutralization of NaOH

Acid added (ml)

Fig. 2 Titration curve of NaOH
3
 MATERIALS

The following materials are provided

1. Alkali solution (containing roughly 3 g/ litre NaOH and 1 g/ litre Na 2CO3).

2. 1 M HCl stock solution
3. Methyl orange indicator (pH range 3.2 – 4.4), 0.05 g dissolved in 50 ml water.
4. Phenolphthalein (pH range 8.3 – 10.0), 0.5 g in 50 ml ethanol and 50 ml
water.

METHOD

Make 100 ml of 0.1 M HCl from stock. Place 25 ml aliquot of unknown alkali
solution (containing NaOH and Na2CO3) into a conical flask.

Titration

Phase 1: Add 2 drops of phenolphthalein indicator to the solution in the

flask. Titrate to end point (solution turns from pink to colorless)
with 0.1M HCl. Note volume (V1).

Phase 2: Add 2 drops methyl orange indicator to the above solution and
continue titration to the new end point. Note volume (V 2).
Above procedure to be done in triplicate.

DATA ANALYSIS:

Table 1: Summary of results from acid-base titrations

(1)
PROCEDURE TITRE

V1 (ml) V2 (ml)
st
1 Procedure

2nd Procedure

3rd Procedure

Mean _______________ml __________________ml

4
(2) In Phase 1: Only NaOH is completely neutralized. Na 2CO3 is
partially neutralized so that only NaHCO3 remains in
solution.

In Phase 2: NaHCO3 is then completely neutralized.

a) Determine the total volume(mean) of acid (HCl) required to completely

Neutralize:
i) NaOH only in phase 1
ii) Na2CO3 only in both phase 1 and 2

b) Using the above data in a) calculate the concentration of NaOH and

Na2CO3 in the unknown in g/litre.

QUESTIONS

a) According to the Bronsted- Lowry theory:

i) What is an acid?
ii) What is a base?

b) i) Define pH
ii) If the hydrogen ion concentration in a blood corpuscle is
6.5 x 10 –5 M, what is the pH? Show your calculations.

c) i) What is a buffer?
ii) Briefly explain the mechanism of buffer action of the
H2CO3 / HCO3- buffer system.

d) What are the major disadvantages of using an indicator to measure the

end-point in an acid – base titration?

References

Information of buffer and buffer systems will be found in most theoretical

and practical biochemistry books.

5
 EXPERIMENT 2

USE OF THE SPECTROPHOTOMETER

The Nature of light: Electromagnetic radiation, which is classified as light extends

over a small part of the electromagnetic spectrum. The following table shows the
name given to the various wavelengths of electromagnetic radiation, which are
commonly classified as light.

Wavelengths Colour Title

(nm)
100-250 - Far ultraviolet
250-400 - Near ultraviolet
400-750 Violet-Red Visible
750-2000 - Near infra-red
2000-6000 - Far infra-red

In the visible region of the spectrum, illumination of particular wavelengths is

perceived as different colours. The word monochromatic, meaning of single colour,
is therefore, used to describe illumination containing wavelengths selected from a
very small part, or band, of the spectrum. A beam of monochromatic light can be
thought of as a stream of photons all of them having closely similar energies. The
energy of photon is directly proportional to frequency and inversely proportional to
wavelength (λ).

The Absorption of Light By Molecules

A beam of Light passing through a substance can be absorbed if the energy of the
photons happen to coincide with a particular energy transition available in the
absorbing substance. So if light of many wavelengths is passed through a
substance, the adsorption of that light will rise to a peak value and then fall again as
the wavelength coinciding with as energy transition is reached and passed. For
some substance several such peaks may be found and may superimpose to form a
broad peak. If the amount of absorption is measured and plotted against wavelength
the resulting curve is called an absorption spectrum and is characteristic of the
absorbing substance. The absorption at a wavelength where absorption is greatest
is termed the peak extinction or extinction maximum (or absorption peak or
maximum).

If absorption is measured at a fixed wavelength, the value obtained will depend on:
(1) the thickness of the absorbing matter through which the light passes, (2) the
concentration of absorbing matter present, and (3) the efficiency with which the
matter can absorb light.
A spectrophotometer measures the intensity of monochromatic light, which has
passed through a sample with the objective of determining how much light was
6
absorbed by the sample. In the ultraviolet and visible regions of the spectrum,
measurements are made on solutions contained in a suitable vessel (the cell,
cuvette or Spectronic tube). Since the sample is a solution in a tube, a reference
measurement must be made to compensate for absorption by solvent and tube.
Alternatively, the instrument may be zeroed using tube containing solvent.
Absorption of light at a given wavelength is related to the concentration of the
absorption species through 2 laws.

Bouguer or Lambert’ Law

The proportion of light absorbed by a transparent medium is independent of the

intensity of the incident light, and that each successive unit layer of the medium
absorbs an equal fraction of the light passing through it.

Mathematically:-

I = e-αx I = intensity of transmitted light

Io Io = intensity of incident light
x = thickness
a = the linear absorption coefficient
in log form;

In Io = αx
I

to the base 10;

Log Io = αx
I 2.303
Light is absorbed only when a photon collides with a molecule. Thus the amount of
absorbed, or the probability of absorption is proportional to the number of molecules
in the light beam. This is thus dependent on the concentration of the solution and the
length of the light path.

This is Beer’s Law, which states:

The light absorption is proportional to the number of molecules of absorbing

substance through which the light passes. So we can combine Lambert’s and Beer’s
Law to give what is commonly known as Beer’s Law or the Beer-Lambert Law.

Log Io = abc
I
Where a is the absorptivity or specific absorption coefficient of the species
(analogous to α), b is the path length and c is the concentration. When the units are
moles per litre, a is called the molar absorptivity or the molar absorption coefficient
and is denoted, ‫ ع‬or E

The absorption law is also written in terms of absorbance A i.e:

7
 A = Log Io = ‫ع‬bc
I

This absorbance is a linear function for any given wavelength if Beer’s Law is
obeyed.

Transmittance is a term which is also used and is the ratio I

Io
The reading one obtains from modern single and double beam spectrophotometers
is:
Log Io i.e Absorbance
I

REAGENTS

0.00625 mM Fast Green

0.2 M Cobalt Chloride
0.12 M HCl

Experimental Procedure

1. Set zero and infinity absorbance on the Spectronic 20 as given in instruction

on the instrument. Always put the Spectronic tube in the instrument with
perpendicular alignment mark in line with the mark on the instrument. Always
wipe the outside of the tube carefully to remove spillage and finger prints
before placing it in the instrument. A minimum of 3ml solution must be placed
in the tube.

2. Place a 0.00625 mM solution of Fast Green in the instrument. Read the

absorbance against water at 25 nm intervals over the wavelength range 400-
650 nm. Take extra absorbance reading around the absorption maximum so
as to determine accurately the wavelength of absorption maximum. Plot your
values against the wavelength to obtain the absorption spectrum of Fast
Green.

3. Dilute the 0.2 M cobalt chloride solution provided with an exactly equal
volume of 0.12 M HCl to give a 0.1 M concentration. Obtain the absorption
spectrum and maximum of CoCl2 using 0.12 M HCI as your blank.

4. Use a 5.0 ml graduated pipette to add to 4 test tubes the following volumes of
0.2M CoCL2 solution: 0.50, 1.00, 2.50, 3.50 ml. Rinse the pipette with distilled
water and add the exact volume of 0.12 M HCl to give a total of 5.0ml in each
tube. Calculate the molarity of COCl2 in each tube. Measure the %
transmission of each solution and of 0.2M COCl 2 at the peak absorption
obtained above, using 0.12M HCl as a blank. With the aid of log tables
convert your % values to absorbance values. Plot absorbance and % against
the molarity of the solution.
8
Present your results in the following manner:

Result:

Table 1: Absorbance of 0.00625 mM Fast Green and 0.1 M CoCl 2 at different

wavelengths

Absorbance
Wavelength (nM)
0.00625 mM Fast 0.1 M CoCl2
Green

Absorption peak……………..……….nm ….……nm

9
Questions and Exercises
1. Draw a schematic diagram of the instrument, labelling the important parts.
2. Which wavelength would you use to measure the concentration of K 2Cr2O7?
Of CoCl2.
3. What colour is light of the above wavelengths?
4. What is the relationship between the above colours and the colours of the
corresponding solutions?
5. What are the shapes of your plots of % T against molarity and of absorbance
against molarity for the cobalt chloride solutions? Which is the most useful
plot? Why?
6. Does the solution of cobalt chloride obey the Beer-Lambert Law?

16th February 1976

10
 EXPERIMENT 3

PROPERTIES OF PROTEINS

This practical will demonstrate some properties of protein, which will have been
talked about during lectures.

REAGENTS:

0.5% Egg albumin solution

1% Acetic acid
2% Sodium nitroprusside (freshly made by the student)
95% Ethanol
2% Lead acetate
2% Silver nitrate
4% Mercuric chloride
20% Sulphosalicylic acid

Esbach’s Reagent: 10 g Picric acid and 20 g citric acid dissolved in 1000 ml of

water.
10% Trichloracetic acid
Saturated ammonium sulphate
Solid ammonium sulphate
1% Copper sulphate
5 M sodium hydroxide
Conc. Nitric acid
1% -naphthol in alcohol (95%)
Bromine water (freshly made on the morning of the practical).
Million’s Reagent: 15% HgSO4 in 15% H2SO4
1% Sodium Nitrite

1. Denaturation by heat and extreme pH

(a) Place 5 ml of 0.5% albumin in three tubes labeled A, B and C. Add 0.5
ml 1 M HCl to A, 0.5 ml 1M NaOH to B, and 0.5 ml of water to C.
Place the tubes in a boiling water bath for 10 min. and cool to room
temperature. Adjust the acid and alkaline tubes to neutrality.
Comment on your observations.

(b) To 3 ml of egg albumin solution add 1 drop of 1% acetic acid. Mix and
boil for 10 seconds. A thick coagulum will be formed. Acetic acid is
being added to bring the reaction closer to the isoelectric point of the
denatured protein, which coagulates immediately after the solution has
been boiled. This test has an important clinical application.

2. Increased accessibility of –SH groups after denaturation

11
 Place 2 ml of 0.5% egg albumin in two test tubes A and B. Add 5 drops of 5%
NaOH to each tube. Mix, boil the contents of tube B for 10 seconds and cool
thoroughly. Add 0.5 ml freshly prepared 2% sodium nitroprusside solution to
each tube. Comment on your observation.

3 Precipitation by alcohol

Into each of two test tubes A and B place 2 ml of 95% ethanol. To each tube
add 5 drops of plasma. Note the dense precipitate. Add 10 ml of water to A
and mix. The precipitate dissolves. Allow tube B to stand for 45 minutes and
add 10 ml of water. Comment on your observation.

4 Precipitation by heavy metal cations

In each of three test tubes place 2 ml of 0.5% egg albumin solution. To one
tube add 3 drops of 2% lead acetate; to another tube 3 drops of 2% silver
nitrate and to the third tube 3 drops of 4% mercuric chloride. Note the
formation of heavy precipitates in all three tubes.

The precipitates produced by heavy metals are frequently soluble in the

presence of excess metallic salt, probably because the excess ions are
absorbed by the particles giving them a stabilizing positive charge.

5 Precipitation by anions

(a) Add a few drops of 20% sulphosalicylic acid to about 5 ml of 0.5% egg
albumin solution. Note the results.
This is a very sensitive test for albumin and is used for detecting it in
urine.

(b) Treat 3 ml of 0.5% egg albumin solution with an equal volume of

Esbach’s reagent (solution of picric and citric acids in water). Note the
result. This reaction has been used for estimating albumin in urine.

(c) To 3 ml of 0.5% egg albumin solution add 0.5 ml of 10% trichloracetic

acid. Note the result.
Trichloracetic acid is a reagent very frequently used for the quantitative
precipitate of proteins.

6 Salting out with ammonium sulphate

Perform the following experiment on plasma diluted ten times with water

(a) To 2 ml of plasma add an equal volume of saturated ammonium

sulphate. Mix and note the precipitate of globulin. Add 4 ml of water and
mix. The precipitate will dissolve.

12
 (b) To 10 ml of plasma add 10 ml of saturated ammonium sulphate. Mix
and filter the precipitated globulin. (if the filtrate is cloudy re-filter
through the same paper until a clear filtrate is obtained). The solution
is now 50% saturated with ammonium sulphate.

(c) Test part of the filtrate for protein by adding 1 drop of 1% acetic acid
and boiling for 10 seconds. A coagulum will show the presence of
albumin.

Saturate 5 ml of the filtrate with ammonium sulphate as follows: Add a

small spoonful of (NH4)2SO4 crystals and shake. Repeat until some
undissolved crystals remain at the bottom of the tube. Filter and test
the filtrate for protein. Comment on your observation.

7. Biuret test for substances containing two or more peptide bonds

To 2 ml of 0.5% egg albumin solution add 5 drops of 1% CuSO 4 solution,

followed by 4 ml of 5 M NaOH. Mix and note the colour produced.

REACTIONS OF INDIVIDUAL AMINO ACID RESIDUES PRESENT IN

PROTEINS (Use egg albumin solution for the following tests)

8. Xanthoproteic Reaction

To 2-3 ml of protein solution in a test tube add a few drops of conc. nitric
acid. Heat cautiously to boiling or until any precipitate which formed on
addition of the acid is dissolved. Cool the solution and add 6 M sodium
hydroxide in excess. The yellow colour deepens into orange.

Amino acids containing a phenyl group in the molecule (phenylalanine,

tyrosine, tryptophane) give yellow nitro-derivatives. This colour changes to
deep orange in excess alkali. This reaction is identical with the reaction
caused by a drop of conc. nitric acid on the skin.

9. Sakaguchi test for arginine

Place 3 ml of protein solution in two test tubes, A and B and add 1 ml of 5 M

NaOH to each tube. Mix and boil contents of tube A for 10 seconds. Cool
thoroughly. Add 2 drops of 1% -naphthol in alcohol and 4-5 drops of
bromine water to each tube. Comment on your observation.

10. Millon’s Reaction for tyrosine

Add 5 drops of Millon’s reagent (15% HgSO4 in 15% H2SO4) to 1 ml protein

solution. Heat in a boiling water bath for 10 minutes. Cool to room
temperature. Add 5 drops of 1% NaNO 2 solution. A positive result is
indicated by a brick red colour.

13
QUESTIONS

1. What are proteins? Give some examples.

2. Referring to proteins, what does “Denaturation” mean?

14
 EXPERIMENT 4

ENZYMOLOGY

PURPOSE: Determination of the Effect of pH On Enzymatic Activity

1. INTRODUCTION

a) Enzymes as Biological Catalysts

Living organisms are able to obtain and use energy very rapidly because of
the presence of enzymes. Enzymes increase the rate of specific biochemical
reactions and, therefore, are the catalysts of biological systems. Nearly all
known enzymes are globular proteins that have specially designed regions
that attract and bind substrate molecules. These regions, called active sites,
contain catalytic groups that are residues that directly participate in the
making and breaking of bonds.

The active site is a three-dimensional entity formed by groups that come from
different parts of the linear acid sequence. The catalytic efficiency of an
enzyme usually depends on this conformation. Consequently, changing the
optimal 3-dimensional structure of an enzyme can sometimes cause distortion
of its active site and reduce its efficiency as a biological catalyst.

b) Influence of pH on Enzymatic Activity

One of the factors that play a significant role in maintaining an optimal and
stable 3-dimensional structure of an enzyme is the overall charge of the
surface of the enzyme protein. For this reason, pH conditions can greatly
influence the activity of an enzyme. If the pH of the medium containing the
enzyme becomes either too high or too low, the activity of the enzyme is often
reduced. Any enzyme will, therefore, show catalytic function within a specific
pH range. The variation of activity with pH is due to the change in the state of
ionisation of the enzyme protein (and other components of the reaction
mixture in some cases). In mammalian cells, various biochemical and
physiological processes maintain optimal pH conditions.

Objectives
The main objective of the following experiment is to demonstrate this in vitro and to
make you appreciate the significance of pH on enzyme – catalysed reactions using
-amylase as a model enzyme. Saliva contains -amylase which hydrolyses (1-4)-
glycosidic linkages at random resulting in a mixture of glucose and free maltose.
The effect of pH on the activity of -amylase will be demonstrated.

15
 2. MATERIALS

a) Salivary amylase

Preparation: Warm 200 ml of distilled water to body temperature. Rinse the

mount twice with warmed distilled water, which you subsequently discard.
Swirl 40 – 50 ml of warmed distilled water around the mouth a few times to
collect the saliva and collect the washing in a clean beaker. Dilute to 100 ml
warmed distilled water and filter the diluted saliva through glass wool into a
conical flask.

b) 0.06 M Na2HPO4
c) 0.06 M KH2PO4
d) 1% Starch solution in 0.01 M NaCl
e) 20% NaOH
f) 5% Methylamine Hydrochloride

PROCEDURE

Table 1: Preparation of buffer solutions. Set up a series of tubes with 10 ml of

phosphate buffer as follows:

TUBE BUFFER SOLUTION pH

0.06 M Na2HPO4 (ml) 0.06M KH2PO4 (ml)
A 0.4 9.6 5.5
B 1.2 8.8 6.0
C 2.6 7.4 6.4
D 5.0 5.0 6.8
E 7.2 2.8 7.2
F 9.4 0.6 7.6
G 9.7 0.3 8.0

Table 2: Reaction mixture for the determination of enzyme activity

Prepare another set of 8 test tubes using the previously prepared
(Table 1) buffer and a 1% starch solution in 0.01 M NaCl as a
substrate.

TUBE NO. BUFFER (ml) STARCH (ml)

1 2 of A 2
2 2 of B 2
3 2 of C 2
4 2 of D 2
5 2 of E 2
6 2 of F 2
7 2 of G 2
8 2 of D 2

16
Tube 8 will be the blank of the colorimetric measurements.

Incubate tubes 1 – 8 in a water bath at 37 oC for about 5 minutes to bring the

contents of the tubes to 37oC. Add 1 ml of diluted saliva to each of tubes 1 – 7 and 1
ml of distilled water to tube 8 at 30 seconds intervals and mix well. Stop the reaction
after exactly 10 minutes with 5 drops of 20% NaOH and mix well. Remove the tubes
from the bath.

Add 0.2 ml of 5% Methylamine hydrochloride to each test tube, mix and

immediately place the tubes to a boiling water bath and heat for 2 minutes. At the
end of this time, transfer the tubes to a water-bath at room temperature and leave to
cool for about 3 minutes.

Set a spectrophotometer at wavelength 520 nm and zero it with the blank solution in
tube 8. Read the intensity of the carmine colour in each of the test tubes 1–17. The
spectrophotometer recording for each tube is proportional to the amount of maltose
formed and, therefore, is a measure of the velocity of the amylase action.

NB: Please note the optimum pH for your saliva amylase for further use in
Experiment 10

3. DATA ANALYSIS

a) Record your result in the following table.

Table 3: Effect of pH on amylase activity.

TUBE pH REACTION VELOCITY

(Colorimeter reading)
1 5.5
2 6.0
3 6.4
4 6.8
5 7.2
6 7.6
7 8.0

b) Plot reaction velocity, i.e., spectrophotometer readings (absorbance) against

pH on graph paper. Deduce the optimal pH of salivary amylase from your
graph.

17
 4. QUESTIONS

a) NaOH was used to stop the reaction. Explain concisely the effect exerted by
NaOH in stopping the reaction.
b) Explain briefly the choice of 37oC for assaying salivary amylase activity.
c) What purpose was served by the blank (i.e., control tube No. 8)? Why was it
necessary to include such a control in the above experiment?
d) Would you expect the optimal pH of pepsin (e.g., in the human gut) to be
above or below that of salivary amylase. Give reasons for your answer.
e) Which of the following conditions in mammalian tissues is most likely to affect
the activity of salivary amylase;
(i) either acidosis or alkalosis
(ii) or both
Briefly explain your reasoning.

5. REFERENCES:

(a) Plummer, D.T. (1978). Introduction to Practical Biochemistry, 2 nd edition,

page 274 – 275.
(b) Martin, D.W., (1981): Harper’s Review of Biochemistry, 18 th Edition, page 77
(c) Reed, R., Holmes, D., Weyers, J. and James, A. (1998) Practical Skills in
Bimolecular Sciences, pages 192 – 195.

18
 EXPERIMENT 5

ENZYMOLOGY

PURPOSE: To demonstrate the Effect of Enzyme Concentration on Reaction

Velocity

1. INTRODUCTION

The rate of an enzyme-catalysed reaction is dependant on a number of factors such

as substrate and enzyme concentration, pH, temperature and the presence of
activators or inhibitors. The relationship between these factors and the reaction
velocity comprises the field of enzyme kinetics.

As is true for any catalyst, in the presence of excess substrate, the rate of an
enzyme-catalysed reaction is directly proportional to the concentration of the
enzyme.

An enzyme-catalysed reaction may be written as:

K+1 K+2 K+3

E+S ES EP E+P

K1 K2 K3

Where E = free enzyme S = free substrate

P= free product ES = enzyme- substrate complex
EP = enzyme-product complex K = rate constant

The interconversion of substrate to product on the enzyme surface is usually the

slowest step (rate determining step) in the process. If one starts with [P] = 0, then
there is no backward reaction, and the rate of the overall process is given by
V = K+2 [ES]

It is clear from the above that if [S] remains constant, [ES] will increase as [E]
increases up to the point of depletion of S. Beyond that point the rate of the reaction
will no longer be proportional to the concentration of the enzyme. Fig. 1 depicts the
relation between the rate of a reaction and an increasing enzyme concentration in
the presence of an excess of the compound, which is being transformed (also called
the substrate).

19
 4x

3x
Rate of Reaction
2x
1x

Time of reaction

Fig. 1: Effect of enzyme concentration on reaction rate assuming substrate

concentration is in saturating amounts.

Objectives
The major objective of this experiment is to demonstrate the effect of
increasing enzyme concentration on the rate of reaction catalysed by
the enzyme salivary amylase.

2. MATERIALS

a) Salivary amylase
Preparation: exactly as described in experiment 9

b) 0.06 M Na2HPO4
c) 0.06 M KH2PO4
d) 1 % starch solution in 0.01 M NaCl
e) 20 % NaOH
f) 5 % Methylamine hydrochloride

PROCEDURE
Prepare a set of 6 labelled test tubes as shown I Table 1, first mixing 0.06 M
Na2HPO4 and 0.06 M KH2PO4 in appropriate proportions too give a buffer
of the pH found to be optimum for the action of the enzyme (see your results of
previous experiment)

20
Table 1: Reaction mixture for assaying salivary amylase activity

Tube No. Buffer (ml) Starch (ml) Water (ml)

1 1.0 3.0 0.8

2 1.0 3.0 0.6

3 1.0 3.0 0.4

4 1.0 3.0 0.2

5 1.0 3.0 -

6 1.0 3.0 1.0

Incubate these tubes in a water bath at 37˚C for approximately 5 minutes. Add the
following volumes (see Table 2) of diluted saliva to each of tubes 1 – 5 at 30s
intervals and mix well. Tube 6 will be the blank of the colorimetric measurements.

Table 2: Amounts of salivary amylase added to the reaction mixtures

Tube No. Amount of enzyme (ml)

1 0.2

2 0.4

3 0.6

4 0.8

5 1.0

Stop the reaction after exactly 10 minutes by adding 5 drops of 20 % NaOH and
mixing well. Add 5 drops of 20 % NaOH to the blank (tube 6). Remove the tubes
from the water bath. Add 0.2 ml 5 % methylamine hydrochloride to each tube (1 – 6)
and immediately place the tubes in a boiling water bath and heat for 2 minutes.
Transfer the tubes to a water bath at room temperature and leave to cool for about 3
minutes. Read the absorbance at 520 nm as before against the blank (tube 6).

21
3. DATA ANALYSIS
Concentration of salivary amylase in the reaction mixture is directly proportional to
the volume of the diluted saliva added to each tube, can, therefore, be taken to
represent enzyme concentration in that tube. Record your results in the following
table (Table 3).

Table 3: Effect of enzyme concentration on an enzyme catalysed reaction.

Tube No. Enzyme concentration Reaction velocity

(ml) (Colorimetric reading)
1 0.2

2 0.4

3 0.6

4 0.8

5 1.0

a) Plot reaction velocity, i.e colorimeter readings against enzyme

concentration (or amount of enzyme,ml ) on graph paper. Deduce
the slope and clearly state the units.

4. GENERAL QUESTIONS
(a) Why was it necessary to pre-incubate the reaction mixtures (tubes 1- 5)
before the addition of salivary amylase to initiate? How would the initial
reaction velocity of salivary amylase be affected if the pre-incubation stage
were omitted.
(b) What is the importance of the reaction catalysed by α-amylase?
(c) What would be the effect of incubation α-amylase with
i) glucose and protein solution?
ii) maltose and protein solution?
iii) starch and protein solution?
In each case clearly state your reasons and explain briefly what would be the effect
of increasing the enzyme concentration.

5. REFERENCES
a) Conn, E.E. and Stumpf, P.K. (1976) Outlines of Biochemistry, 4 th edition, John
Wiley & Sons Inc., Canada and USA, page 158.
b) Martin, D. W., Mayes, P.A. and Rodwell, V.W. (1981) Harper’s Review of
Biochemistry, 18th edition. Lange Medical Publications, California, USA page
73.
c) Plummer, D.T. (1978) Introduction to Practical Biochemistry. 2 nd edition,
McGraw –Hill Book Company (UK) Limited, page 248-249.

22
 EXPERIMENT 11

DETERMINATION OF SUGAR IN BLOOD

METHOD 1 (FOLIN AND WU)

INTRODUCTION
Glucose is essential as an energy source for survival of mammalian tissues. It is
normally transported to different tissues of the body. Its concentration in the blood
varies between narrow limits. Normal fasting blood sugar range:65 – 95 mg
glucose/100 ml blood. However, in certain disease conditions the level may rise
(Hyperglycemia) due to over dosage with insulin in the treatment of diabetes. Insulin
– secreting tumors of the pancreas, hypothyroidism (Addison’s disease) are all
known to produce hypoglycemia. Liver malfunction usually affects the level of
glucose in the blood. Hence, it is seen that accurate determination of blood glucose
is useful in diagnosis of body malfunction.

REAGENTS

1. Isotonic Sodium Sulphate: 30g of Na2SO4H2O (AR) in 1 litre of water. (Or

13.2g of Na2SO4 anhydous)
2. Sodium Tungstate solution: 100g of Na2WO4.2H2O, (AR) in 1 litre of water
3. 0.3 M H2SO4: 16.5 ml concentrated H2SO4, (Sp. Gr. 1.84, AR) per litre of
water.
4. Copper Sulphate solution: 12g CuSO4.5H2O, (AR) in a litre of water.
5. Harding’s Solution: Dissolve 12g of Sodium Potassium Tartrate, (AR), 20g
of Na2CO3, (AR) of NaHCO3, (AR) and 18g of Potassium Oxalate, (AR), in
1 litre of water.
6. Nelson’s Solution: Dissolve 50g of ammonium molybdate, (AR), in 900ml
of water. Slowly add 42 ml of H2SO4, (Conc., AR, Sp. Gr. 1.84), mixing
during the addition. Dissolve 6g of sodium arsenate (Mono-H)
(Na2HA3O4.7H2O)., in 50 ml of water, and add this to the solution. Mix.
Allow the solution to stand in a 370C incubator for two days then keep in a
brown bottle at room temperature.
7. Stock Standard Glucose Solution: 100mg/100ml. Dissolve 250mg of D (+)
glucose, anhydrous (AR), in 250 ml of reagent 1.
8. Glucose Working Standard: Dilute 5 ml stock standard solution in 100ml
with 0.1% benzoic acid.

METHOD

(a) PRETREATMENT OF BLOOD SAMPLES

Samples of blood for the determination of glucose will be provided. However it is
important to realize that haemolysis (breaking down of red blood cells) is to be
avoided and that the oxidation of glucose must be prevented. The usual practice
is to add potassium or sodium oxalate as an anti-coagulant plus sodium fluoride

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 to inhibit glycolysis and therefore preserve sugar. Finally, the samples should be
stored at 50C and analysed as soon as possible.

For every 10 ml of freshly collected blood, and 0.2 ml of a solution containing

10% w/v potassium oxalate and 3.3% w/v sodium fluoride. The technician may
either add this to the blood directly with gentle mixing or alternatively spread the
anti-coagulant NaF mixture as a thin film on the walls of the test tube before
adding the blood, and mixing gently. Store in the refrigerator.

(b) DEPROTEINISATION
The first step is removal of protein. For this purpose many substances can be
used, which include heavy metals, salts, acids and certain organic solvents.
Tungstic acid and trichloroacetic acid are the most often used.

In a centrifuge tube, place:-

Na2SO4 - 3.6 ml Mix well after each addition

Blood - 0.2 ml Let stand for 5 mins and centrifuge 10

Na2WO4.2H2O - 0.1 ml mins at 200 rpm.

0.3 M H2SO4 - 0.1 ML Retain the SUPERNATANT

(c) SPECTROPHOTOMETRIC ANALYSIS

BLANK STD (1) STD (2) STD (3) STD (4) SAMPLE (1)
SUPERNATANT
Isotonic Na2SO4 2.0 1.5 1.0 0.5 - 1.0
Glucose Working - 0.5 1.0 1.5 2.0 -
Standard
Supernatant - - - - - 1.0
Harding’s Solution 2.0 2.0 2.0 2.0 2.0 2.0
Cu2SO4 2.0 2.0 2.0 2.0 2.0 2.0

Place for 10 minutes in a boiling water bath and then cool to room temperature
under running tap water. To each test tube add 2 ml of Nelson’s Solution, mixing
well after every addition. Dilute the contents to 25 ml with distilled water and allow
the test tube to stand at room temperature for 6 minutes, then record the
absorbance readings of the solutions at 580 nm.

STANDARD GLUCOSE CURVE

In colorimetric analysis, it is usually wise to prepare a standard curve, which is
usually linear, or may sometimes only be linear between certain limits of
concentration of the substance being analysed.

Note:

(1) Determine the glucose levels in the samples provided.

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 (2) Write the chemical equation of all the reactions of the reactions
involved in this method of sugar estimation.
The flow diagram to help in understanding the calculation of blood glucose is as
follows:-

BLOOD SAMPLE GLUCOSE STOCK STANDARD

(100 mg/100ml or 1mg/1ml)

0.2 ml blood 5 ml STOCK STANDARD SOLUTION

Plus precipitating agents

0.4 ml total volume

100 ml Working Standard –
Contains 0.05 mg glucose/ml

SUPERNATANT

1.0 ml of analysis

Base on the volumes of solutions used in standard 2 and sample 1 above:-

(GLUCOSE) in mg/100 ml Blood

= 100 x absorbance of sample

Absorbance of glucose standard

Reference:

NELSON’S modification of the SOMOGYI Method: J. Biol. Chem., 153 (1944), 375
and J Biol. Chem. 195 (1952) 19.

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