UN
NESCO-NIG
GERIA TEC
CHNICAL &
VOCATIO
ONAL EDUC
CATION
REVIITALISATIION PROJE
ECT-PHASE II
NATIIONAL
L DIPLO
OMA IN
I
SCIENC
S
CE LAB
BORATORY TECH
HNOLO
OGY
BIO
OCH
HEMISTRY
Y
CO
OURSE CODE:
C
STC2222
YE
EAR II- SE MES
STER III
ACTICA
AL
PRA
V
Version
1:: Decembeer 2008
1
TABLE OF CONTENTS
Week 1.
Molecular organization of the living cells
Experiment 1. Differential centifiguration……………………..4
Week 2.
Concepts of pH and buffers
Experiment 2. pH measurement……………………..…………7
Week 3.
Properties of carbohydrates
Experiment 3. General test for carbohydrates…………………9
Week 4.
Optical activities of monosaccharides
Experiment 4. Measurement of optical activity
of sugars using polarimeter. ……………………………………11
Week 5.
Reducing and non-reducing properties of sugars
Experiment 5. Identification of reducing and
non-reducing sugars……………………..………………………12
Week 6.
Qualitative reactions of lipids
Experiment 6. Test for lipids……………………..…………….15
Week 7.
Qualitative reactions of fats/oils
Experiment 7. Test for fats/oils…………………………………18
Week 8.
Properties of proteins
Experiment 8. Qualitative (colour) reactions
for protein……………………..………………………………...21
Week 9.
Reactions of amino acids
Experiment 9. Qualitative test for amino acids………………..23
Week 10.
Chromatographic methods for amino acid separation
Experiment 10. Partition chromatography…………………….. 25
Week 11.
Nature of enzymes
Experiment 11. Study of the rate of an enzymes
catalysed reaction (as exemplified by catalase) ……………….28
2
Week 12.
Nature of enzymes
Experiment 12. Determination of isoelectric
point of a protein (as exemplified by casein) …………………30
Week13.
Nature of enzymes
Experiment 13. Kinetics of enzyme reaction
(as exemplified by salivary α-amylase) ………………………...32
Week 14.
Nature of enzymes
Experiment 14. Kinetics of enzyme reaction
(as exemplified by salivary α-amylyse) ………………………..34
Week 15.
Quantitative test for a water soluble vitamin
Experiment 15. Determination of ascorbic
acid (vitamin C) …………………………………………………35
3
WEEK 1. MOLECULAR ORGANIZATION OF THE LIVING CELLS.
EXPERIMENT 1
TITLE: DIFFERENTIAL CENTRIFUGATION
AIM:
TO
FRACTIONATE
CELLULAR
ORGANELLES
BY
DIFFERENTIAL SEDIMENTATION
Introduction
Centrifugation techniques are used to separate particles either on the basis of their densities or
sizes, using a gravitational (centrifugal) force field. One of the major use of centrifugal methods
in biochemistry is in the separation of cell organelles from tissue homogenates. The use of
centrifugal methods to separate cell organelles is also referred to as cell fractionation. Both the
density gradient centrifugation and differential centrifugation are used to separate sub-cellular
organelles.
Materials/Apparatus
Experimental animal (Albino Rat), dissecting set, scissors, weighing balance, beaker, buffer
solution, ice, 0.25cm3 sucrose solution, tissue slicer, potter homogenizer, mortar/pestle,
measuring cylinder, centrifuge, chloroform, plastic jar, cotton wool.
Procedure
1.
Sacrifice the experimental animal (Albino rats) by suffocating in a jar of chloroform.
2.
Dissect the animal and remove the tissue of interest (e.g. liver tissue).
3.
Cut the tissue into pieces of about 0.5cm3 thick using a scissors and weigh.
4.
Add about 8cm3 of cold buffer solution to each gram of tissue piece.
5.
Slice the tissue into smaller pieces and homogenize in a potter homogenizer at 1000g for
2 minutes or in a mortar using pestle
4
6.
Transfer quantitatively on to a cooled measuring cylinder and make up to the volume
with buffer solution to obtain a 10% suspension by weight.
7.
Centrifuge a sizeable aliquot (5cm3), 600 – 800g for 10 minutes, though the actual speed
may depend on the design of the centrifuge may depend on the design of the centrifuge
may depend on the design of the centrifuge.
8.
Remove the clear solution at the top of the tube (supernatant A) and re-suspend the
deposit (pellet) and re-homogenize as before for 1 minute only.
9.
Centrifuge the re-homogenized pellet again at 800g for 10 minutes. Remove supernatant
B and re-suspend the deposits in 5cm3 of buffer to obtain the “nuclear fraction”.
10.
Centrifuge both supernatant at 10,000 – 17,000g for 15 minutes and discard the
supernatant B.
11.
Retain supernatant A, pool the pellets and re-suspend in 5cm3 of buffer to obtain the
“mitochondrial fraction”.
12.
Centrifuge the supernatant A, obtained from step 11, at 60,000 – 100,000g for 1hour.
13.
Remove the supernatant A, which gives the “soluble fraction”. The pellet is suspended in
5ml of buffer to obtain the “microsomal fraction”.
5
Separation of cell components by differential centrifugation is illustrated schematically in the
figure below.
Tissue homogenate (in 0.25cm3 sucrose)
800 x g (10min)
Pellet (nuclear fraction)
Supernatant
16,000 x g (15min)
Pellet
(microsomal fraction)
supernatant 60, 000 x g (1hr)
Pellet
(microsomal fraction)
supernatant (soluble fractioncytosol)
Figure1. Fractionation of cellular organelles by differential sedimentation.
6
WEEK 2. CONCEPT OF pH AND BUFFERS.
EXPERIMENT 2
TITLE: pH MEASUREMENT
AIM: (I) TO LEARN TO CALIBRATE THE pH METER
(II) TO MEASURE/DETERMINE THE pH OF SOLUTIONS
Introduction
The term pH is used to measure the amount of hydrogen ion concentration [H+] of a solution. It
is therefore described as a measure of the acidity or alkalinity of the solution. The most
convenient and reliable method of measuring pH is by the use of a pH meter.
pH = -log 10[H+]
The pH is of special importance in Biochemistry because changes in pH will affect the
conformation of proteins and in consequence will have profound effect on many biochemical
parameters including the rates of enzymatic reactions
Materials/Apparatus
pH meter, buffer 7 solution, buffer 4 solution, buffer 9 solution, phosphate buffer solution, tris –
buffer solution, phosphate-citrate buffer, beakers, cotton wool.
Procedure
Calibration
1.
Insert the pH meter electrode in a pH 7 buffer solution.
2.
If the test sample is expected to be acidic, insert the pH meter electrode in a pH 4 buffer
solution.
7
3.
If the test sample is expected to be basic, insert the pH meter electrode in a pH 9 buffer
solution.
4.
After calibration, insert the pH meter electrode into the test solutions. Take the pH
readings
Carry out the above procedure using solutions of prepared buffers, weak acids, weak bases, and
salts.
8
WEEK 3. PROPERTIES OF CARBOHYDRATES
EXPERIMENT 3
TITLE: GENERAL TEST FOR CARBOHYDRATES
AIM: TO IDENTIFY CARBOHYDRATES
Introduction
Carbohydrates are a group of universally occurring compounds with general formula (CH2O)n.
Carbohydrates are the aldehyde or ketone derivatives of polyhydric alcohols. Carbohydrates and
their derivatives are found in all animal tissues, blood and milk. A variety of carbohydrates,
which are taken in the food are converted to simply sugars e.g. glucose which is the primary
carbohydrate utilized by body tissues.
Materials/Apparatus
Carbohydrate solutions; starch, glycogen, glucose, sucrose, inulin, filter paper (cellulose), 1%
alpha – napthol in ethanol, test tubes, anthrone reagent, test tubes, beakers, iodine solution.
Procedure
Molisch Test
Principle
This reaction is based on the formation of a purple condensation product, with alpha-naphthol, of
the furfural derivatives – yielded by carbohydrate radicals when treated with concentrated
H2SO4.
Method
1.
To 3cm3 of the carbohydrate solution, add 3 drops of 1% alpha – napthol in ethanol.
9
2.
Carefully run 3cm3 of conc. H2SO4 under the fluid. Agitate very gently to mix the fluids
at the boundary and cause slight warming.
3.
Leave for few minutes and observe for a positive test, which is indicted by a purple ring
at the interface. A green ring is disregarded.
Anthrone Reaction
Principle
This is another general test for carbohydrates. Concentrated H2SO4 hydrolyses glycosidic bonds
to give the monosaccharides, which are then dehydrated to furfural, which reacts with anthrone
to give a blue-green complex.
Method
1.
Add 10 drops of the test solution to 1.5cm3 of anthrone.
2.
Mix thoroughly and observe for dark bluish colour for a positive reaction.
Iodine Test (For Starch & Glycogen)
Principle
Iodine forms blue coloured absorption complexes with starch while glycogen gives red brown
colour.
Method
1.
To 1cm3 of sample/test solution, add 5 drops of iodine solution.
2.
Observe colour formed.
10
WEEK 4. OPTICAL ACTIVITIES OF MONOSACCHARIDES.
EXPERIMENT 4
TITLE:
MEASUREMENT
OF
OPTICAL
ACTIVITY
OF
SUGARS
USING
POLARIMETER
AIM: TO DETERMINE/MEASURE ISOMERISM IN SUGARS
Introduction
One very important characteristic of sugars is their ability to rotate rays of polarized light. The
presence of an asymmetric carbon atom in a sugar (or organic substance) confers on them the
power of turning the plane of a beam of polarized light either to the left or to the right. If a beam
of polarized light is passed through equally concentrated solution of two optical isomers, one
will rotate the plane of polarization to the right, dextrorotatory (+) and the other by an equal
amount to the left, laevorotatory (-).
Materials/Apparatus
Distilled water, solution of sugars; D – glucose, L – fructose D – galactose, Polarimeter, test
tubes, beakers.
Procedure
1.
Zero the Nicolprism of the polarimeter using distilled water
2.
Place the sample in the sample cell/tube
3.
Observe the rotation of the plane of polarized light to either the right of left
11
WEEK
5.
REDUCING
AND
NON-REDUCING
PROPERTIES
OF
CARBOHYDRATES
EXPERIMENT 5
TITLE:
REDUCING
AND
NON-REDUCING
PROPERTIES
OF
CARBOHYDRATES
AIM: TO IDENTIFY REDUCING AND NON-REDUCING SUGARS
Introduction
carbohydrates may be classified as either reducing or non-reducing sugars. The presence of free
or potentially free aldehyde or ketone groups in the sugar molecule enables them to function as
reducing agents. The reducing properties of reducing sugars are usually observed by their ability
to reduce metal ions, notably copper or silver, in alkaline solution.
Materials/Apparatus
Benedict’s reagent, Barfoed’s reagent, boiling water bath, Fehling’s solution, test tubes, solutions
of monosaccharides (glucose, fructose, galactose) and disaccharides (sucrose, maltose, lactose)
measuring cylinder, beakers.
Procedure
Benedict’s test
Principles
Carhoydrates free or potentially free aldehyde or ketone groups have reducing properties in
alkaline solutions. In addition monosaccharides act as reducing agents in weakly acid solution.
12
If a suspension of copper hydroxide in alkaline solution is heated, then black cupric oxide is
formed; Cu (OH)2 Æ CuO + H2O. But with the presence of a reducing substance, the rust brown
cuprous oxide is precipitated.
Method
1.
Add 5 drops of the test sample (carbohydrate solution) to 2cm3 of Benedict’s reagent.
2.
Place in a boiling water bath for 5 minutes.
3.
Record your observation
Barfoed’s test
Principle
Barfoed’s reagent is weakly acidic and is only reduced by monosaccharides. It can therefore be
used to differentiate between reducing monosaccharides/ disaccharides. However, prolonged
boiling may hydrolyze disaccharides to give a false positive reaction.
Method
1.
Add 2 – 5cm3 of the test solution 1.5cm3 of Barfoed’s reagent.
2.
Boil for 5minutes.
A brick-red precipitate of cupious oxide is a positive result for reducing monosaccharides.
Fehling’s test
Principle
This test is based on the ability of reducing sugars to reduce Cu2+ to Cu+ in alkaline solution.
Method
1.
Add 2cm3 of fehling’s solution (1, 3 of Fehling’s solution 1 + 1cm3 of test carbohydrate
solution.
13
2.
Boil in a boiling water bath for five minutes.
A brick-red precipitate indicates the presence of a reducing sugar.
14
WEEK 6. QUALITATIVE REACTIONS OF LIPIDS.
EXPERIMENT 6
TITLE: TEST FOR LIPIDS
AIM: TO CARRY OUT QUALITATIVE TEST FOR FATS/OILS (OR
LIPIDS)
Introduction
Lipids are group of fatty substance, which are insoluble in water but soluble in non-polar
solvents like ether and chloroform. Fats and their derivatives are collectively known as lipids,
they include fats, oils, waxes and related compounds. Lipids are classified as simple and
complex lipids. Like carbohydrates, lipid are also composed of carbon, hydrogen and oxygen,
the carbon atoms being arranged as small carbon-chains of various lengths.
Materials/Apparatus
Distilled water, ethanol, chloroform, oil samples, household detergent solution, bile salt solution,
filter paper, KHSO4, test tubes, measuring cylinder heating device
Procedure
Solubility Test
1.
Take, three dry test tubes and add to the first, 2cm3 of ethyl alcohol/ethanol, and to the
third 2cm3 of distilled water, to the second 2cm3 of ethyl alcohol/ethanol, and to the third
2cm3 of chloroform.
2.
To each of the three test tubes, add 3 drops of the oil sample
3.
Shake gently and observe.
Note in which of the three solutions is oil completely and sparingly soluble.
15
Emulsification Test (For Oils)
1.
To three clean test tubes add 5 cm3 of distilled water and add 5cm3 of bile salt solution to
the second.
2.
Add 5cm3 of household detergent solution to the third test tube. Then add 3cm3 of the oil
to each of the test tubes. Shake vigorously and observe.
3.
Record your observation and note in which of the solutions oil forms a stable and in
stable emulsion.
Grease Spot Test
1.
Place one drop of an ether solution of the sample (s) on a filter paper
2.
Leave to dry.
Note any translucent spots formed.
Glycerol (Acrolein) Test (For Triglycerides)
When glycerol is heated with potassium bisulphate, dehydration occurs and the aldehyde
acrolein is formed, which has a characteristic odour. This test is given by glycerol, which is
either free or combined as an ester.
CH2OH
CHOH
CHO
CH2OH + KHSO4
GLYCEROL
HEAT
CH
+ H2O
CH2
ACROLEIN
Method
1.
Place a layer of KHSO4 (about 0.5cm deep) in a test tube
2.
Add about 5 drops of the test solution or the equivalent amount of solid
16
3.
Cover with further KHSO4 and heat slowly.
Note the characteristic pungent odour of acrolein.
17
WEEK 7. QUALITATIVE REACTIONS OF FAT/OILS
EXPERIMENT 7
TITLE: TEST FOR LIPIDS
AIM: TO CARRY OUT QUALITATIVE TEST FOR FATS/OILS
Introduction
Lipids are compounds found in living of organisms which are insoluble in water, but soluble in
fat solvents such as chloroform, hot ethyl alcohol/ethanol, ethyl ether, petroleum ether, benzene,
carbon tetrachloride and acetone. Lipids may be divided into three major classes: simple lipids,
(e.g. fatty acids, lipids alcohols, esters of lipid alcohol e.g. cholesterol and neutral fats e.g.
triglycerides, phosphoglycerides, and sphingolipids.
Materials/Apparatus
Fat/lipid samples (e.g. butter), Dam’s iodine, alcoholic potassium hydroxide (10%), chloroform,
Pasteur pipette, beaker, test tubes, 1% phenolphthalein, potassium hydroxide (0.5 mol/dm3 in
90% ethanol), pure olive oil (of any pure oil sample), round bottom flasks, pipette, pipette filler,
burette, water bath and reflux condensers.
Procedure
Principle
The iodine number of fat is the number of grams of iodine taken up by 100g of fat. The iodine
number is an indication of the degree of unsaturation of the fat.
The basis of the reaction is:
-CH=CH-+I2 -
CH-CH18
I
I
Dam’s Iodine Test (For Degree Of Unsaturation)
1.
Add 1 drop of method fat (or lipid) to 1cm3 of chloroform.
2.
Shake
3.
Using a Pasteur pipette, add drop wise dam’s iodine solution to the mixture until a
permanent brown colour is obtained.
4.
Record the number of drops of dam’s iodine solution required to effect the colour
change in each case.
The numbers obtained will be directly proportional to the iodine values of the lipids.
Saponification Number
Principle
The saponification number of fat is the number of mg of potassium hydroxide that can be
neutralized by the fatty acid content of 1g of fat. If a fat contains (C18 and above) or a sterol, the
number will be small.
Procedure
1.
Wright out accurately between 1 and 2g of olive oil into a 100cm3 round-bottom
flask.
2.
Using the pipette filler, add exactly 25cm3 of 0.5 mol/dm3 ethanolic potassium
hydroxide to this and to a control flask.
3.
Position the condensers and lower the flasks into the water bath and boil until the
contents are clear (about 50 minutes with occasional shaking).
4.
Cool
19
5.
Titrate against 0.25 mol/dm3 sulphuric acid using phenolphthalein as indicator (end
point: discharge of pink colour).
20
WEEK 8. PROPERTIES OF PROTEINS.
EXPERIMENT 8
TITLE: QUALITATIVE (COLOUR) REACTIONS FOR PROTEINS
AIM: TO IDENTIFY FUNCTIONAL GROUPS OF PROTEINS
Introduction
Proteins are high-molecular nitrogen – containing organic compounds composed of amino acids
linked through peptide bonds. Proteins are subdivided into two groups; simple proteins and
conjugated (or compound) proteins.
Amino acids and proteins undergo a number of colour – forming reactions that can be used to
determine the presence of peptide bonds of specific amino acids. The most common qualitative
tests for proteins are biuret, millons and ninhydrin reaction.
Materials/Apparatus
Biuret reagent million’s reagent, protein solutions, amino acid solutions, test tubes , test tube
racks.
Biuret’s reaction (for peptide linkage in proteins)
Principles
When a protein is mixed with a solution of sodium hydroxide and a weak solution of copper
sulphate, a violet colour is produced. This is a test of peptide linkage and will be positive when
two or more peptide linkage present.
Procedure
1.
Mix 1cm3 of a protein solution with 1cm3 of sodium hydroxide
21
2.
Drop wise, add 0.1% copper sulphate solution with mixing
A violet – pink colour indicates the presence of peptide bonds
Millon’s reaction (for the presence of phenolic groups in proteins)
Principles
When a protein is heated with millon’s reagent, a red colour is produced. A positive test is due to
the presence of phenolic groups in the protein molecule. This test is positive for tyrosine.
Procedure
1.
Prepare millon’s reagent by dissolving 1 part of mercury in cold funning nitric acid.
2.
Dilute with twice the volume of water and decant the clear solution after several hours.
3.
Place a small amount of powdered protein on a sport plate.
4.
Add five drops of millon’s reagent.
Observe on standing red colour, which indicates the presence of phenolic amino acids
22
WEEK 9. REACTIONS OF AMINO ACIDS
EXPERIMENT 9
TITLE: QUALITATIVE TESTS FOR AMINO ACIDS
AIM: TO IDENTIFY SPECIFIC AMINO ACIDS
Introduction
Amino acids are organic compounds that contain amino and carboxyl groups and therefore
posses both acidic and basic properties. There are about 22 amino acids found in proteins and
nearly all of them are α-amino acids.
Materials/Apparatus
0.5% aqueous solution of ninhydrin, conc. HNO3, egg white solution, 40% NaOH, α-amino acid
samples, test tubes.
Ninhydrin Reaction (For α-Amino Group)
Ninhydrin, which is a powerful oxidizing agent, reacts with all α-amino acids between pH4 and
8 to give a purple coloured compound. The reaction is also given by primary amines and
ammonia but without the liberation of carbon dioxide. Proline and hydroxyproline react to give a
yellow/orange colour.
Method
1.
To 1cm3 of the sample, add 3 drops of ninhydrin solution.
2.
Boil for 1 minute
3.
Observe for any colour development
Xanthoprotaic Reaction (For Cyclic Amino Acid Aromatic Ring)
Principle
23
Amino acids, which contain an aromatic nucleus, form yellow nitro-derivatives on heating with
conc. HNO3.
Method
1.
Add 0.5cm3 conc. HNO3 to about 0.5cm3 of the sample solution
2.
Cool and observe the colour change.
3.
Add 0.5cm3 of 40% NaOH to make the solution strongly alkaline
A yellow colour in acid solution, which turns bright orange with alkali, is a positive result
24
WEEK 10. CHROMATOGRAPHIC METHOD FOR AMINO ACID
SEPARATION.
EXPERIMENT 10
TITLE: PARTITION CHROMATOGRAPHY
AIM: TO SEPARATE MIXTURE OF AMINO ACIDS USING PAPER
CHROMATOGRAPHY
Introduction
Chromatography is an effective method for separation of amino acids which is widely used in
biochemical studies. The most simple and accessible chromatographic technique is partition
paper chromatography. It is implemented through the use of a system of solvents constituting the
mobile and stationary phases whose proper choice is a decisive factor for effective separation of
amino acids.
Materials/Apparatus
Thermostat with a temperature setting of 37 – 380C; a drying cabinet with a temperature setting
of 100 – 1050C, equipped inside with a horizontal rod with clips or hooks on its for suspending
chromatography paper strips; large test tubes (2.0 – 2.5cm in diameter and 18 – 20cm long) with
tightly fitting corks; a test tube stand for the large test tubes; chromatography paper, a pencil and
a ruler; a threaded needle; a micropipette; a Petri dish or a pulverizer; straight forceps; scissors; a
pipette of 5ml capacity. Mixture of L-alanine, leucine, and glutamic acid, 0.04 mol/litre aqueous
solution of.
Procedure
Principle
The method is based on different migration rates of amino acids on the chromatography paper
depending on the amino acid partition coefficient for the stationary (phenol-admixed water) and
the mobile (water-saturated phenol) solvent phases.
1.
Take a paper strip by its any end using the forceps (care should be exercised not to touch
the paper strip with the fingers!)
2.
Perforate it with the threaded needle and tie thread to make a loop 5-6cm long.
25
3.
Draw a transverse line with the pencil on the opposite end of the paper strip at a distance
of 2cm from the strip edge
4.
Mark a circle of 3-4mm diameter at the line midpoint for applying the amino acid
mixture solution.
5.
Place the glass test tubes on the table surface horizontally side by side and put across
them a chromatograpjhy paper strip.
6.
Apply 0.2ml of amino acid mixture solution with the micropipette to the circled site, not
by whole, but portions.
7.
After each portion applied, allow the micropipetted spot to dry so as to prevent its
spreading beyond the penciled circle.
8.
Transfer 2ml of water-saturated phenol in a dry test tube with the 5ml pipette avoiding to
touch the inner wall of the test tube. (caution! Care should be exercised in handling
phenol since it may cause chemical burns to the skin. Be sure not to fill the pipette by
applying suction from the mouth! Use a rubber blowing bulb to fill the pipette)
9.
Put the test tube in the test tube stand strictly upright.
10.
Take the paper strip by the thread and carefully introduce it into the test tube so as to
submerge the bottom end of the thread outside and pressing the thread against the test
tube mouth with a tightly fitted cork.
11.
Place the test tube stand with the test tube in the thermostat (at 37 – 380C) for 1.5hours.
12.
At the end of this time, remove the test tube stand from the thermostat.
13.
Take the paper strip out of the test tube and suspend it by the thread loop in the drying
cabinet; allow it to dry at 100 – 1050C for minutes.
14.
To develop the chromatogram, catch the paper strip by the end with the forceps and wet it
in the Petri dish containing 15ml of ninhydrin solution by letting the paper strip pass
across the solution layer; replace the wetted paper strip in the drying cabinet at the same
temperature for 5 minutes.
Exercise
1.
Make a sketch of the paper chromatography device used in the experiment.
2.
Measure the distance for each amino acid using a ruler
26
3.
Calculate the respective Rf values.
27
WEEK 11. NATURE OF ENZYMES.
EXPERIMENT 11
TITLE: NATURE OF ENZYMES
AIM: TO STUDY THE RATE OF AN ENZYME-CATALYZE REACTION (AS
EXEMPLIFIED BY CATALASE)
Introduction
Enzymes are catalysts responsible for driving chemical reactions within the cell. Enzymes
shotten the time required for equilibrium to occur in a reaction. Catalase is an example of
enzyme. It catalyses the decomposition of hydrogen peroxides liberating oxygen and water.
Materials/Apparatus
Enzyme solution, substrate solution, buffer solutions, 2N H2SO4, 0.5N KMn04, 1.5%
NaBO3.4H20, stopwatch, distilled water, conical flask, burette, rat liver homogenate.
Procedure
Principle
Catalase decomposes hydrogen liberating oxygen. Its function may be to project calls by
destroying hydrogen peroxide
2H2O2 Catalase
2H2O+O2
Method
1.
Pipette (into a boiling tube) 8.0cm3 of 1.5% NaBO3 solution and 1.5cm3 of 0.1m buffer
solution (the buffer chosen must not react with any of the re-agents used for the enzyme essay).
2.
Place in a water bath at the appropiate temperature for a few minutes, to allow for
temperature equilibration
3.
Start the reaction by adding 0.5cm3 of liver homogenate and start timing (mixing
thoroughly)
28
4.
After 5 or 10 minutes, stop the reaction by adding 10.0cm3 of 2N H2SO4 and mix well.
5.
Wash the contents of the tube into a cornical flask with distilled water.
6.
Determine the amount of NaBO3 remaining by titrating with 0.05N KmnO4. The end
point is indicated by a pink colour which remains for longer than 30 seconds.
7.
Prepare a suitable blank (with reagents, but no enzymes), incubate and titrate using the
procedure as above.
8.
The difference between this and the sample with enzyme will be equivalent to the amount
of substrate used up by the enzyme.
Exercise
Calculate enzyme velocities as migomoles of substrate used per milligramme of liver
homogenate per minute.
29
WEEK 12. NATURE OF ENZYMES
EXPERIMENT 12
TITLE: DETERMINATION OF PROTEIN ISO-ELECTRIC POINT
AIM: TO PRECIPITATE PROTEIN (CASEIN) FROM SOLUTION AT ITS
ISO-ELECTRIC POINT
Introduction
Protein as an amphoteric polyelectrolyte carries both positive and negative charges, whose ratio
is defined by the number of acidic and basic amino acids in the protein molecule. The charge on
a protein molecule is a factor of protein stability in solution, since it prevents the agglomeration
of protein particles and their precipitation. Each protein is characterized by a pH value at which
the sum of positive and negative charges on the protein is equal to zero. This states of proteins is
referred to as isoelectric point. At the isoelectric point, protein solutions are unstable and are
prone to easily deposit as a precipitate, especially in the presence of dehydrating agents (ethanol,
acetone and others).
Materials/Apparatus
0.2 MA acetic acid, 0.2M sodium acetate, 96% ethanol, 0.1M casein, pipettes, test tubes, test
tube rack.
Procedure
Principle
This method is based on the ability of dissolved protein (casein) at its isoelectric point to transit
to an unstable state and to form a precipitate, which outwardly shows up as a distinct clouding of
the solution. Addition of ethanol (as dehydrating agent) accelerates protein precipitation.
1.
Make up 6 buffer solutions of different pH value ranging over 3.4-5.7 in 6 test tubes.
30
2.
Shake the content of each test tube and add 0.5cm3 of casein solution to each test tube.
3.
Shake the test tube contents again and note the solution to cloud.
4.
Add 2cm3 of ethanol to each test tube.
5.
Make visual estimation of the solution clouding degree.
Exercise
1.
Make estimation of the degree of solution clouding on a five-point scale before and after
adding ethanol: 1- no clouding, 2- weak, 3- moderate, 4- strong, 5- very strong clouding.
2.
At which pH value, do you observe very strong clouding
3.
State the iso-electric point of casein based on the pH with strongest clouding.
31
WEEK 13. NATURE OF ENZYMES
EXPERIMENT 13
TITLE: KINETICS OF ENZYME REACTION (AS EXEMPLIFIED BY
SALIVARY α-AMYLASE)
AIM: TO STUDY THE EFFECT OF TEMPERATURE ON THE
VELOCITY OF ENZYME CATALYZED REACTION
Introduction
Kinetics of enzymic reactions is a branch of enzymology which is concerned with the study of
relationships between the rates of enzyme – catalyzed reactions and the chemical nature of
reactants involved under different reaction conditions such as temperature, concentration of
components, medium pH, medium composition, effects due to modifying agents present, etc.
Materials/Apparatus
Human saliva sample, water bath, laboratory thermometer, test tubes, ice, iodinated potassium
iodide solution, stop watch, teat pipette/dropper
Procedure
Principle
This method is based on the measurement of the rate of starch hydrolysis by salivary α-amylase
as a function of temperature.
1.
Transfer 5 drops of saliva solution in to three test tubes, place one test tube in boiling
water bath for 1 or 2 minutes and then allow to cool.
2.
Add 10drops of starch solution to each of the three test tubes.
3.
Place the first test tube and another test tube in a water bath at 380C.
4.
Place the third test tube in a beaker of ice or 3 minutes.
5.
At the end of the time, add 1 drop of iodinated potassium iodide solution to each test
tube.
6.
Note the difference in colour developed (intensity) in the samples tested.
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Exercise
Record the results in a tabulated form as shown below, point out the dependence of enzymic
reaction rate on temperature
Enzyme
Substrate
Iodine test
Effect observed
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Temperature
WEEK 14. NATURE OF ENZYMES
EXPERIMENT 14
TITLE: KINETIC PROPERTIES OF ENZYMES
AIM: TO STUDY THE EFFECT OF MEDIUM pH ON ENZYME
REACTION RATE (AS EXEMPLIFIED BY SALIVARY α-AMYLASE)
Introduction
Kinetics of enzymic reactions is a branch of enzymology which is concerned with the study of
relationships between the rates of enzyme catalyzed reactions and factors affecting the rate of
enzyme reaction.
Material/Apparatus
Human saliva sample, starch, phosphate – citrate buffer solution, iodinated potassium iodide
solution, test tubes, dropper, water bath, stop watch, teat pipette.
Procedure
Principle
This method is based on a comparison of starch hydrolysis rate at different medium pH (the
hydrolysis is induced by salivary α-amylase and estimated by iodine test).
1.
Transfer 10drops of each phosphate – citrate buffer solution with pH values of 5 – 6, 6.4,
6.8, 7.2, and 8.0 into five test tubes.
2.
Add 5 drops of 10 – fold diluted saliva sample and 10 drops of starch solution to each test
tube.
3.
Place the test tubes in water bath maintained at 380C.
4.
Wait 1 or 2 minutes, then sample 1 drop from each test tube into five clean test tubes.
5.
Add 1 drop of iodinated potassium iodide solution.
Note the time required for the red colouration (stage of erythro-dextrin formation) to develop in
each sample.
Exercise
Record the results in the form of a graph by plotting the medium pH as abscissa versus the time
required for starch hydrolysis to reach the erythro-dextrin stage (minutes) as ordinate.
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WEEK 15. QUANTITATIVE TEST FOR A WATER-SOLUBLE VITAMIN
EXPERIMENT15
TITLE: DETERMINATION OF ASCORBIC ACID (VITAMIN C)
AIM: TO DETERMINE ASCORBIC ACID USING TITRATION METHOD
Introduction
Vitamins are organic compounds required by the body in trace amounts to perform specific
cellular functions. Vitamin C is an example of a water soluble vitamin. Vitamin C exists in the
body in equilibrium between the reduced form, ascorbic acid and the oxidized form
dehydroascorbic acid, with only a small fraction in the latter state. Both forms are active.
Materials/Apparatus
0.1% 2 – 6-dichlorophenol, indophenol, 5% glacial acetic acid, standard ascorbic acid
1mg/100cm3, red pepper (or any fresh vegetable), burette, beakers, retort stand, volumetric flask,
measuring cylinder
Procedure
1.
Weigh one small fresh pepper and grind with few drops of glacial acetic acid in a mortar.
2.
Transfer quantitatively with distilled water in a 50cm3 flask, and make up the volume to
the mark.
3.
Add 1cm3 of the dye in a test tube and add 1 drop of dilute acetic acid.
4.
Titrate with the extract from a burette and note the volume of extract use to decolourise
the dye.
5.
Repeat the titration using a standard ascorbic acid solution in place of pepper extract.
Exercise
1.
Calculate the amount of ascorbic acid per 100g of pepper
Ascorbic acid (mg/100g pepper) = V x C
Where V = cm3 of dye used in titration
C = mg ascorbic acid/cm3.
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