The University of the West Indies

St. Augustine

Laboratory manual
MDSC1001, MDSC1101, MDSC1102, and OPTM1062

Academic year (2019-2020)

Biochemistry Unit
Department of Preclinical Sciences
Faculty of Medical Sciences
Lab cover page

Date (dd/mm/yy) ______________________________________________

Course ______________________________________________

Student ID number ______________________________________________

Experiment number ______________________________________________

Title of experiment ______________________________________________

Marks obtained ______________________________________________

Comments:

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Biochemistry Unit, Department of Preclinical Sciences, Faculty of Medical Sciences 2019-2020
CONTENTS

PREFACE 4

INTRODUCTON 5

SAFETY AND LABORATORY RULES 8

EXPERIMENT 1 Laboratory techniques 11

EXPERIMENT 2 Reactions of amino acids and proteins 18

EXPERIMENT 3 3A. Demonstration of the specificity of glucose oxidase

and determination of blood glucose 23

3B. Classification of sugars and determination

of glucose in urine 29

EXPERIMENT 4 Determination of cholesterol 32

REFERENCES 38

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Biochemistry Unit, Department of Preclinical Sciences, Faculty of Medical Sciences 2019-2020
PREFACE

Biochemistry is taught using laboratory sessions (Labs), lectures, and Problem Based Learning
(PBL) classes as part of an integrated basic science programme that includes physiology and
anatomy.

Lectures
There will be approximately eighty-five biochemistry lectures over the course of the first two
years. The number provided each week will vary from one to four depending on the course
module.

Laboratory sessions
The Biochemistry Unit aims to conduct two Labs per semester. Due to large numbers,
students will be divided into groups, each of which will have a Lab approximately once every
four weeks. Group listings and Lab schedules will be posted on our bulletin board and on
Myelearning at the beginning of each semester.

Lab marks
Lab marks contribute towards continuous assessment, which contributes to final marks for each
module. Biochemistry topics may be included in spotter examinations.

Changes
This Lab manual was last updated July 2019. Please email any suggestions for its improvement
to [email protected].

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Biochemistry Unit, Department of Preclinical Sciences, Faculty of Medical Sciences 2019-2020
INTRODUCTION
This booklet contains protocols for experiments, assessment exercises, and safety guidelines that
must be adhered to during Labs.

Experiments
The aims of our experiments are to:
• reinforce concepts introduced in lectures;
• introduce biochemical concepts best taught in a practical setting;
• provide experience of common biochemical techniques.

Except for those exempted, all students are required to complete the experiments presented in
this Lab book.

Exemption from Labs

Lab exemption forms are available on Myelearning. The requirements for exemption from Labs
are presented in the table below.

Category Institution Documents required

A BSc degree in biochemistry A recognised university A completed exemption form plus a
copy of your degree certificate or
transcript
A BSc degree in a biological UWI A completed exemption form plus a
subject with one or more copy of your transcript
biochemistry laboratory
component A recognised university A completed exemption form plus a
transcript providing detailed
descriptions of relevant biochemistry
courses including Labs

Students repeating MDSC1001, UWI A completed exemption form

MDSC1101, or MDSC1102

A mark of 80% will be awarded for all exempted Labs for non-repeaters. Repeaters will be
assigned marks previously attained. The advantage of an exemption is that you have more time
to devote to other areas; the disadvantage is that you do not have an opportunity to improve your
Lab mark.

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Biochemistry Unit, Department of Preclinical Sciences, Faculty of Medical Sciences 2019-2020
Lab notebook
Before the first Lab all students must possess a hardcover notebook and a permanent marker for
labeling. As you will only be doing four experiments you do not require a thick notebook.

All results and workings must be entered directly into your Lab notebook in pen (no pencil).
Recordings made on scraps of paper or loose sheets will not be accepted. Please ensure that the
page containing your results is stamped at the end of each Lab by one of our technicians or
demonstrators. It is your responsibility to get your book stamped.

Depending on numbers, students will work in pairs, individually, or in groups of three. Each student
must provide an individual Lab report that includes answers to questions in the assessment section
of each experiment.

Lab reports
For each experiment, you are required to submit a report for marking, which must consist of a
cover page (page two of this manual), a copy of your signed and stamped results, and the
answers to all questions set. Each class will be notified of deadlines for the submission of
reports. Failure to meet any of these will result in the deduction of marks. Any lab report or
part of a lab report submitted that is plagarised from another student or another source such as the
Internet will be penalised.

For information on The UWI policy on plagiarism please refer to:

http://sta.uwi.edu/resources/documents/Exam_Regulations_Plagiarism.pdf.

Preparation
Students are required to prepare thoroughly for labs by studying the objectives, protocol, and
rationale of each experiment. As part of your preparation, you are required to construct and bring
to class a flow chart and tables to record your results. Your flow chart should be a diagrammatic
representation of the sequence of operations involved in conducting the experiment. If you are
unsure, you are advised to find out. Marks will be deducted from lab reports submitted without
flow charts.
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Biochemistry Unit, Department of Preclinical Sciences, Faculty of Medical Sciences 2019-2020
Code of behaviour
To avoid mishaps or damage, you are expected to be dressed and behave in a manner appropriate to
a Lab environment.

Students must not:

1. Enter the lab in the absence of a lecturer, technician or demonstrator.
2. Attempt to use a piece of equipment unless they are familiar with how it works.
3. Attempt to use any equipment that is not directly related to the experiment being conducted.
4. Play in the lab.
5. Eat, drink, or smoke in the lab.

Students must:
1. Report all accidents immediately to a laboratory supervisor.
2. Clear up all minor spills immediately. Ask the lecturer, a technician or a demonstrator about
the proper cleanup procedure if you are unsure.
3. Report all broken or faulty equipment (including glassware) to a laboratory supervisor.
4. Dispose of broken glassware, syringes, and needles into specified bins.
5. Wear long sleeved lab coats in the lab.
6. Wear safety glasses while working in the laboratory (not provided).
7. Wear shoes that cover your entire foot.

Students without a lab coat or proper footwear will not be allowed in Labs.

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Biochemistry Unit, Department of Preclinical Sciences, Faculty of Medical Sciences 2019-2020
SAFETY AND LABORATORY RULES

A lab can be a dangerous place. In order to prepare yourself for a safe year in the laboratory,
please read over and adhere to the safety rules listed below.

A. Dress code
1. Laboratory coats must be worn in the laboratory at all times.
2. Tie back long hair in order to keep it away from any chemicals, burners, and candles, or other
laboratory equipment.
3. Any article of clothing or jewelry that can hang down and touch chemicals and flames should
be removed or tied back before working in the laboratory. Sleeves should be rolled up.
4. Neither sandals nor opened-toe shoes are permitted to be worn in the laboratory.
5. Some experiments may require that you wear safety goggles, which are not provided.

B. General safety rules

1. Read and understand procedures for experiments. Follow directions as they are written. If
you are in doubt about any part of an experiment, ask the lecturer, a technician, or a
demonstrator for assistance.
2. Never perform activities that are not authorised by the lecturer, technician or demonstrator.
3. Never handle any equipment unless you have specific permission to do so.
4. Take extreme care not to spill any material in the laboratory. If spills occur, ask the lecturer,
technician or demonstrator immediately about the proper clean-up procedure. Never simply
pour chemicals or other substances into the sink or trash container.
5. Never eat or drink in the laboratory.
6. Wash your hands before and after each experiment.
7. There should be no loud talking or horseplay in the laboratory.
8. When performing a Lab, make sure the work area has been cleared of purses, books, jackets,
etc.
9. Know the location and use of all safety equipment (goggles, aprons, eyewash, fire blanket,
fire extinguishers, etc.)
10. Read your assignment before coming to class and be aware of all safety precautions.
11. Never work alone or unsupervised in the lab.

C. Heating and fire safety

1. Never use a heat source such as a candle or burner without wearing safety goggles.
2. Never heat any chemical that you are not instructed to heat. A chemical that is harmless
when cool may be dangerous when heated.
3. Always maintain a clean work area, and keep all materials away from flames.
4. Never leave a flame unattended.
5. Never reach across a flame.
6. Make sure you know how to light a Bunsen burner. (Lab staff will demonstrate the proper
procedure for lighting a burner.) If the flame leaps out of a burner towards you, turn the gas
off immediately. Do not touch the burner as it may be hot.
7. Always point a test tube that is being heated away from yourself and others. Chemicals can
splash or boil out of a heated test tube.

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Biochemistry Unit, Department of Preclinical Sciences, Faculty of Medical Sciences 2019-2020
8. Never heat a liquid in a closed container. The expanding gases produced may blow the
container apart, injuring you or others.
9. Never pick up any container that has been heated without first holding the back of your hand
near it. If you can feel the heat on the back of your hand, the container may be too hot to
handle. Always use a clamp or tongs when handling hot containers.

D. Using chemicals safely

1. Never mix chemicals for the "fun of it." You might produce a dangerous, possibly explosive
substance. No unauthorised experiments should be performed.
2. Never touch, taste, or smell any chemical (unless instructed by Lab staff). Many chemicals
are poisonous. If you are instructed to note the fumes in an experiment, always gently wave
your hand over the opening of a container and direct the fumes toward your nose. Do not
inhale the fumes directly from the container.
3. Use only those chemicals needed for the activity. Keep all lids closed when a chemical is not
being used. Notify Lab staff of any chemical spillage.
4. Dispose of all chemicals as instructed by Lab staff.
5. Be extra careful when working with acids or bases. Pour such chemicals over the sink, not
over your workbench.
6. When diluting an acid, always pour the acid into water. Never pour water into the acid.
7. Rinse any acids off your skin or clothing with water. Immediately notify Lab staff of any
acid spill.
8. Never pipette by mouth.
9. Be sure you use the correct chemical. Read the label twice.
10. Do not return any excess back to the reagent bottle.
11. Do not contaminate the chemical supply.
12. Keep combustible materials such as alcohol, carbon disulfide, and acetone away from open
flames.
13. Do NOT use the same spatula to remove chemicals from two different containers.
14. When you remove the stopper from a bottle, do not lay it down on the desk. Hold the bottle
so the label is in the palm of your hand, and hold the stopper between your fingers. Be sure to
rinse and wipe off any drips that may have gotten on the outside of the bottle.
15. Be careful not to interchange stoppers from two different containers.
16. Replace all stoppers and caps on bottles and other containers as soon as you finish using
them.
17. Mercury spills must be cleaned up immediately. Alert Lab staff if there is a spill. Do not
touch mercury.

E. Using glassware safely

1. Glass tubing should never be forced into a rubber stopper. A turning motion and lubricant
will be helpful when inserting glass tubing into rubber stoppers or rubber tubing. Lab staff
will demonstrate the proper way to insert glass tubing.
2. When heating glassware, use a wire or ceramic screen to protect glassware from the flame of
a Bunsen burner.
3. Never use broken or chipped glassware. If glassware breaks, notify Lab staff, and dispose of
the glassware in the proper container.
4. Always thoroughly clean glassware before putting it away.
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Biochemistry Unit, Department of Preclinical Sciences, Faculty of Medical Sciences 2019-2020
 F. Using sharp instruments
1. Handle scalpels or razor blades with extreme care. Never cut any material towards you:
always cut away from you.
2. Notify Lab staff immediately if you are cut in the laboratory.

G. Electrical equipment rules

1. Batteries should never be intentionally shorted. Severe burns can be caused by the heat
generated in a bare copper wire placed directly across the battery terminals. If a mercury type
dry cell is shorted, an explosion can result.
2. Turn off all power when setting up circuits or repairing electrical equipment.
3. Never use metal articles such as metal rulers, metal pencils or pens, rings, metal watchbands,
or bracelets when doing electrical work.
4. When disconnecting a piece of electrical equipment, pull the plug and not the wire.
5. Use caution in handling electrical equipment that has been in use and has been disconnected.
The equipment may still be hot enough to produce a serious burn.
6. Never connect, disconnect, or operate a piece of electrical equipment with wet hands or while
standing on a wet floor.

H. End-of-experiment rules
1. When an experiment has been completed, clean up your work area and return all equipment
to its proper place.
2. Wash your hands after every experiment.
3. Make sure all candles and burners are turned off before leaving the laboratory. Check that the
gas line leading to the burner is off as well.

I. Other Safety Rules

Do not use hair spray or hair mousse during or even before coming to laboratory class. These are
highly flammable and might cause automatic ignition when in close proximity to a heat source.

(http://www.sanbenito.k12.tx.us/teachers/science_safety/Safety_And_Lab_Rules.html)

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Biochemistry Unit, Department of Preclinical Sciences, Faculty of Medical Sciences 2019-2020
EXPERIMENT 1: Laboratory techniques
The aim of this experiment is to allow students to become familiar with some basic operations that
are routinely performed in a biochemistry laboratory. Procedures include centrifugation, volumetric
measurements, dilutions, spectrophotometry, and pH measurement. The concept of buffer action is
also introduced.

1A. Preparation of mitochondria from calf liver

In biochemistry is it often necessary to isolate specific cell organelles for investigation of
particular biomolecules, enzymes, metabolic pathways etc. Such separation or fractionation of
cell components can be accomplished by differential centrifugation after the cells have been
broken, by subjecting the tissue to one of several cell-rupturing procedures.

Procedure
A 10% (w/v) liver homogenate in 0.25M sucrose, 0.001M EDTA, pH7.2 has been prepared for
you.

1. Obtain approximately 20 ml of homogenate, and add equal amounts into two plastic tubes for
centrifugation. Make sure the tubes are balanced and counter poised when placed in the
centrifuge.
2. Centrifuge at 2300 rpm (750 x g) for 10 min. Carefully remove the tubes at the end of the
spin and immediately pour off the supernatants (S1) into two new centrifuge tubes. Do not
transfer any of the loose fluffy layer on top of the pellet (P1).
3. Balance the tubes as in (1), above. You may use some sucrose medium to add weight to one
of the tubes. Centrifuge at 5400 rpm (4000 x g) for 10 min. Pour off the supernatants (S2)
into a single test tube and save. Note the appearance of the pellets (P2) i.e. crude
mitochondria.
4. Add 8 ml of cold sucrose to each of the P2 pellets and re-suspend using a glass rod. Balance
the tubes and centrifuge at 5400 rpm (4000 x g) for 10 min. Discard the supernatants (S3).
Note the appearance of the pellets (P3) containing mitochondria.

N.B. Cleaner mitochondria may be obtained by further resuspensions and centrifugations.

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Biochemistry Unit, Department of Preclinical Sciences, Faculty of Medical Sciences 2019-2020
1B. Spectrophotometry: Beer-Lambert Law
The measurement of absorbance is the final step in many quantitative determinations. The
compound p-nitrophenol in the dissociated form (i.e. in alkaline solutions), absorbs light in the
visible range of the EM spectrum with an absorption maximum centered at 405 nm (λmax).

Procedure
1. Collect approximately 10 ml of solution N, a 0.30mM p-nitrophenol solution, and prepare a
stock solution (O). To prepare O, take 8 ml of solution N and make up the volume to 40 ml
using 0.02M NaOH.
2. Using your stock solution O, prepare the dilutions X2 to X5 below using graduated cylinders
and repeat the dilutions using automatic pipettes. Prepare between 2 to 5 ml of each dilution.
a. Χ2: a one in two dilution i.e. 1 part O: 1 part 0.02M NaOH
b. Χ3: one in three dilution i.e. 1 part O: 2 parts 0.02M NaOH
c. Χ4: one in four dilution i.e. 1 part O: 3 parts 0.02M NaOH
d. Χ5: one in five dilution i.e. 1 part O: 4 parts 0.02M NaOH
3. Zero the spectrophotometer at 405 nm using 0.02 M NaOH as a blank.
4. Record the absorbances for the 2 sets of dilutions and for solution O.

1C. Buffer action

In biological systems a constant pH is essential for most cellular processes. The maintenance of a
narrow range of pH is accomplished by buffer systems, which resist changes in pH that would
otherwise occur during metabolism. A buffer solution is characterized by the pK value of the
weak acid (base) and the ratio of conjugate base to acid as shown by the Henderson-Hasselbalch
equation below.

pH = pKa + log10 ( ["#$%&'()* ,(-*]

["#$%&'()* ("/0]
)

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Biochemistry Unit, Department of Preclinical Sciences, Faculty of Medical Sciences 2019-2020
Procedure
In this section, you will test the buffering capacity of 4 vials, where vials 1 and 2 each contain 10
ml distilled water, and vials 3 and 4 each contain 10 ml of any one of solutions A, B, C, or D
(which are already measured).

1. Draw a table as shown, and use it to record your results.

Vial pH Before pH After

1
2
3
4

3. Measure the pH of vials 1 and 2. Record. Add 0.1 ml of 0.2M HCl (to vial 1), mix well, and
record the pH. Add 0.1 ml of 0.2M NaOH to vial 2, mix well, and record the pH.
4. Measure the pH of vial 3 and 4. Record. Add 0.1 ml of 0.2 M HCl (to vial 3), mix well, and
record the pH. Add 0.1 ml of 0.2M NaOH to vial 4, mix well, and record the pH.
5. Obtain results for the three other solutions from other members of the class (eg. if your
solution was A, obtain results for solutions B, C and D).
6. The compositions of the test solutions are:
A. 500 ml of 0.1M NaH2PO4 added to 500 ml of 0.1M Na2HPO4.
B. 500 ml of 0.1M HCl added to 500 ml of 0.2M Na2HPO4.
C. 50 ml of 0.1M acetic acid added to 950 ml of 0.2M sodium acetate.
D. 50 ml of 0.1M NaH2PO4 added to 50 ml of 0.1M Na2HPO4 and diluted to 1 L with H2O.

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Biochemistry Unit, Department of Preclinical Sciences, Faculty of Medical Sciences 2019-2020
Assessment (25 marks)
(No flow chart minus 2 marks)

1. What is the role of 0.25M sucrose as the medium for the fractionation process? (2 marks)

__________________________________________________________________________

__________________________________________________________________________

__________________________________________________________________________

__________________________________________________________________________

2. List the major components that are present in (a) pellet P1, and (b) supernatant S2. (2 marks)

a. _________________________________________________________________________

b. _________________________________________________________________________

3a. State the Beer-Lambert Law. (2 marks)

__________________________________________________________________________

__________________________________________________________________________

__________________________________________________________________________

__________________________________________________________________________

__________________________________________________________________________

__________________________________________________________________________

3b. Briefly explain why absorbance has no units. (1 mark)

__________________________________________________________________________

__________________________________________________________________________

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Biochemistry Unit, Department of Preclinical Sciences, Faculty of Medical Sciences 2019-2020
4. The molar extinction coefficient of p-nitrophenol at 405 nm is 18.8 x 103 Lmol-1cm-1. Use this

value to calculate the concentration of solution O. (show your calculations in the box below). (3

marks)

5. Plot a graph of absorbance vs. concentration for both sets of dilutions (use the concentrations

calculated from your dilutions, do not use the concentrations obtained by using Beer-Lambert

law). Attach the graph to your report. (4 marks)

Which dilution appears to be more accurate? Comment on the spread of values. (2 marks)

__________________________________________________________________________

__________________________________________________________________________

__________________________________________________________________________

__________________________________________________________________________

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Biochemistry Unit, Department of Preclinical Sciences, Faculty of Medical Sciences 2019-2020
6a. Tabulate the data for the pH measurements for all solutions in the space below. (2 marks)

6b. State whether each of the test solutions did or did not exhibit buffering capacity. (2 marks)

__________________________________________________________________________

__________________________________________________________________________

__________________________________________________________________________

__________________________________________________________________________

6c. Explain your observations based on the composition of the solutions. (2 marks)

__________________________________________________________________________

__________________________________________________________________________

__________________________________________________________________________

__________________________________________________________________________

__________________________________________________________________________

__________________________________________________________________________

__________________________________________________________________________

__________________________________________________________________________

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Biochemistry Unit, Department of Preclinical Sciences, Faculty of Medical Sciences 2019-2020
__________________________________________________________________________

__________________________________________________________________________

__________________________________________________________________________

__________________________________________________________________________

7. List the major buffer systems in the body of mammals and show the equations for each

system? (3 marks)

__________________________________________________________________________

__________________________________________________________________________

__________________________________________________________________________

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Biochemistry Unit, Department of Preclinical Sciences, Faculty of Medical Sciences 2019-2020
EXPERIMENT 2: Reactions of amino acids and proteins

This experiment illustrates some of the general reactions of amino acids and proteins, which are
important in the study of protein chemistry.

A. Quantitative determination of protein

The Folin-Lowry assay is very sensitive, and is one of the most commonly used methods for
determining protein concentration. The final colour develops as a result of two reactions.
First, the Biuret reaction, based on the reduction of Cu2+ which then binds to protein molecules
forming a Cu1+ peptide complex. Second, the subsequent reduction of the Folin–Ciocalteu
reagent by this complex. A blue-green colour is produced, the intensity of which is proportional
to the amount of tyrosine and tryptophan as well as cysteine, cystine, and histidine residues in the
protein molecules.

Procedure
1. To prepare a standard curve:
a. Add 0.2, 0.4, 0.6, 0.8 and 1.0 ml of the standard protein solution (20 mg/100 ml) to 5 test
tubes, so that each receives a different aliquot.
b. Make up the volumes of each of the 5 test tubes to 1.0 ml with distilled H2O.
c. Set up a blank tube with 1.0 ml of H2O.
d. Into separate test tubes pipette 1.0 ml each of the two unknown protein samples.

2. Mix (just before using) 50 ml of Reagent X (2% Na2CO3 in 0.1M NaOH) with 1.0 ml of
Reagent Y (0.5% CuSO4 in 1% sodium citrate), and add 4 ml of the prepared solution to all
tubes and mix. Leave standing at room temperature for exactly 10 min.

3. Add 0.5 ml of Folin-Ciocalteu’s reagent to all tubes, mix well, and leave standing at room
temperature for 30 min.

4. Read the absorbance of all tubes against the blank at 750 nm.

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Biochemistry Unit, Department of Preclinical Sciences, Faculty of Medical Sciences 2019-2020
B. Ninhydrin reaction
The most common means of detecting amino acids is by reaction with ninhydrin, which is a very
strong oxidizing agent that reacts with a–amino acids to produce a blue-purple compound, CO2,
H2O, and an aldehyde. Proline gives a yellow colour. Under controlled conditions, the reaction is
used for quantitative estimation of amino acids, but it is not as sensitive as newer methods that
yield fluorescent products.

Procedure
Limit of detection
1. Add 1 ml of neutral glycine solution (1g/L) to a test tube.
2. Prepare six 50% sequential dilutions of the neutral glycine solution (1g/L). A technician or
demonstrator will explain how to do this.
3. Add five drops of ninhydrin solution #1 to all tubes.
4. Place all tubes in a boiling water bath for 5 min to develop the blue-purple reaction complex.
5. Determine the approximate limit of detection of this quantitative test.

Qualitative test
1. Pipette 0.70 ml of 0.5mM glycine into a test tube. Do the same with 0.5mM tyrosine and
0.5mM proline so that you have 3 test tubes each with only 0.70 ml of solution.
2. Set up a blank test tube with 0.70 ml of distilled H2O.
3. Add 0.8 ml of ninhydrin solution #2 to all tubes and mix well.
4. Place the tubes in a boiling water bath for 15 min.
5. Cool the tubes, and add 4 ml of 50% aqueous n-propanol to each.
6. Mix and leave at room temperature for 10 min.
7. Read the absorbance of the amino solutions against the blank at 570 nm and at 440 nm for
proline (you will have to blank the instrument at 570 nm and 440 nm).

C. Protein precipitation and denaturation

A wide variety of chemicals, including certain organic acids, concentrated neutral salts, heavy
metal ions and certain organic solvents, can precipitate proteins. In many cases, the precipitation
results in irreversible denaturation of the protein.

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Biochemistry Unit, Department of Preclinical Sciences, Faculty of Medical Sciences 2019-2020
Procedure
1. With a small measuring cylinder, put approximately 3 ml of protein solution (5mg/ml) into 4
test tubes. To each add only one of the following:
• a few drops of 0.1M CuSO4;
• 2 ml of 10% trichloroacetic acid;
• 2 ml of saturated (NH4)2SO4 solution;
• 10 ml of cold ethanol.

2. Place the tubes, except the one with ethanol, in a boiling water bath for 5 min. Note the
results.

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Biochemistry Unit, Department of Preclinical Sciences, Faculty of Medical Sciences 2019-2020
Assessment (20 marks)
(No flow chart minus 2 marks)

1a. Was there any significant difference in the absorbance values for glycine and tyrosine in the
qualitative Ninhydrin test? (1 mark)
__________________________________________________________________________

1b. Explain. (3 marks)

__________________________________________________________________________
__________________________________________________________________________
__________________________________________________________________________

2. State an important use of the Ninhydrin test in a clinical or research laboratory, other than
quantification. (2 marks)
__________________________________________________________________________
__________________________________________________________________________

3. Plot a graph of your standard curve for the Folin-Lowry assay. Determine the concentration of
your two unknowns in mg/ml. Attach the graph to your report. (6 marks)
__________________________________________________________________________
__________________________________________________________________________
__________________________________________________________________________
__________________________________________________________________________

4. Define what is meant by the denaturation of proteins, and state any two denaturing agents
commonly used in the laboratory. (4 marks)
__________________________________________________________________________
__________________________________________________________________________
__________________________________________________________________________
__________________________________________________________________________
__________________________________________________________________________
__________________________________________________________________________
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Biochemistry Unit, Department of Preclinical Sciences, Faculty of Medical Sciences 2019-2020
5. Although (NH4)2SO4 precipitates protein, at lower concentrations of the salt protein solubility
is increased. Explain this phenomenon. (4 marks)
__________________________________________________________________________
__________________________________________________________________________
__________________________________________________________________________
__________________________________________________________________________
__________________________________________________________________________
__________________________________________________________________________

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Biochemistry Unit, Department of Preclinical Sciences, Faculty of Medical Sciences 2019-2020
EXPERIMENT 3A: Demonstration of the specificity of glucose oxidase, and the
determination of blood glucose

Glucose oxidase catalyses the oxidation of β-D-glucose to gluconic acid in the presence of oxygen
thus:
glucose oxidase
β-D-glucose + H2O + O2 D-gluconic acid + H2O2

In the presence of the enzyme peroxidase and a hydrogen donor (DH2), hydrogen peroxide (H2O2)
is converted to water while DH2 is converted to D as follows:

peroxdase
H2O2 + DH2 2H2O + D

Where there is a difference in colour between DH2 and D the reaction may be used to measure
glucose concentration by coupling the two reactions above. A change in the amount of a compound
that absorbs light such as a dye, can be used to determine the amount of D-glucose oxidized by
direct proportionality. The absorption spectrum of D formed from DH2 has a broad peak centered
around 450 nm. For optimal accuracy, conditions need to be such that the oxidation of glucose goes
to completion. A standard curve of absorbance plotted against concentrations of glucose is required
to convert absorbance values to concentrations.

In this experiment the concentration of glucose in blood will be determined using the coupled
reactions above. In addition, the specificity of glucose oxidase will be examined by comparing its
activity on the substrates glucose, fructose, and lactose.

Normal ranges of plasma glucose:

• fasting glucose 60-100mg%
• post-prandial glucose 90-140 mg%
• random glucose 90-150 mg%

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Biochemistry Unit, Department of Preclinical Sciences, Faculty of Medical Sciences 2019-2020
Clinical significance of blood glucose
• An excess of glucose in blood is called hyperglycemia.
• Blood glucose level is increased in uncontrolled diabetes mellitus. Increased levels may also be
caused by hyper-function of the anterior pituitary and adrenal cortex.
• In hyperthyroidism, fasting blood sugar level may be normal but there is pronounced
hyperglycemia in the fed state.
• A deficiency in blood glucose is referred to as hypoglycemia (< 40 mg%). This condition is
seen in insulin secreting tumors of the beta cells of the pancreas.
• An overdose of insulin also causes hypoglycemia.

Procedure
Enzyme cocktail:
• glucose oxidase 12.5 mg
• peroxidase 4.0 mg
• O-dianisidine dihydrochloride (1% in ethanol) 0.5 ml

1. Add 0.5 M sodium phosphate buffer pH 7.2 to make 100 ml.

Preparation of test sample (blood supernatant):

1. Pipette 0.1 ml of blood into a centrifuge tube containing 1.5 ml distilled water.
2. Add 0.2 ml Ba(OH)2 solution (4.7%), mix well, and add 0.2 ml ZnSO4 solution (5%).
3. Prepare a reagent blank by pipetting 1.6 ml water into a centrifuge tube.
4. Add 0.2 ml Ba(OH)2 solution (4.7%), mix well, and add 0.2 ml ZnSO4 solution (5%).
5. Mix thoroughly, and centrifuge for 5 min at 500g. The supernatant should be clear.

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Biochemistry Unit, Department of Preclinical Sciences, Faculty of Medical Sciences 2019-2020
Setting up test tubes
Eleven test tubes are prepared as specified in the table below, after which the enzyme cocktail is
added, after which they are incubated at 37°C for 45 min.

Reagents Test tubes (all quantities are in ml)

Tube no * 1 2 3 4 5 6 7 8 9 10 11
Tube label B S1 S2 S3 S4 S5 S6 T1 T2 L F
glucose - 0.0 0.1 0.2 0.3 0.4 0.5 - - - -
(0.3mM)
supernatant - - - - - - - 0.3 0.3 - -
lactose - - - - - - - - - 0.5 -
(0.3mM)
fructose - - - - - - - - - - 0.5
(0.3mM)
reagent 0.3 - - - - - - - - - -
blank
distilled 0.2 0.5 0.4 0.3 0.2 0.1 - 0.2 0.2 - -
water

Be sure to use 0.3mM glucose solution to prepare the standard curve NOT 0.1M glucose.

* Tube 1 is your blank B.

* Tubes 2 to 7 (S1 to S6) are used to prepare your standard curve using glucose as a substrate.
* Tubes 8 and 9 (T1 and T2) are duplicate sample tubes to be tested for glucose.
* Tube 10 (L) is used to test the specificity of glucose oxidase on lactose.
* Tube 11 (F) is used to test the specificity of glucose oxidase on fructose.

6. Set up 11 test tubes as specified in the table above.

7. Add 2.0 ml enzyme cocktail to each of the eleven tubes.
8. Mix each tube briefly using a vortex.
9. Incubate at 37°C for 45 min.
10. After using your blank B to zero the spectrophotometer, read the absorbance of the other 10 test
tubes at 450 nm.

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Biochemistry Unit, Department of Preclinical Sciences, Faculty of Medical Sciences 2019-2020
Assessment (20 marks)
(No flow chart minus 2 marks)

1. Calculate the concentration of glucose in test tubes S1 to S6, and plot a standard curve of
absorbance (y-axis) versus concentration of glucose in mmoL (x-axis). Attach the graph to your
report. (6.0 marks)

2. Use the standard graph to determine the concentration of glucose in your sample of blood in
mmol/L then express your results in mg/dL. Show your calculations in the box below. (4.0
marks)

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Biochemistry Unit, Department of Preclinical Sciences, Faculty of Medical Sciences 2019-2020
3. Name the two enzymatic methods commonly used in laboratories to determine blood glucose.
(1.0 mark)
__________________________________________________________________________
__________________________________________________________________________

4. What are the two advantages of enzymatic methods used to analyse blood glucose? (1.0 mark)
__________________________________________________________________________
__________________________________________________________________________

5. Briefly describe the purpose of sodium fluoride in blood collection tubes, where glucose
measurements are to be done. (1.0 mark)
__________________________________________________________________________
__________________________________________________________________________
__________________________________________________________________________

6. Which hormone is deficient in diabetes mellitus? (0.5 marks)

___________________________________________________________________________

7. List any three actions of the hormone through which it regulates blood glucose. (1.5 marks)
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________

8. What is glycated hemoglobin (HbA1c)? (1 mark)

___________________________________________________________________________
___________________________________________________________________________

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Biochemistry Unit, Department of Preclinical Sciences, Faculty of Medical Sciences 2019-2020
9. Briefly describe the clinical importance of HbA1c. (3 marks)
____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________

10. List 4 clinical symptoms that may occur if hypoglycemia is not treated? (1 mark)
____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________

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Biochemistry Unit, Department of Preclinical Sciences, Faculty of Medical Sciences 2019-2020
EXPERIMENT 3B. Classification of sugar and determination of glucose in urine

Simple carbohydrates may be classified as either reducing or non-reducing sugars. Reducing sugars
have a free aldehyde or ketone group or a potentially free aldehyde group as in the hemi-acetal
forms present in the cyclic molecule. These monosaccharides reduce alkaline solutions of copper
(Cu2+), with the formation of a coloured (usually brick-red) precipitate of cuprous oxide (Cu2O).
Benedict's solution contains cupric sulphate (CuSO4), sodium carbonate (Na2CO3), and sodium
citrate.

The Benedict's test is utilized in the detection and quantitation of monosaccharides, such as glucose,
in biological fluids such as urine. You may recall that patients suffering from diabetes mellitus have
abnormally high blood glucose levels, which may be detected in their urine.

Procedure
1. Carry out Benedict's test (as outlined below) on 0.1M solutions of the glucose, fructose, lactose
and sucrose solutions provided.
2. Examine the sensitivity of the test on glucose by diluting the 0.1M glucose solution to produce
solutions of concentrations 0.05M, 0.025M, 0.01M, 0.001M and 0.0001M, and carrying out
Benedict's test on each.
3. Obtain a sample of urine and carry out the Benedict's test. Do not dilute the urine sample.

The Benedict's test

1. Pipette 2.0 ml of the solution to be tested into a test tube.
2. Add 2.0 ml Benedict's reagent to test tubes.
3. Place in a boiling water bath for 5 min.
4. Let the tube cool slowly (do not place under running water). A precipitate (green, red or yellow)
is indicative of a positive reaction.

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Biochemistry Unit, Department of Preclinical Sciences, Faculty of Medical Sciences 2019-2020
Assessment (15 marks)
(No flow chart minus 2 marks)

1. Briefly explain your results. (7.0 marks)

_________________________________________________________________________
_________________________________________________________________________
_________________________________________________________________________
_________________________________________________________________________
_________________________________________________________________________
_________________________________________________________________________
_________________________________________________________________________
_________________________________________________________________________

2. Name the non-reducing sugar that does not give a positive Benedict’s test and explain why? (2.0
marks)
_________________________________________________________________________
_________________________________________________________________________
_________________________________________________________________________
_________________________________________________________________________
_________________________________________________________________________

3. What is renal glycosuria and what are its causes? (2.0 marks)
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________

4. What is the renal threshold value for glucose? (1.0 mark)

________________________________________________________________________
________________________________________________________________________
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Biochemistry Unit, Department of Preclinical Sciences, Faculty of Medical Sciences 2019-2020
5. What is the clinical significance of performing oral glucose tolerance tests? (2.0 marks)
_______________________________________________________________________
_______________________________________________________________________
_______________________________________________________________________
_______________________________________________________________________
_______________________________________________________________________

6. What is gestational diabetes? (1.0 mark)

_______________________________________________________________________
_______________________________________________________________________
_______________________________________________________________________

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Biochemistry Unit, Department of Preclinical Sciences, Faculty of Medical Sciences 2019-2020
EXPERIMENT 4: Determination of cholesterol
Lipids are a very diverse group of biomolecules utilised for a wide range of biological purposes.
They are insoluble in water, and consequently need to be transported in plasma in association
with various lipoprotein particles. Plasma lipoproteins can be separated into five major classes:
chylomicrons, very-low-density lipoproteins (VLDL), intermediate-density lipoproteins (IDL),
low-density lipoproteins (LDL), and high-density lipoproteins (HDL). Each lipoprotein has
specific roles in lipid metabolism, and contains different amounts of cholesterol, cholesterol
esters, triglycerides, phospholipids and fatty acids. Abnormal proportions of lipoproteins in
blood are usually indicative of a disease state.

In this experiment total cholesterol and triglycerides in serum, normal, and pathogenic samples
will be determined using commercially available enzymatic preparations. Concentrations of
HDL-cholesterol in different samples are predetermined and are provided.

Reagents provided
• cholesterol standard (CH standard, 200 mg/dL)
• cholesterol enzyme cocktail (CHE cocktail)
• triglycerides standard (TG standard, 200 mg/dL)
• triglycerides enzyme cocktail (TGE cocktail)
• sample serum (Sample serum1 and Sample serum2)
• sample normal (Sample N)
• sample pathogenic (Sample P)

Preparation of serum
Serum can be prepared by allowing a sample of blood to clot for about 15 min in a centrifuge
tube. The fibrin clot is then ruptured and centrifuged at 6000 rpm for 5 min. The supernatant
(serum) is then removed with a Pasteur pipette, and diluted for use.

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Biochemistry Unit, Department of Preclinical Sciences, Faculty of Medical Sciences 2019-2020
Procedure
1. Set up test tubes and carry out assays as outlined in Table 1 and Table 2 below. Sample
serum1 and Sample serum2 are duplicates. In laboratories it is routine practice to assay
samples in duplicate or triplicate (even better).

Table 1. Assay cholesterol using cholesterol enzyme cocktail (CHE cocktail).

Reagents Blank CH Sample Sample Sample Sample
standard N P serum1 serum2
water 1 ml - - - - -
sample - 1 ml 1 ml 1 ml 1 ml 1 ml
CHE cocktail 1 ml 1 ml 1 ml 1 ml 1 ml 1 ml
Vortex and incubate for 10 min at room temperature
Read absorbance at 500 nm against the blank

Table 2. Assay triglyceride using triglyceride enzyme cocktail (TGE cocktail).

Reagents Blank TG Sample Sample Sample Sample
standard N P serum1 serum2
water 1 ml - - - - -
sample - 1 ml 1 ml 1 ml 1 ml 1 ml
TGE cocktail 1 ml 1 ml 1 ml 1 ml 1 ml 1 ml
Vortex and incubate for 10 min at room temperature
Read absorbance at 500 nm against the blank

33
Biochemistry Unit, Department of Preclinical Sciences, Faculty of Medical Sciences 2019-2020
The amount (mg/dL) of total cholesterol and triglycerides in each sample can be calculated using
the change in absorbance and the concentration of analyte in the standard.

Sample calculation:
∆2 -(345*
C sample = ∆2 -)($0(60 ´ C standard = mg/dL

Where C standard refers to CH standard (200 mg/dL) or TG standard (200 mg/dL).

For calculations requiring HDL-cholesterol concentration, use the following values:

Sample N: 55 mg/dL
Sample P: 30 mg/dL
Sample serum: 47 mg/dL

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Biochemistry Unit, Department of Preclinical Sciences, Faculty of Medical Sciences 2019-2020
Assessment (20 marks)
(No flow chart minus 2 marks)

1. Tabulate your results and calculate the concentration of cholesterol and triglycerides in serum,
normal, and pathogenic samples given. Show your working in the space below. (5 marks)

35
Biochemistry Unit, Department of Preclinical Sciences, Faculty of Medical Sciences 2019-2020
2. State the Friedewald Formula and use it to estimate the concentrations of VLDL and LDL in
serum, normal, and pathogenic samples. Show your working in the space below. (3 marks)

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Biochemistry Unit, Department of Preclinical Sciences, Faculty of Medical Sciences 2019-2020
3. List the lipoproteins that constitute a lipid profile, and briefly discuss the clinical significance
of the profile. (4 marks)

4. Write the normal blood cholesterol level in a healthy adult, and list any four clinical conditions
in which you may find high blood cholesterol. (2 marks)
____________________________________________________________________________

____________________________________________________________________________

____________________________________________________________________________

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Biochemistry Unit, Department of Preclinical Sciences, Faculty of Medical Sciences 2019-2020
5. Why is the total cholesterol/HDL ratio considered an atherogenic index? (3 marks)

6. Which of your samples could be considered atherogenic? (3 marks)

___________________________________________________________________________

___________________________________________________________________________

___________________________________________________________________________

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Biochemistry Unit, Department of Preclinical Sciences, Faculty of Medical Sciences 2019-2020
REFERENCES
1. Burtis, CA and Bruns DE. 2014. Tietz Fundamentals of Clinical Chemistry and Molecular
Diagnostics 7th edition, Saunders, USA
2. Nayak, S. 2013. Manipal manual of Clinical Biochemistry, 4thedition, Jaypee Medical
publishers.
3. Gaw A, Murphy MJ, Srivastava R, Cowan RA and O’Reilly D St. J. 2013. Clinical
Biochemistry: An Illustrated Colour Text, Churchill Livingstone, New York

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Biochemistry Unit, Department of Preclinical Sciences, Faculty of Medical Sciences 2019-2020