Biochemistry

1
& Genes Prac Manual
BIOS2042 Biochemistry
School of SciencePractical Manual
2022 Autumn
UNIT CO-ORDINATOR:
Dr Roland Gamsjaeger
Email: [email protected]
Biochemistry Practical Manual 2
Contents
Important information for Autumn 2022 3
Aims of the Practical Component 4
Laboratory book and Practical Skills Quiz 5
Prac 1: Pipetting Techniques & Standard Curve 7
Prac 2: Chemistry of Carbohydrates 14
Prac 3: Lipids 18
Prac 4: Properties of Amino Acids and Proteins 21
Prac 5: Enzyme Kinetics of Alkaline Phosphatase 26

Biochemistry Practical Manual 3
Important information for Autumn 2022
There will be 5 face-to-face practicals for Biochemistry in the Autumn 2022 session.
Please note that only in very special circumstances (such as students located
overseas) will students be exempt from these practicals and allowed to undertake
the online practical modules
(modular videos that introduce practical material,
demonstrate techniques and the use of equipment, data that students will be able to
analyse and interpret similar to that collected within live practical classes).
Please note that due to Covid regulations masks are mandatory in all lab classes.
Lab coats, appropriate shoes and gloves are to be worn at all times in the practical
classes.
This practical manual is an important resource in this session. Background information,
materials and methods sections are relevant as demonstrations were carried out as per
the instructions shown. It is recommended that students
answer all possible questions,
such as
pre-lab questions and discussion questions.
Engagement with questions in the practical manual will help you understand the practical
material and how this links to lecture content, as well as learn skills used in the laboratory.
Knowledge of practical content and skills will be assessed in various ways. Firstly, there
will be an
online post-lab quiz for all pracs 1-4 that will focus on key concepts and
interpretation. Secondly, a
lab book is to be submitted following Prac 1-4 (you will be
assigned a lab)
– this is to be submitted online but should follow a hard-copy lab book
format. Finally, a
Practical Skills Quiz (which will also include specific questions
about lab 5)
is to be completed online which assess knowledge of basic techniques,
dilutions, calculations, and analysis. Details, including due dates, for all tasks can be found
in the Learning Guide.
If you have any questions or would like some support, then please reach out to staff via
email or ask a question in the online workshops.

Biochemistry Practical Manual 4
Aims of the Practical Component
The experiments have been selected with the following aims in mind.

i. To demonstrate a range of basic techniques used in the biochemical analysis of
biomolecules.
To provide training in experimental design and knowledge of procedure.
To provide training in the critical evaluation of experimental results.
To provide links between what are taught in lectures and what are happening in the real world
ii.
iii.
Iv

By the end of this unit students should be able to:
1. Understand basic biochemistry experiments and the methods involved.
2. Be familiar with the use of equipment and instruments commonly used in such
experiments, particularly pipettes, balances and spectrophotometers.
3. Carry out basic biochemical calculations correctly.
4. Construct scientifically correct tables and graphs, including by hand and via software.
5. Interpret experimental data, in a clear and logical way in a laboratory notebook.
6. Understand the components of a legally compliant lab notebook.

Biochemistry Practical Manual 5
Laboratory book and Practical Skills Quiz
Laboratory book (10%) and Practical Skills Quiz (10%) are two assessments for this unit. Keeping
proper notes at a legal standard is one of the graduate attributes for a Science graduate. Such
documents are also used to record self-reflections, interactions with group members and
interactions with the literature.
A lab book is to be submitted online. Write up will concern Practical 1-4 (you will be assigned on
practical), as detailed in the Practical manual. Students must complete the lab book individually.
The lab book will require perfunctory elements and each practical should have Aims, Methods,
Results, Discussion, and Self-Reflection sections. Details on how to prepare your lab book are
outlined below.
Criteria for Laboratory book
Each student will produce a laboratory book to be submitted online (one prac from pracs
1-4).
This will include answers to questions associated with practical classes, calculations,
presentation of results, discussion, self-reflections, and a recording of relevant articles
that have been used to support your work.
You may include a referenced diagram, photograph, or sketch to augment, but
these are not to replace your own work/ provided data.
It will contain a table of contents.
All pages are to be consecutively numbered and blank space is to be at a minimum.
The date is to be clearly shown.
For laboratory experiments it will be used to note:
experimental background, aim, procedures that are proposed
data recorded and tabulated accurately and succinctly
fully labelled diagrams and drawings
potential sources of error noted/discussed
problems that are encountered, and problem solutions
conclusions and summaries
Conclusions should be supported by the scientific literature and referenced in text (i.e. in
discussion section, not at the end of the practical).
At the end of each practical a brief self-reflections section about your progress in laboratory
work will be recorded. Try to include what you have enjoyed, what you have disliked, what
you feel confident in, and most importantly what you have had difficulties with and how you
plan to resolve the difficulty.
Plagiarism will be dealt with according to the latest Policies and Procedures as published
on the Web. The lab book is an individual task.

Biochemistry Practical Manual 6
Marking Criteria:
Lab book write up relates to one assigned practical form labs 1-4. Students are to submit online
(as a PDF or MS Word file) however, the layout should mirror that of a hard copy Lab book.
Submitted Lab books will be marked for completeness/ correctness based on the following criteria.
Lab book – Marking Criteria (10 marks in total):
– Perfunctory elements (1 mark)
Perfunctory elements to include are: descriptive contents page; page numbering; dates; no blank
spaces; experiment titles. These are either present or not and marks will be awarded on
completeness.
– Completion of specific questions associated with practicals (2 marks)
Answer all questions shown in the Practical manual for either practical 2 or 3. Marks will be
awarded on completeness.
– Aims (1 mark)
Each part of a practical should have an appropriate aim, not just an overall aim for the whole
practical. These should reflect your own belief about the purpose of the experimental procedure
undertaken.
– Methods (1 mark)
This section should accurately reflect the methods used in the practical. You should not just copy
the methods from the Practical manual.
– Results (2 marks)
Results should be recorded in an appropriate way, such as being tabulated or graphed properly.
Be mindful of layout and providing units. Note that each table or graph should have an appropriate
descriptor, that is each table requires a descriptive title and each graph requires a descriptive
figure legend. For example, Table 1: Preparation of standard dilutions of DCIP.
– Discussion and Self-reflection (3 marks)
Include a detailed discussion of your results for each practical, e.g. include your thoughts on
accuracy, precision and/or your confidence in the results presented. Where relevant, discussions
should include reference to the broader literature, e.g. “this is similar to many other enzymes, such
as enzyme X and enzyme Y, where there is a narrow pH range and where they have maximum
catalytic activity. However, it contrasts with enzyme A and enzyme B, which have very broad pH
tolerance. The latter come from bacteria, which are subject to a broad range of environmental
conditions, therefore, enzyme A and B are likely required for activity over a wider pH range.”
Include a Self-reflection section for each practical. In this section you need to first describe what
you have learned from your experience engaging with the practical material, and then consider
how you would change your behaviour in light of this new learning. You may also wish to consider
what you have enjoyed (and why) and what areas you need to improve (and why).
Note: Do not copy the work submitted by another individual. Be aware of what constitutes
plagiarism and the consequences that arise if evidence of plagiarism is detected.
Practical Skills Quiz
The Practical Skills Quiz will contain multiple choice questions and short answer questions. These
will have unequivocal answers. Please note that specific questions relating to practical 5 (kinetics)
will be included in the Practical Skills Quiz.

Biochemistry Practical Manual 7
Practical 1: Pipetting Techniques & Standard
Curve
A fundamental scientific skill is to be able to make up solutions correctly and then to be able to
determine that the solution has been made up correctly. This idea will be explored.
Pre-work
Note: Do not look up the answers. Write what you think. If you do not know then write “I do
not know” and bring it to the attention of a supervisor. If you have guessed or are unsure,
bring it to the attention of a supervisor. We need to understand where the issues are so that
we can focus our teaching in these areas.
1. If I weighed out 1 g of water, what volume of water would I have?
2. Write out in words the name for this symbol: µ.
3. If I had 100 µL of water, what percentage of 1 mL of water would this be?
4. Would the answer to Q3 change if the word water were replaced by oil in the question
above?
5. What do you think the term 1 molar (1 M) means? (Do not look this up! We want to know
what you think and be as precise as possible.)
6. If I took a 10 mL sample from 2 litres of a 100 mM solution of NaCl (sodium chloride or
common table salt), what would be the concentration of NaCl in my 10 mL sample?
7. On an electronic balance, what is the purpose of the tare button? What else might it be
called or labelled.
8. Give an example of when you would record experimental data in a table and explain why
this is more appropriate than listing or describing the results.
9. Name 2 common functions that you would use on your calculator (not the simple operator’s
addition, subtraction, division and multiplication).
10. If you saw the scientific term 560 nm, what topic do you think might being discussed?
Explain why you think this.
Part A. Micropipetting Technique
Background
It is typical in modern laboratories to make solutions in small volumes. A micropipetter is a key
scientific device that enables this to be done and therefore it is essential that a graduate has
superior micropipetting skills. This practical is an opportunity to learn how to use a micropipettor
correctly and to develop and test your skills in using a micropipettor.
A micropipette is used to precisely add accurate volumes to make solutions. Volumes from 1
µL to
1000
µL are commonly added this way, but up to 5000 µL can be used.
There are two basic types of micropipettors: (1) air displacement and (2) positive displacement. Air
displacement pipettors are most commonly used and so it is essential that you learn to use these
correctly and skilfully. The correct technique for using a micropipettor will be demonstrated.
In your laboratory book, following the demonstration of pipetting, draw a fully labelled diagram of a
micropipettor including its disposable tip.

Biochemistry Practical Manual 8
To practice and evaluate your skills using a micropipettor, carry out the following experiments.
Make sure that both people in the group participate fully in the pipetting.
Check list for pipetting:
1. Make sure the right volume is selected
2. Use a correctly fitting tip and close the lid on the pipette tip box.
3. Hold the pipettor vertically when sampling and expelling solution.
4. Place the pipettor no more than 5 mm below the surface of the liquid being sampled or
when expelling the liquid.
5. The push button should be operated smoothly and slowly to avoid introducing air and
inaccuracies into the dispensing procedure.
6. Avoid contamination where appropriate by replacing tips
Experiment 1
Pipetting Water (Do not forget to include the aim of and write out your methods for this
experiment in your laboratory book)
Choose either the 1000 µL or the 200 µL pipettor for this experiment.
Choose a volume of water to pipette. Make sure that it is above 50 µL. This could be e.g. 226 µL;
125 µL; 922 µL. The volume is entirely your choice. Make a note of this volume in your laboratory
book.
Using a balance (as many student groups as possible use 4-digit balances for this experiment)
weigh the volume of water pipetted at your chosen volume. Repeat this so that at least a total of 12
recordings are made. Make sure you record which attempts were your partner’s.
Record your observations.
Based on the mass that you would expect the volume of water being pipetted to have, evaluate
your accuracy, and also evaluate your precision. Determine if there is a difference between each
person in the group.
Experiment 2
Repeat the above experiment using oil, but just use the 200 µL pipettor.
Discussion of Procedure
Based on your experience, describe how you would change your procedures to obtain the most
efficient method for testing your pipetting. Would this be different for pipetting oil compared with
water?
Discussion
Comment on your accuracy and precision in pipetting water compared with oil and justify your
answer scientifically.
How did your accuracy and precision compare with your partner’s? Was there a likely reason for
this and how could you test it?
If you required an accurate volume of oil or other viscous substance, what technique would you
avoid and what would you recommend? Explain your reasoning.
Note: when pipetting, try to keep the following in mind:
Biochemistry Practical Manual 9
Choose the correct sized pipettor for the volume to be aliquotted. (Hint: If the volume
desired is less than 20% of the maximum volume for the pipettor, then a different pipettor
should be used.
e.g., 180 µL requires to use a 200 µL variable pipettor rather than a 1000 µ
L variable pipettor.)
Make sure the right volume is selected.
Use a correctly fitting tip.
Understand the function of the “first” and “second” stops when using the pipettor push
button.
Hold the pipettor vertically when sampling and expelling solution.
Place the pipettor no more than 5 mm below the surface of the liquid being sampled or
when expelling the liquid.
Operate the push button smoothly and slowly to avoid introducing aerosols, air and
inaccuracies into the dispensing procedure.
Please note: to avoid contamination, replace tips – use tip ejector to remove tips.
Introduction to Mixing
This sounds trivial, but it is not at all. There are many factors that will affect mixing such as the
nature of the solvent and the solute, the temperature at which the mixing is to be done and the
method of mixing. Overmixing can be critical in biochemistry because mechanical forces and
increased temperature during mixing can break down proteins leading to inaccurate results.
Overmixing can also create aerosols and hence loss of components from the reaction mixture and
release of contaminants or toxins into the air. Undermixing is also a problem because a uniform
mixture is not obtained.
Commonly used methods for mixing are shaking, stirring, inversion, ultrasonification and vortexing.
Each will be demonstrated.
Different mixing methods can be tried while carrying out the experiments dealing with preparation
of a standard curve.
Part B. Using a Standard Curve to determine the concentration of the blue dye (2,6-
dichlorophenol indophenol: DCIP)
Background
You may have heard a standard curve being called a calibration curve previously.
Prework. Do the following exercise.
Biochemistry Practical Manual 10
Here are a set of standard intensity colours: 0 (no colour) to 10. Based on these standards,
estimate the unknown colour (it is in between some of the colours shown)?
Repeat this for the following standards and unknown colour.
Comment on this exercise in terms of how easy it is to determine the colour of the unknown in
each example and what limits the determination of the colour of the unknown.
The colour of a solution is related to its concentration. The more concentrated the solution the
darker the colour. Like the example above, if we have a solution of unknown concentration
(unknown colour above) then we can estimate the concentration (colour above) by comparing it
with a series of solutions of different concentrations. This is what you will be doing in this practical.
You will be given a solution of unknown concentration and be asked to find out what concentration
it is. To do this you will have to make a range of known concentrations to compare with your
unknown.
Making the range of known concentrations is called “constructing a standard
curve”.
Therefore in this exercise you will construct a standard curve to estimate the concentration
of a sample of the blue dye in which the concentration is unknown.
To measure the colour of a solution precisely, we can use a machine called a spectrophotometer.
It works by shining light of a particular wavelength through the solution that you are interested in
and comparing the intensity of light that comes through the solution with the intensity of light before
it passed through the solution. The more concentrated the solution, the less light gets through. We
say that the light is absorbed by the solution. The spectrophotometer allows us to give a number
that represents the amount of light absorbed by the solution compared with the amount of light
before it passed through the solution. For a series of known concentrations of the solution, the
0 1 2 3 4 5 6 7 8 9 10
Unknown colour 1
0 1 4 6 7 8 10
Unknown colour 2
0 1 4 6 7 8 10
Unknown colour 3

Biochemistry Practical Manual 11
amount of absorbance for each solution can be measured and plotted against its concentration.
This series of measurements forms a standard curve which can be used to determine the
concentration of the unknown solution.
One advantage of a spectrophotometer is that besides the visible spectrum, it can use
wavelengths of light that we cannot see such as ultraviolet light. This process is called
spectrophotometry.
From a technical point of view, a spectrophotometer measures how strongly a solution absorbs
light of a particular wavelength relative to a
blank (or reference solution). The blank may not
absorb light or could be coloured and absorb light. The spectrophotometer is designed to account
for any absorbance in the blank.
Absorbance is actually a log scale.
For a blank measurement, it is assumed that the intensity of incident light = the intensity of
transmitted light (= log
10 1 = 0). Absorbance is proportional to concentration.
The relationship between the absorbance
A (which is a ratio, and therefore has no units), the
concentration (or Molarity) of a substance
c (in mol L–1 or molarity) and the length l (in cm) of the
path in the solution through which the light must pass is given by:
Beer-Lambert Law A = εcl
where ε is the molar absorptivity (also called molar extinction coefficient, units M-1). This quantity
is the absorbance of a 1 M solution with a path length of 1 cm. By making the distance that the
light has to travel through the solution to be 1cm, this eliminates the value of this term. The
absorbance value depends on the nature of the solution and on the wavelength used.
NB: this equation can be used for UV, infrared or visible wavelengths, depending upon the
application.
The Beer-Lambert Law predicts a linear relationship between concentration and absorbance, but in
reality the curve eventually asymptotes to a maximum because once all of the light is absorbed by
the solution, increasing the concentration of the solution still means that it absorbs all of the light.
The main components of a
spectrophotometer are a light source, a wavelength selector (filter or
diffraction grating), a slit and a detector (
e.g., a photoelectric cell). The solution is placed in a
special transparent container of square cross section, called a cuvette or cell. The absorbance is
read directly on a meter or digital output. The arrangement is shown diagrammatically below.


=
intensity of the transmitted light
intensity of the incident light
A log
10
light source filter or grating slit
sample
cuvette detector signal readout

Biochemistry Practical Manual 12
Equipment & Materials
Available will be a standard solution of 2,6-dicholorophenol indophenol (DCIP) at a high
concentration (the concentration will be on the bottle; this is known as your stock solution) and two
unknowns (A and B). Your task will be to construct a standard curve from this high concentration
standard and to use this standard curve to estimate the concentration of the two unknowns.
You will be told the minimum volume to make up for each standard solution of your standard curve.
Procedure – Determining the concentration of DCIP
1. Make up a range of concentrations from 0 to the highest available (The concentration of DCIP
stock solution provided is 0.1 mM). There are no rules as to what concentrations to use or
how many. However, the prelab exercise above should give some idea about what a good
range of concentrations might look like. The concentrations that you choose do not have to be
linear (e.g. 2, 4, 6, 8, 10 µM etc) but can be not evenly separated (0.5, 1, 3, 5, 7.5, 10 µM). It
does not matter from a theoretical point of view how many different concentrations that you
use or which concentrations you use because each should fit on the same standard curve.
However, from a practical point of view about 7 different concentrations are recommended
plus your blank (8 in total) To ensure that there are less errors, duplicates of the
concentrations used for the standard curve are usually done.
In setting up your experiment, it is good to construct a table that indicates the amount of water
that you add, the amount of standard solution, the final concentration of the standard solution and
then a column for the absorbance measured at 620 nm for each standard concentration. It is also
good to include a couple of rows for your unknowns in the table as well.
If you decide to use a table to guide your experiment, make sure that the table has a descriptive
label. This by convention is written above the table.
2. Remember to thoroughly mix the solution in each standard because poor mixing will lead to
large errors. Do not forget to describe in your methods how you mixed the solutions.
3. Before measuring your solutions, compare the colour of your unknown solutions with your
series of standard solutions and estimate the concentration of your unknowns. Record these
estimates.
4. The absorbance of the standard solutions and the two unknowns should be measured against
your blank solution (in this case water).
5. Fill one cuvette with the water that you used to make your dilutions – this is called the
blank
and serves to eliminate any absorption due to any dissolved substances present in the water.
The spectrophotometer is ‘zeroed’ using the blank.
6. Fill other cuvettes with your standard solutions and blanks. Measure the absorbance of each
at 620 nm and record it in your table. (Do not forget to zero the spectrophotometer on your
blank before you do this. If you were to forget, then you could take the absorbance reading of
blank away from the standard solutions measures of absorbance to get the true answer.
7. Using a graph paper or Microsoft Excel, plot Absorbance (620 nm) against the known DCIP
concentrations for the standard dye solutions. This is the DCIP
standard curve.
8. Use the DCIP standard curve to determine the concentration of two DCIP solutions of
unknown concentration
Discussion of methods
1. Describe a process for making up your standards that would give the greatest accuracy for
your standard solutions. Explain why you would do this.
2. Should you plot both sets of data for your standard curves (duplicates) or take the mean of
the values? Justify your answer.

Biochemistry Practical Manual 13
3. What do you think would happen to the absorbance if you used 420 nm for the light to be
absorbed? Would the absorbance be greater or smaller?
Discussion of Results
1. Are your standard curves straight lines?
2. If they are not straight lines, explain why they are not.
3. Which part of the curve is best to measure your unknowns in and why?
4. How did your unknown values compare with those obtained by two other groups?
5. Calculate the extinction coefficient for the blue dye. (Think about the units.)
6. Which mixing technique would you use in the future and why is it better than the other
methods?

Biochemistry Practical Manual 14
Practical 2: Chemistry of Carbohydrates:
Monosaccharides and Disaccharides
Background
From the lecture material you will recall that carbohydrates have the general formula (CH2O)n
where n is an integer of 3 or greater. If n=3 the carbohydrate is a triose. If n= 4, it is a tetrose, and
so on. One important group of monosaccharides (or carbohydrates) consisting of only one unit is
the hexoses (n=6).
Biologically important hexoses are: glucose, galactose, mannose and fructose. In aqueous
solutions, they exist predominantly in a ring form, which is in equilibrium with an open chain
aldehyde form in the case of glucose, galactose and mannose, and ketone form in the case of
fructose.
Glucose and other aldehyde sugars (aldoses) form ring structures,
cyclic hemiacetals. When
such cyclic hemiacetals form, two different structures are possible depending upon the orientation
of the OH group formed by ring closure at C1, the anomeric carbon. These two forms (α and β) are
at equilibrium with each other in solution and the interconversion of α and β anomers is called
mutarotation, which can be measured by change in rotation of polarised light.
Disaccharides consist of two sugar units linked together by ether links. This link is called an
Oglycosidic bond. Common linkages are C1 to C4 and C1 to C1. The orientation of the link at C1
often determines the properties of the disaccharide. The link may be either
α or β. Disaccharides
are hydrolysed to their constituents with mild treatment of acid.
Common disaccharides are: maltose (
αD-glucopyranosyl-(14)-D-glucopyranose) found in barley
grain; sucrose (
αD-glucopyranosyl-(12)-βD-fructofuranoside) found in fruit.
Pre-work
1. If I weighed out 1 g of water, what volume of water would I have?
2. Write out in words the name for this symbol: µ.
3. If I had 100 µL of water, what percentage of 1 mL of water would this be?
4. Would the answer to Q3 change if the word water were replaced by oil in the question
above?
5. What is the boiling point of ethanol?
6. At 20˚C is glucose a solid or a liquid?
7. In your laboratory book draw the structures of
αD-glucopyranose; β-D-galactopyranose; α
D-mannopyranose; β-D-fructofuranose; maltose and sucrose (this means draw glucose,
galactose, mannose, fructose, maltose and sucrose in their ring forms).You will notice that
these structures are all very similar. However, it is possible to conduct a series of chemical
tests to distinguish between these structures. This concept will be explored by trying to
identify an unknown sugar. Therefore, a series of chemical reactions will be carried out on
a number of known sugars and the results compared with the same tests on your unknown
sugar. In addition, one of the tests (chromatography) will also be used to explore the nature
of the sugars in a ripe banana.
8. What is the stationary phase in paper chromatography?
9. What is the mobile phase in paper chromatography?
10. For the solvent in the chromatography jar, which component(s) is (are) hydrophilic? Which
one is more hydrophobic?
11. Why can sugars be stained by silver nitrate?

Biochemistry Practical Manual 15
12. What are the monosaccharides to be tested today?
13. What are the disaccharides to be tested today?
14. Which sample contains polysaccharides?
Each group will be allocated an unknown sugar to investigate. Record the letter of the unknown
sugar e.g. unknown sugar = C
Part A. Paper Chromatography
General Background
Chromatography is a general technique where different substances are identified or separated by
the degree to which they prefer one environment or phase over another. In this case, one
environment is a stationary phase (chromatography paper), and the other phase is a liquid which is
mobile. A small amount of the sample sugar is spotted onto the paper and allowed to dry. This
means that the sample sugar is incorporated in the stationary phase. The mobile phase is then
placed at one end of the chromatography paper and allowed to absorb into the paper. It then flows
over the sample sugar towards the other end of the paper. If the sample sugar is soluble in the
mobile phase, it will move along with the mobile phase. If it is not at all soluble, then it will remain
where it was originally on the filter paper. Some sugars are more soluble in the mobile phase than
others and therefore move with the mobile phase to different degrees. This can be used to identify
an unknown sugar or the sugars in fruit by comparing their degree of movement with that of known
sugars. This is what will happen in this practical exercise, but first the sugars in the fruit must be
extracted.
Materials
1x ripe banana per bench
Purified water for dilution
10% Standard sugar solutions: glucose, galactose, fructose, sucrose, lactose and maltose
10% Unknown sugar solutions 1, 2 or 3 (choose one of these).
10 mL ethanol
Solvent – the mobile phase (isopropanol:acetic acid:water (3:1:1 v/v/v))
Stain (saturated silver nitrate in acetone)
Fixative (1g sodium hydroxide in 10 mL of water diluted with 90 mL with ethanol)
Procedure
Extraction of sugars from a ripe banana (Steps 1-4)
1. Weigh 5 g of banana.
2. Pulp the banana with a fork, and place in a tube.
3. Add 10 mL of ethanol and mix thoroughly by vortexing and manual shaking. Heat the tube
to 80
°C for 20 min.
What problem must be overcome with this step? Hint – refer to pre-work.

Biochemistry Practical Manual 16
4. Filter the suspension through a fluted filter paper into a new tube. The filtrate is your ripe
banana sugar solution. Do not dilute this.
Paper Chromatography
Dilution, spotting and development (Steps 5-9)
5. Aliquot 1 mL samples of each standard sugar solution and your unknown into separate
tubes. Dilute these using purified water to obtain a final concentration of 5%. Record your
calculations.
6. Take a sheet of Whatman No. 1 chromatography paper as supplied (15 cm x 28 cm) and
mark a line in pencil 2.5 cm from the bottom. Mark nine points on this line (with small
crosses) that are 1.5 cm apart.
7. Onto each of the crosses apply 2 µL of one of the diluted standard sugar solutions; the
diluted unknown sugar solution and the undiluted filtrate from the banana (each solution will
be on a different spot).
8. Record the locations of the sugars. Label in pencil the top part of the filter paper with your
initials or name.
9. You will be shown how to prepare and place the chromatography paper into the developing
tank. The tank is then sealed as this assists development. Allow the chromatogram to
develop for about 1.5 hours. Develop the filter paper with isopropanol:acetic acid:water
(3:1:1 v/v/v) solvent mixture.
10. After developing, measure the distance the solvent front has travelled from each original
spot (where you applied the sugar solutions). Think about the best way to display these
data.
Staining and calculation of RF (Steps 11-14)
Please note: the steps 11-13 will be carried out by lab Supervisor/demonstrator (in order to prevent any
spill of silver nitrate, which could result in indelible stains)
11. Dip the paper in the staining solution (5 mL saturated silver nitrate in 200 mL acetone). Use
fresh solution
.
12. Dry the paper in the fume hood and dip it into the solution (1g of sodium hydroxide in 10 mL
of water and then diluted with 90 mL ethanol).
Use fresh solution (~10 mL).
13. Dry the paper at 100
°C for 1 min, mark the location of the centre of the sugar spots.
14. Calculate the Retardation/Retention Factor (R
F) for each sugar spot. The RF value is the
ratio of distance travelled of the sugar compared to the distance travelled of the solvent.
Thus, the
formula for RF =
Discussion of Procedure
Write down your thoughts about the following to demonstrate that you have an understanding of
the procedure. Make sure that you do not use any reference sources to compile your answers. We
need to know how you are thinking and why.
Do you think that the ripeness of the banana might affect the outcome of the practical?
What made you come to this conclusion?

Biochemistry Practical Manual 17
Do you think that the amount of banana weighed out was important and why?
Is the mobile phase used here hydrophilic or hydrophobic? Why is it relevant to separation
of sugars?
What would happen if the samples that were applied to the chromatography paper were
placed below the level of the developing solution?
In some cases, the development front was not parallel to the initial line where the sugar
samples were applied. How would the R
F values be determined and would they be valid?
Results
Determine the most likely sugars to be present in the ripe banana and the most likely sugar
to be your unknown based on the
RF values.
Part B. Benedict’s Test for reducing/non-reducing sugars
Materials
As above
1mL Benedict’s solution (alkaline cupric citrate)
Procedure
1. Aliquot 1 mL samples of each standard sugar solution and your unknown into separate
tubes.
2. Add 2 drops of Benedict’s solution.
3. Heat tubes in a boiling (100
°C) water bath for 2-5 min.
4. Record your observations for each of your sugar solutions.
A positive test (red/yellow precipitate) indicates the presence of a reducing sugar that
reduces cupric ions in the Benedict’s solution to the cuprous state.
The test is frequently used clinically to test sugar in the urine.
Discussion of Procedure (lab notebook)
As previously described, write down your thoughts about the following to demonstrate that you
have an understanding of the procedure.
What is the underlying principle for the colour change when Benedict’s reagent is mixed
with a reducing sugar?
Results (lab notebook)
Which sugar/s is/are a non-reducing sugar?
What is the main structural feature that differentiates them from a reducing sugar? Illustrate
this with chemical drawings of specific sugars.

Biochemistry Practical Manual 18
Practical 3: Lipids
Lipids are essential in Biochemistry and are the building blocks of cell membranes. In this section
we will do a set of experiments to characterise the properties of lipids.
Pre-work:
1. What functional group is present in a triglyceride?
2. What functional group is present in a fatty acid?
3. Draw the structure of oleic acid.
4. Draw the structure of glyceryl triolein.
5. What do lipids have in common?
6. What type of solvent would be needed to remove an oil spot? Why?
7. The melting point of stearic acid is 70°C, and the melting point of oleic acid is 4°C. Explain
in detail why their melting points are so different.
8. Which oil is more unsaturated, safflower oil or olive oil? Explain.
9. Which should have a higher melting point, safflower oil or olive oil? Explain your reasoning.
10. What components are present in a phosphoglyceride?
11. What reaction takes place with unsaturated lipids and bromine solution?
12. What the is the chemical principle of saponification?
Background
Lipids are a class of biological molecules that are insoluble in water and soluble in nonpolar
solvents. There are many different categories of lipids and each category has different components
present in its structure.
Fatty acids are components of many types of lipids. Fatty acids are carboxylic acids with very long
hydrocarbon chains, usually 12-18 carbon atoms long. Even though these carboxylic acids can
hydrogen bond with water, they are insoluble because of the length of their hydrocarbon chains.
Fatty acids can be saturated or unsaturated. A saturated fatty acid contains no carbon-carbon
double bonds, so it is “saturated” with hydrogen. Unsaturated fatty acids contain one or more
cis
double bonds. (Very few naturally occurring fatty acids contain trans double bonds.) The presence
of
cis double bonds has an important effect on the melting point of the fatty acid. Cis double bonds
form rigid kinks in the fatty acid chains (remember that there is no rotation around a double bond),
and the result is that unsaturated fatty acids cannot line up very well to give a regularly arranged
crystal structure. Saturated fatty acids, on the other hand, line up in a very regular manner. The
result of this is that saturated fatty acids have high melting points and are usually solids at room
temperature. Unsaturated fatty acids, however, have low melting points and are usually liquids at
room temperature.
Waxes are lipids that are used in nature as protective coatings. Structurally, a wax molecule is an
ester of a long-chain alcohol and a long-chain fatty acid. Naturally occurring waxes are mixtures of
different molecules. There are natural waxes present on the surfaces of many fruits and leaves, in
beeswax, and on the feathers of aquatic birds.
Fats and oils both belong to a class of molecules called triacylglycerols or triglycerides. Fats
usually come from animal sources and are solids at room temperature, and oils are generally from
plant sources and are liquids at room temperature. Triglycerides are triesters of glycerol and three
fatty acid molecules. The fatty acids in the triglyceride can be the same or different. Naturally
occurring fats and oils are typically mixtures of different triglycerides. The melting point of a
particular fat or oil depends on the proportions of saturated and unsaturated fatty acid components
present. For example, butter (which is a fat) contains about 30% unsaturated fatty acids and about
70% saturated fatty acids and cholesterol. Corn oil contains about 88% unsaturated fatty acids and
about 12% saturated fatty acids. In general, the higher the degree of unsaturation, the lower the
melting point of the fat or oil.
Cholesterol is a steroid and has a very different structure from other types of lipids. It is classified
as a lipid because it is nonpolar and therefore insoluble in water.

Biochemistry Practical Manual 19
Phospholipids contain a charged phosphate and a charged amino alcohol in addition to having
long nonpolar chains. Therefore, they have a dual nature – one end of the molecule is charged and
therefore compatible with water, and the other end is nonpolar and therefore compatible with
nonpolar substances. Phospholipids are the main components of cell membranes, where they are
arranged in a lipid bilayer. The charged ends face the solvent (water), and the nonpolar ends face
each other in the interior of the membrane.
Part A: Physical Properties of Lipids and Fatty Acids
Materials
Olive oil, safflower oil, stearic acid, lecithin (L-α-Lecithin, Soybean), cholesterol, vitamin A
capsules
Dichloromethane (DCM)
Procedure
1. Label 7 clean, dry test tubes. Put a small sample of each of the following lipids in separate
test tubes: olive oil, safflower oil, stearic acid, oleic acid, lecithin, cholesterol and vitamin A.
If the lipid is a solid, use a very small amount on the tip of a spatula. If the lipid is a liquid,
use 5 drops. If the vitamin A is given as a capsule, you will need to puncture it and squeeze
out some of the liquid inside to test it.
2. Classify each of the lipids as a triglyceride, fatty acid, steroid, or phospholipid.
3. Describe the appearance and odour, if any, of each of the lipids.
4. Add 20 drops of dichloromethane (DCM) as solvent to each tube and shake each of the
tubes to mix the solutions well. Determine whether each of the lipids is soluble or insoluble
in DCM. Record your observations in the lab book. Save these solutions for the next part.
5. Label 7 clean test tubes. (The tubes do not have to be dry this time.) Put a small sample of
each of the lipids in separate test tubes, as you did in step 4.
6. Add about 2 mL of deionized water to each test tube and shake each tube to mix
thoroughly. Determine whether each of the lipids is soluble or insoluble in water. Record
your observations in your lab book.
Part B: Saponification
Materials
Olive oil
5M NaOH
Ethanol
1% CaCl2 solution
NaCl
Procedure
1. In a 250 mL beaker add about 5 mL of the olive oil.
2. Add 15 mL of ethanol and 15 mL of 5M NaOH to the beaker. (Be very careful when pouring

Biochemistry Practical Manual 20
the NaOH solution, don’t let it splatter and wear gloves and eye protection at all times.)
Add a small magnetic stir bar to the beaker and heat and stir the mixture on a magnetic
stirrer-hotplate
inside a fume hood. Cover the beaker with a watch glass.
3. Heat the mixture (with constant stirring) for approximately 30-60 minutes inside the fume
hood, until the solution no longer has two separate layers. The mixture will slowly become
smoother and opaquer; it should thicken to a pudding-like consistency (semi-solid mass).
Do not let the mixture overheat or foam over, and do not allow it to boil to dryness! If this
happens, you will need to start over.
Caution: the mixture of oil and ethanol will be very
hot and may splatter or catch fire. Have a watch glass nearby to smother any flames.
Wear goggles at all times, because NaOH can cause permanent eye damage!
4. Only if there is no semi-solid mass formed, prepare a saturated salt solution (15 g of
NaCl in 50 mL of water) and pour the content with mixing of the 250 mL beaker into a 500
mL beaker containing the saturated salt solution (‘salting out’ the soap’).
5. Put a small amount of the soup mass in a test tube containing about 5 ml of water and
shake well; a froth is obtained indicating the presence of soap.
6. In another test tube, prepare a soap solution by dissolving a small amount of the formed
soap in water. Add few drops of CaCl
2 to this solution and shake well; note the formation of
insoluble Ca soaps without any froth. This explains why soap is not effective in hard water
which contains calcium (or magnesium); for soap to be effective, it must be soluble in
water.
Discussion
Why are some lipids soluble in DCM, whereas other are not?
What is the degree of saturation for each of the used lipids?
What is the function of the ethanol for saponification?
Write down the chemical reaction of the soap with the Ca ions.
Biochemistry Practical Manual 21
Practical 4: Properties of Amino Acids and
Proteins
Amino acids and proteins are key biological molecules, and their chemistry and structure are vital
to function. In this practical we will explore the chemical and structural nature of amino acids and
proteins.
Pre-work:
1. What ways could amino acids be grouped?
2. What is the definition of pH? (Do not use the words ‘acid’ or ‘acidity’ in your answer!)
3. What is an acid? What different ways can it be defined?
4. What is a base?
5. What is a zwitterion?
6. What reaction joins amino acids together to form a protein? Can you draw the bond that
joins the amino acids?
7. What are the four levels of protein structure?
8. What types of chemical bonds or intermolecular forces hold proteins together?
9. You did paper chromatography in practical 2 – what other types of chromatography are
there and how could they be used to separate mixtures of biomolecules?
Part A. Cation exchange chromatography to separate amino acids
Firstly, let’s consider common table salt – NaCl, an ionic compound. When you put salt into water it
dissociates and there are Na
+ and Clions floating around. A similar thing can happen to organic
molecules, for instance a carboxylic acid such as acetic acid, CH
3COOH, in water can form
CH
3COOand H+, this is called ‘ionisation’ or ‘dissociation’ and the acid part (-COOH) is called an
ionisable group’. But there is a catch, unlike salt, for an organic molecule like acetic acid, the pH
plays a very important role. At low pH acetic acid will stay as CH
3COOH, but at high pH it will
dissociate. The pH at which 50% of acetic acid CH
3COOH is dissociated to CH3COOis called the
pK
a of acetic acid. The pKa of acetic acid is in the acidic region (pH<7). Bases do the same thing, a
base like an amine (-NH
2) is protonated as N+H3 at low pH, and N+H3 is deprotonated to NH2 + H+
at higher pH. It is just that, unlike acids, the pH at which this dissociation occurs is in the basic
region (pH >7). The exact pH at which an ionisable group dissociates is different for different acids
or bases – for instance acetic acid and propionic acid (CH
3CH2COOH) will dissociate at different
pHs (so have different pK
as).
Now let’s think about an amino acid. Each one has a
α-amino group and a α-carboxylic acid.
Some also have ionisable groups on their side-chain. Each individual group will have its own pK
a
which means that there can be different charges on an amino acid at different pHs. When the
individual charges are added up an amino acid can have an
overall negative or positive charge, or
it can have an overall charge of zero. When it is zero at a certain pH, this pH is called the amino
acid’s isoelectric point (pI) and is defined as the
pH at which there is no net charge (i.e net charge
is zero)
. For glutamic acid (below) this is at pH = 3.22.
Using the figure as a guide, draw (on the graph to the right) the change in charge on glutamic acid
as the pH increases.

Biochemistry Practical Manual 22
At different pHs an amino acid will have a different overall charge (+ve, 0 or –ve). This means we
can purify an amino acid using
ion exchange chromatography. In cation exchange
chromatography we use a resin that has SO3- groups (anions) on its surface which can bind to
positively charged amino acids. If we then increase the pH of the buffer surrounding the resin and
amino acid then there will be a pH = pI and the amino acid becomes overall neutral. Increasing the
pH further the amino acid will become negative, will not bind to the resin and therefore be washed
off. Because the pI is often different between amino acids then we can use cation exchange
chromatography to separate mixtures of amino acids.
In this experiment we will be separating a mixture of three amino acids – glutamic acid, histidine
and arginine.
Important information: the isoelectric point (pI) of glutamic acid is 3.22, histidine is 7.58 and
arginine is 10.76
Before you start: try to explain to your lab partner what these numbers mean and why it is
important for your experiment. If you can’t then ask a demonstrator.
Materials
Water bath (80°C)
Column packed with sulphonic acid cation exchange resin equilibrated with pH 3.75 citrate buffer
Solution containing arginine, histidine, glutamic acid (3 mg/mL each) in citrate buffer
C
COOH
COOH
CH
2
CH2
H H
3N+
pH<3.22 pH=3.22 (=pI) pH>3.22 pH>9.5
C
COOCOOH
CH
2
CH2
H H
3N+ C
COOCOOCH
2
CH2
H H
3N+ C
COOCOOCH
2
CH2
H2N H
pKa 9.7
pK
a 2.2
pK
a 4.3
H
Overall
charge +
ve
-ve
0

pH

Amino acid
SO
3
SO3
+ amino acid
amino acid
X
Cation Zwitterion Anion
Biochemistry Practical Manual 23
Citrate buffer, pH 3.75 (0.35 M NaCl in 0.1M citrate, pH 3.75)
Borate buffer, pH 8 (0.1 M borate buffer, pH 8)
0.1 N NaOH (~pH 13)
Ninhydrin reagent (0.4% (w/v) in acetone
Procedure
1. Open the tap at the base of the column and allow the citrate buffer (pH 3.75) sitting above the
resin to flow through into a container until it has just reached the top of the resin. Close the tap
do not allow the column to run dry – if it does consult your demonstrator.
2. Carefully add 1mL of the amino acid solution to the top of the column.
3. Open the column tap and collect flowthrough into a clean falcon tube. Flow column until the
amino acid solution you added is just getting into the resin (do not let the column dry). Close
the tap.
4. Add 2 mL citrate buffer to the top of the column. Open the tap and allow it to drain to the top of
the resin while collecting the eluate into a fresh 12 mL tube. Close the tap.
5. Fill the column slowly with 3mL citrate buffer, open the tap and collect 3 mL into a falcon tube.
6. Repeat two more times.
7. Switch to the 0.1 M borate buffer (pH 8.0) by adding 3 x 3mL to the top of the column and
collect each 3 mL fraction into new, numbered 12 mL tubes.
8. Repeat the elution using 0.1 M NaOH (pH 13) collecting another 3 x 3 mL fractions into new,
numbered 12 mL tubes.
9. Test each fraction from the 3 different pH buffer elutions for the presence of amino acids using
the ninhydrin test (see below) and record your results.
Discussion of procedure
Why is the resin equilibrated with citrate buffer (pH 3.75)?
Ninhydrin test
The purpose of this test is to indicate which fractions (tubes) from the column contain amino acids.
Ninhydrin is a powerful oxidising agent that carries out the oxidative deamination of the
α-amino
group of primary amino acids, liberating ammonia, carbon dioxide, the corresponding aldehyde,
and a reduced form of ninhydrin. The ammonia reacts with an additional atom of ninhydrin and the
reduced ninhydrin to yield a bluish colour. Therefore, the appearance of a colour is an indication of
an amino acid or a protein. The colour may change at high pH.
1. Add 1 mL of ninhydrin reagent (0.4% (w/v) in acetone) to each elution fraction and heat in
an 80°C water bath in the fume hood for 5-10 min.
2. Allow to stand for 5 min and record which tubes show a positive test.
Consult the supervisor, demonstrator or technical staff about cleaning the column.
Cleaning Procedure to re-use the cation exchange resin
1. Wash resin with 6 mL water
2. Wash resin with 6 mL NaCl 0.5 M

Biochemistry Practical Manual 24
3. Wash resin with 6 mL water
4. Re-equilibrate resin with 6 mL Citrate buffer.
Discussion
Indicate which of the three amino acids has eluted in the citrate buffer, which amino acid
has eluted in the borate buffer, and which amino acid has eluted with NaOH.
Describe in your own words and diagrams why the separation has occurred. Explain how
the structure of the amino acids (you may need to draw them) contributes to the
separation?
Why did you collect flowthrough in step 3?
Why did you collect three fractions for each elution? What might happen if you collected
only one fraction? Can you collect more than 3 fractions?
Are there any other separation techniques you could use to analyse a mixture of amino
acids?
Part B. Denaturation of an enzyme by heat and other factors
As you know from lectures, proteins are large molecules consisting of amino acids joined together.
The important thing about proteins is their structure because structure allows function. When the
structure is disrupted (the protein is
denatured) then function can be compromised. Proteins are
held together by different forces – from strong covalent bonds to weak hydrophobic interactions,
and these forces can be affected by different environmental conditions. In this experiment you will
treat an enzyme to different conditions and link the observed effect to a specific force holding a
protein together.
Protein we will use: the enzyme
rennin
Its normal function: rennin catalyses the splitting off of portions of the milk protein casein.
Observable result: Precipitation of a milk clot.
Materials
Water baths (80°C, 37°C)
Plastic disposable pipettes
15 mL Falcon tubes (blue cap)
pH indicator paper
¼ Rennet tablet (contains the enzyme rennin)
Milk
Urea (solid)
NaOH (1M)
HCl (1M)
Procedure
1. Make up 10 mL of rennin solution by dissolving ¼ Rennet tablet in 10 mL of water in a 15 mL
Falcon tube.
2. Divide 2 mL portions of this solution into 4 x 15 mL Falcon tubes (label tubes 1-4).

Biochemistry Practical Manual 25
3. Leave one tube as a control (no treatment).
4. Heat the second tube in a water bath (80°C) for 2 min.
5. To the third tube add solid urea until it is saturated (= no more urea can be dissolved).
6. To the fourth tube add 0.1 mL of concentrated hydrochloric acid (carefully), and incubate for 5
min on the bench. Re-adjust the pH to 7.0
drop-wise with NaOH. Use pH indicator paper to
monitor the pH of your solution after the sequential addition of a few drops.
7. To each of the 4 tubes add 5 mL of milk and incubate for 15 min at 37
°C.
8. Keep your samples – you may need them for the next experiment.
Discussion
Make a table containing the treatments and your observations after each treatment.
Explain your observations in terms of how heat, urea, and pH denaturation affect the
function of the rennin enzyme. It is not appropriate to just say that you have denatured the
enzyme. You need to specifically describe how each treatment has altered intramolecular
bonds that contribute to protein structure.
Hints: Do you know the structure of urea? To explain how urea works you might want to draw its
structure and compare to the peptide bond. How are they similar/different? You may also want to
think about what heat and acids do at the molecular level.
In step 6 why do we bring the sample back to neutral pH before we add milk in step 7?
In step 7, why do we do the incubation at 37 °C?
Biochemistry Practical Manual 26
Practical 5: Enzyme Kinetics of Alkaline
Phosphatase
Purpose: This practical demonstrates how scientists measure two important parameters that
define enzyme function. These are the maximum speed
(rate or velocity), Vmax and the Km. Vmax
tells us how fast an enzyme can convert substrate into product and Km describes how tightly a
substrate binds to an enzyme. This area of biochemistry is called
enzyme kinetics.
In addition, you will learn to construct tables, draw and use a standard curve, construct basic
graphs used in the interpretation of enzyme kinetic data, understand professional literature and
practise critical thinking. These skills are expected of all science graduates.
The background knowledge for this practical has been presented in lectures. Please review
this before coming to class.
Pre-work:
1. How would you define an ‘enzyme’?
2. When related to enzyme activity, what does the term ‘rate of reaction’ mean to you?
3. Let’s say an enzyme can convert 100 apples to juice in one hour, how many apples could it
convert in just one second?
4. Let’s say a different enzyme can convert 1200 mmoles glucose to glucose-6-phosphate in
one hour, how many mmoles glucose can it convert in one second?
5. If you knew that glucose was colourless, but glucose-6-phosphate was a bright purple how
could you tell if a glucose kinase was present in a solution?
6. What are two ways we can measure the rate of an enzyme reaction in the laboratory?
Background:
In this prac, the basic principles of enzyme kinetics will be studied using an enzyme called alkaline
phosphatase
. This enzyme will dephosphorylate (i.e. remove a phosphate) many biological
molecules (the biochemists would say it
hydrolyses organic phosphate esters with the production
of
orthophosphate). Its name is alkaline phosphatase so it is not unreasonable to predict it works
best at alkaline pHs. It is of clinical interest because it is one of the few intracellular enzymes that
is detected in the
serum. Increased levels of serum alkaline phosphatase are found in diseases of
liver and bone, and are of diagnostic value. The enzyme is also found in intestine, placenta, kidney
and leukocytes.
Alkaline phosphatase is assayed by measuring the rate of hydrolysis of a synthetic phosphate
ester (substrate), using a spectrophotometric method to detect the coloured product. A synthetic
substrate is used because most naturally occurring phosphate esters do not have suitable
absorption spectra, nor do the hydrolysis products derived from them. The synthetic substrate,
pnitrophenyl phosphate (pNPP), also has no suitable absorption spectrum, and neither does the
product derived from it,
p-nitrophenol (pNP). However, in alkaline conditions, p-nitrophenol ionises
very rapidly, and the resulting
p-nitrophenolate ion (pNPi) does absorb visible light quite intensely.
The p
Ka of p-nitrophenol is about 7.1. Thus, at pH 9.2 or higher it is virtually all in the ionised form.
Biochemistry Practical Manual 27
The dephosphorylation of pNPP and subsequent ionisation of pNP are shown below:
Hydrolysis of p-nitrophenyl phosphate (pNPP) catalysed by alkaline phosphatase and subsequent
ionisation of the product p-nitrophenol (pNP) to p-nitrophenolate (pNPi)
By this stage of semester, you will have developed some biochemical literacy.
Ask yourself what is enzyme, what is substrate? What is cofactor or coenzyme
or prosthetic group? What is initial velocity? What is Vmax and what is Km?
What is competitive inhibitor? What is a non-competitive inhibitor? If you don’t
think you have a good answer, or your Google search just makes you more
confused, or your discord pals are hopeless, then ask a lecturer/demonstrator in
a prac or in the workshops.
Experiment 1. Standard curve for p-Nitrophenol
Before you start:
What is the role of p-Nitrophenol (pNP) in the enzyme assay?
How do you measure it?
Why are we doing a standard curve for this compound (Aim)
In order to determine the rate at which pNP is produced we must be able to accurately monitor the
amount of
pNP present in the reaction tube over the course of the assay. There are two options: a)
the amount of pNP released during the alkaline phosphatase enzyme assay may be calculated
using the molar absorbance coefficient (if you have forgotten what this is see the Mathbench
module called “Measurement”) of the
p-nitrophenolate ion or b) via a standard curve for pNPi at the
appropriate pH.
For this practical, you will be given a table with the absorbance values for the standard curve for
the
pNPi:
Biochemistry Practical Manual 28

concentration pNPi (µM) absorbance
10 0.17
20 0.37
30 0.52
40 0.74
50 0.91
60 1.06

These data were obtained by preparation of a standard curve for pNPi from 0 to 60 µM in 0.1M
Borate buffer, pH 9.5 using 1 cm cuvettes. You will need to plot these data and fit a linear function
to calculate the absorbance coefficient of pNP [M
-1cm-1] or [µM-1cm-1].
Main Experiment. Determining the initial velocity (Vo) of alkaline phosphatase at different
substrate concentrations.
What is the purpose of measuring enzyme velocity? What does initial velocity (Vo) mean?
In this experiment you will set up five reactions in which enzyme concentration, temperature and
pH will be held constant but substrate concentration will be altered. We can determine how
substrate concentration affects V
o by monitoring the formation of product over time (3 min). We do
this by running the reaction in a cuvette placed within a spectrophotometer and measuring the
absorbance at 405 nm every 15 s. This data will be used to determine the initial enzyme velocity
(V
o) at each substrate concentration. This data will be used for a Lineweaver-Burk plot to
determine K
m and Vmax.
Materials & Materials
Materials:
1.5 mM pNPP in 0.1 M borate buffer (pH 9.5)
0.1 M borate buffer pH 9.5 containing 5 mM Magnesium sulphate
Alkaline phosphatase enzyme (0.5 mg/mL, 10 U/mg) – store on ice prior to use.
What does U/mg mean? Why do we store the enzyme on ice? Hint: Think back to
the protein practical earlier in the semester
Method:
1.
Prepare 5 different concentrations of pNPP from 0.075 to 1.5 mM in borate buffer, pH 9.5
in a final volume of 5 mL.
Draw up a table indicating the volume of stock solution and buffer to make up the solutions.
Biochemistry Practical Manual 29
2. To run an assay, pipette 1.25 mL from one of the substrate concentrations into a clean
plastic cuvette. Use this solution to blank the spectrophotometer at 405 nm. Start the
reaction by adding 0.25 mL of enzyme. Record the absorbance every 15 s for 3 min.
Discard the assay mix and repeat with another substrate concentration.
Before you start draw up a table where you can record the absorbances of the assays every 15 s
for 3 min. Use the one table for all five assays. Indicate the
final concentration of the substrate in
the assay as column headings for your table. Hint: Once you add enzyme to the assay mix, you
will have diluted the substrate concentration in the assay. You need to calculate the final
concentration.
Pre-warm the enzyme to room temperature before you add it to the assay mix.
Why?

Substrate
(1.25 mL)
Substrate + enzyme
(1.5 mL)

When the enzyme is added to the cuvette, the substrate concentration decreases because of the
additional volume. For example, if you have 1.25 mL of 1 mM substrate, then after the enzyme is
added, the [S] decreases to 1.25/1.50 x 1 mM = 0.83 mM.
Remember: The enzyme will not convert substrate to product until it is in added to the cuvette.
Place the cuvette containing the substrate into the sample holder of the spectrometer. Pipette the
enzyme into the cuvette, pipetting up and down a couple of times to mix the enzyme with the
substrate. Shut the lid of the spectrophotometer and record absorbance. It may be slightly
negative (why?) and record this value as 0 s and record every 15 s.
3.
Plot the data from all five assays on the one graph and determine Vo.
Once identifying the dependant and independent variable of the experiment, plot this data on
graph paper or Excel. The units of initial velocity (V
o) are Absorbance/time. This can be obtained
from each graph where the enzyme is converting substrate into product at the maximum rate (i.e.
the steepest part of each graph). You will need to measure the tangent of the graph at this point to
get a quantitative value for this rate or use Excel. You should be able to determine 5 different V
o
values; one for each substrate concentration.
4. Using the standard curve from above, convert Vo from having the units of Absorbance/time
to [pNP]/time (using the calculated absorbance coefficient of pNP)
Using Vo with units of Abs/time as an unknown, determine [pNP]/time from the standard curve
using the calculated absorbance coefficient. Repeat for all five V
o values.
5.
Plot [pNP]/s v [S]
Biochemistry Practical Manual 30
This is known as a Vo vs [S] plot. From this graph we can get an estimate for Vmax and Km.
Estimate these values.
6. Plot a Line-weaver Burk plot from the Vo and [S] data
Draw a table with four columns. One column will be used to indicate the [S] used and another will
be used for the [pNP]/s measured (V
o). A third column will indicate 1/[S] while the fourth will be
1/V
o. These two later columns will be used to plot the LB plot. You need to plot 1/Vo on the y-axis
and 1/[S] on the
x-axis. Draw a line of best fit and calculate Km and Vmax.
Please attempt to draw your graphs in the practical class. Your demonstrators and
supervisors will assist you.
Discussion
1. List your Vmax and Km values determined for alkaline phosphatase, and compare to
literature values. Remember your units for these parameters, you may have to convert to
be able to compare to literature values. Are your values higher or lower than literature
values and what does it mean for the function of your enzyme compared to the literature
one (e.g. is yours faster or slower?). Compare your values with a friend, are the values
close? If not, what did they do differently?
2. Compare the V
max and Km values estimated from the Michaelis-Menton plot with the values
obtained from the Line-Weaver Burk plot. Are they similar or not? Why might there be
discrepancies between them?
3. Discuss the effect of a pH change or the inclusion of phosphate on the enzyme function.
What has happened to the V
max or Km? What does a literature search say about the effect
of phosphate? Are there any experimental limitations to be aware of when conducting these
experiments.
Appendix: Measuring reaction rates
The rate (velocity) of an enzyme-catalysed reaction can be measured either by the rate at which
product is formed, or the rate at which substrate is used up. As substrate is converted to product
the rate decreases, and equilibrium is approached. Therefore, it is important for both accuracy and
reproducibility to measure this rate during the period when formation of product (or disappearance
of substrate) is linear with time. This
initial linear reaction rate is known as the initial velocity
(Vo) of the enzyme reaction.
Enzyme activity can be calculated as:
amount of product formed (or substrate used) per unit time for a given enzyme at a specific
temperature and pH,
e.g., µmoles product formed per minute.
Enzyme Assays
To measure an enzyme’s activity one must first have a suitable enzyme assay i.e., a specific test
for measuring the activity of the enzyme one is interested in.
An enzyme assay requires:

Biochemistry Practical Manual 31
knowledge of the reaction catalysed by the enzyme;
a method of reproducing this reaction in vitro;
a substrate or product which can be easily detected and quantitated.
Such methods must be thoroughly tested to ensure they give reproducible results before they are
used routinely. In this week’s experiment you will examine the kinetics of the enzyme, alkaline
phosphatase.
Enzyme Kinetics
Enzyme kinetics is the study of enzyme reaction rates, the parameters, which determine these
rates, and the ways in which these parameters may be affected by the presence of various
inhibitors. Such information is useful because it helps us understand how the rates of cellular
reactions are normally controlled, as well as how this control may be altered by the presence of
drugs. In this week’s laboratory session you will carry out a series of reactions to estimate two
basic parameters of enzyme reactions:
Km and Vmax.
Significance of Km and Vmax
At low substrate concentrations, with a fixed amount of enzyme, the rate of a simple enzyme
reaction is linear with substrate concentration. As substrate concentration increases further, the
rate of the enzyme reaction increases to a lesser degree, i.e. the relationship is no longer a linear
one. At high substrate concentrations the rate of the enzyme reaction approaches a maximum –
(V
max). See Figure below. This occurs when the active site of the enzyme is saturated with
substrate i.e. when the substrate concentration is high enough to ensure that the active site is not
left empty for any significant length of time.

Vmax
½ Vmax
Note that the curve would have to be
extended considerably before
reaching the actual
Vmax value.

Km [S]
The relationship of the initial velocity,
Vo, of a simple enzyme reaction to the substrate concentration,
[S], and to
Vmax, is expressed by the Michaelis-Menten equation:

Vo = Vmax [S]
[S] + Km

Km is defined in terms of various rate constants (see your lectures and textbook). However, it is
mathematically equal to the substrate concentration at which the reaction rate is half of the
maximum velocity.
Vmax is the maximum velocity for a defined set of reaction conditions i.e., pH, temperature etc. The
highest value of
Vmax will be obtained when the reaction conditions are optimal.
Biochemistry Practical Manual 32
It is usually not practical to estimate Vmax from a substrate saturation curve, since saturation may
be experimentally very difficult to achieve. A number of other graphical methods have been
developed for measuring
Vmax and Km. Of these; the Lineweaver-Burk plot is widely used. This is a
double-reciprocal plot of 1/
V vs 1/[S], which is based on the reciprocal of the Michaelis-Menten
equation:

1 = Km . 1 + 1
V Vmax [S] Vmax

The values of Vmax and Km are derived from their reciprocals, which are equal in magnitude to the
intercepts on the ordinate (vertical axis) and abscissa (horizontal axis), respectively, as shown in
the Figure above. To obtain the data to prepare this plot,
Vo (initial velocity) must be measured at
various substrate concentrations.
However, not all enzymes obey simple Michaelis-Menten kinetics. Allosteric enzymes, for example,
exhibit sigmoid kinetics and require more complex equations.
A typical Lineweaver-Burk plot
Inhibition
A molecule that binds to the active site of an enzyme, but is not a substrate for the enzyme or is a
different substrate for the enzyme, will be capable of competitively inhibiting the reaction. Such a
molecule will continually associate with, and dissociate from, the enzyme. During the time that it is
bound to the enzyme no product can be formed as the active site will be occupied. The
concentration of competitive inhibitor relative to substrate affects the extent of activity reduction.
The more molecules of inhibitor that are present the greater the chance that the enzyme active site
will be blocked, which results in fewer substrate molecules being converted to product.
Competitive inhibition can be overcome by increasing the concentration of substrate. If the number
of substrate molecules in the solution far outnumber the molecules of competitive inhibitor, then
the chances of an enzyme molecule binding an inhibitor molecule rather than a substrate molecule
are considerably reduced. Thus,
Vmax can still be achieved, but the substrate concentration needed
to achieve
Vmax is greater than in the absence of an inhibitor, i.e., ½ Vmax is greater and so there is
an apparent increase in the
Km. This will be detected by obtaining a Lineweaver-Burk plot for the
Biochemistry Practical Manual 33
uninhibited enzyme and for the enzyme in the presence of inhibitor. For the inhibited enzyme,
1/
Vmax will be seen not to change but 1/Km will be less than in the absence of inhibitor.
In non-competitive inhibition an inhibitor molecule binds to the enzyme at a site other than the
active centre, causing it to function defectively. In this case, the
Km may be unchanged, but the
achievable
Vmax is reduced. Therefore, on a Lineweaver-Burk plot 1/Vmax will be seen to increase
due to the reduced denominator – Vmax, but 1/
Km may be unchanged.
Alternative graphical methods
Double-reciprocal plots have certain disadvantages. Two alternative graphical methods have been
developed to measure
Km and Vmax:

the Eadie-Hofstee plot (V vs. v )
[s]
Hanes-Woolf plot ( [s] vs. [s])
v

Each has particular advantages. However, for analysis of enzyme inhibition, the Lineweaver-Burk
plot remains the preferred method.