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Introduction to Biochemistry Laboratory 3

General Laboratory Principles 6

Laboratory Requirements 10
Laboratory Notebook 10
Laboratory Report 11
Answers to Research Questions 13

Laboratory Activity 01: Biochemical Systems 14

Research Questions 21
Structure-Property/ Structure-Activity Relationship Question 21

Laboratory Activity 02: Bradford Assay and Image Analysis 23

Research Questions 35
Structure-Property/ Structure-Activity Relationship Question 35

Laboratory Activity 03: Isolation, Quantification and Kinetics of Yeast Invertase 37

Research Questions 44
Structure-Property/ Structure-Activity Relationship Question 44

Laboratory Activity 04: Lipid Molecules and Radical Scavenging Activity 46

Research Questions 58
Structure-Property/ Structure-Activity Relationship Question 58

Laboratory Activity 05: E-ring Modified Steroids and

17β-Hydroxysteroid Dehydrogenase Type 1 Inhibition:
An In Silico Analysis 60
Research Questions 68
Structure-Property/ Structure-Activity Relationship Question 68

Laboratory Activity 06: Structure-Property Relationship of Saccharides 70

Research Questions 78
Structure-Property/ Structure-Activity Relationship Question 78

Laboratory Activity 07: Plant Metabolites and α-Amylase Inhibition 80

Research Questions 85
Structure-Property/ Structure-Activity Relationship Question 85

Laboratory Activity 08: DNA Extraction and Gel Electrophoresis 86

Research Questions 91
Structure-Property/ Structure-Activity Relationship Question 91

Laboratory Activity 09: In Silico Analysis of Cytotoxic Nucleoside Analogues 92

Research Questions 97
Structure-Property/ Structure-Activity Relationship Question 97

Laboratory Activity 10: Lipase Inhibition of Dipeptides 98

Research Questions 104
Structure-Property/ Structure-Activity Relationship Question 104

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Laboratory Activity 11: Phenylalanine Derivatives and
Tryptophan Hydroxylase-1 (TPH1) Inhibition 105
Research Questions 110
Structure-Property/ Structure-Activity Relationship Question 110

Glossary 111

Appendix 1: Toulmin Argumentation Pattern 114

Appendix 2. Rubric for the Evaluation of Laboratory Notebook and Laboratory Report 115

Appendix 3: Evaluation Guide for Arguments using Toulmin Argumentation Pattern 116

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 Congratulations! You are now officially enrolled in the biochemistry laboratory!

You are reading this introduction because (1) you have gained a learning experience about
fundamental chemistry concepts in general inorganic and organic chemistry, (2) you are prepared
to learn fundamental principles and concepts related to the structure, property and function of
biomolecules, 3) you are capable of applying chemistry concepts to health concepts and (4) your
laboratory teacher probably instructed you to read this page before proceeding to the first activity.
Seriously, you want to be on the right track when performing activities in this laboratory manual.

In the health sciences, learning biochemistry concepts is relevant in understanding bodily

functions in a molecular level. Since all processes in the body has a molecular basis, the choice
of health decisions are also based on molecular processes and biochemistry principles. Hence, it
is important that you do maximize your time in the laboratory in performing laboratory procedures.

There are differences in learning biochemistry and general inorganic and organic chemistry.
First, you need to consider that chemical processes in living organisms are quite complex. What
you will be doing in the laboratory are simulations of what happens inside a cell, for example. Of
course, the chemical environment inside a cell is much more complex compared to a typical
aqueous environment. Second, you will be performing activities in a microscale level. This means
that you will be performing laboratory activities using very small amounts of chemicals – in the
milligram or even microgram measurements.

Some reagents and chemicals are also very sensitive to contaminants. You don’t want your
enzyme to be deactivated simply because you forgot to rinse your glassware, do you? You also
don’t want to submit a blank laboratory report because you contaminated your stock solutions.
Third, the activities require you to analyse data, present them in an organized manner and discuss
results using correct biochemistry principles. This is a little bit trickier to do because you are using
minute amounts of reagents. Lastly, you will be using your data to understand health concepts.
Quite challenging, right?

Now, let me orient you about what you are going to expect in using this biochemistry manual.
The laboratory activities are classified as in silico, in vitro and ex vivo procedures. In silico
procedures involve computer software such as online freeware and statistical tools. In vitro
procedures will involve observation of chemical reactions, while ex vivo procedures involve
studying living organisms in laboratory settings.

For in silico analysis, you will be using a molecular visualization online software (or freeware),
MolView. In addition, you will also use freeware which provide molecular properties such as
ChemDes and ChemMine. The in vitro procedures are used to gather quantitative data in
quantifying biomolecules, determine enzyme activity or their inhibition, and analyze structure-
property or structure-activity relationships. The ex vivo procedures will be focused on studying a
simple organism to observe the processes in living organisms. In some activities, you will be
performing in silico-in vitro procedures. Some procedures involve in silico-ex vivo procedures.
Lastly, some activities are purely performed in silico.

In addition, you will be required to analyze data using correlation analysis, simple linear
regression and multiple linear regression. Don’t worry, these statistical tests are laid out as a
tutorial so that you can perform the analysis on your computer. I have also included the procedure

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for image analysis using the ImageJ software as an alternative to spectrophotometric methods.
The data are originally expressed as mean gray values, but you can convert this into quantifiable
data, similar to the absorbance values in spectrophotometry.

The laboratory activities in this manual are divided into three: biochemistry procedures for
analysis of biomolecules (such as extraction, electrophoresis or chromatography), quantitative
biochemistry assays (e.g. Bradford Assay), enzyme assays (lipase inhibition assay) and in silico
quantitative structure-activity relationship analysis. Some activities will require two to three
laboratory meetings so you need to keep in track with your observed data.

The laboratory manual is divided into several sections: Introduction, Aims, Materials,
Equipment, Tools and Databases, Experimental Section, Research Questions and Structure-
Property/ Structure-Activity Relationship Questions.

In addition, there are icons which are used solely in this manual. Here are some of them:

This icon indicates “suggestions” or “pointers.” There are procedures which will
require you to solve. When you see this icon, it means that you can use the tips
provided to perform a step or solve for data.

This icon indicates “REMINDER.” Some results need to be written either in your
laboratory notebook or laboratory report. Your laboratory instructors will be
checking these reminders in your report, by the way.

This icon means “CAUTION.” It tells you in advance some hazards related to the
procedure or to the reagent being used.

This icon refers to “research questions.” Since these are questions which require
research, you need to read reliable journal articles, books or other materials to
justify your answers. Your answers to research questions will be recorded as part
of your laboratory reports.

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 This icon indicates CHALLENGE QUESTIONS. It simply means that you will be
assessed based on prior concepts you have learned in general inorganic and
organic chemistry. It may also be an evaluative test to measure what you have
learned from the laboratory activity. Your score will be recorded as a quiz or
seatwork.

This icon is the “STRUCTURE-PROPERTY or STRUCTURE-ACTIVITY” question.

When you see this icon, it means that you are required to make an argument using
the Toulmin Argumentation Pattern. Your answers will be recorded as a laboratory
assignment.

This icon indicates summary of concepts you have learned from the activity. Write
all relevant concepts you learned in bulleted form.

This icon represents “supplementary discussions or concepts” which can provide

additional information about the topic in the activity that you are doing.

I wish you success in learning biochemistry concepts using this biochemistry laboratory
manual.

Enjoy learning biochemistry!

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 Apparatus and surroundings in the laboratory must be clean. Keep your laboratory
areas clutter-free.

 Use water and detergent in cleaning your glassware. In doing assays in the laboratory,
you need to make sure that the reaction vessels are not contaminated. Rinse the
vessel with distilled water, then the solution to be used prior to assays.

 Apparatus should be cleaned promptly after use.

 If any substance is spilled, clean it up immediately. DO NOT return spilled reagents to

the stock reagents.

 Never leave the laboratory in a mess.

 Organize your working area.

 All wastes should be segregated accordingly.

 In every experiment, follow the procedures carefully and intelligently. If you are in
doubt, ask your laboratory teacher.

 Double check the label in bottles to be certain that you have the correct reagent.

 Wear a laboratory gown over your clothes.

 When heating substances in a test tube, never point it towards yourself or your
neighbors.

 Never look directly down in the mouth of the flask, beaker, crucible, or any container
which is being heated because of the danger of spattering or bumping.

 To protect your eyes, wear goggles throughout the duration of the laboratory period.

 Never taste chemicals!

 To determine the odor of the liquid, fan the vapors with your hands towards your nose
and sniff cautiously.

 Never hold a test tube or container directly under your nostrils because of the danger
of choking or of heated liquid bubbling up to your nose.

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 When mixing acid and water, always pour the acid into the water with constant stirring.
Never reverse to prevent spattering.

 Make sure that all set-up of apparatus are well constructed and sturdy so that they do
not break down at a crucial moment or becomes hazardous.

 Particular care should be exercised when dealing with corrosive acids, alkalis,
poisonous gases, and flammable materials.

 All procedures that produce noxious and toxic fumes and gases must be performed
under the hood.

 Eating or chewing is strictly prohibited.

 Avoid playing with chemicals, knocking over apparatus set-up, or breakage of

glassware.

 No unauthorized experiments should be attempted.

 Avoid contact of corrosive chemicals with any part of the body since this can often
produce burns, stains, and other disagreeable reactions.

 Wash your hands before leaving the laboratory.

 Students must assume personal responsibility in keeping reagents pure.

Contaminations will lead to inaccurate and misleading experimental results of a large
number of students.

 Avoid causing contamination of the reagents through the covers or stoppers. When
taking any chemical, either hold the stopper between your fingers or if it is flat topped,
lay it on the table top down. Also, lay the stopper directly at the back of the bottles in
the same row of the reagents used to avoid mixing of the stoppers.

 For removing solid reagents from bottles, use a clean, dry spatula.

 When weighing chemicals, only dry, non-hygroscopic substances may be weighed on

paper; otherwise, use a watch glass.

 Always grasp the bottle to cover the label with the palm of your hand so that when
pouring any liquid running down, the side will not deface the label.

 Use clean test tube or beakers for carrying liquids.

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 Always take only the amount of the reagent indicated in the procedure. Small
quantities of reagents are easy to manipulate and takes less time to handle. In
addition, lesser amount of waste in the lab is generated if you use small amounts of
reagents.

 Do not carry reagent bottles or stock solutions to your desk.

 After the performance of the experiment, do not pour material back into the reagent
bottle. This is to prevent possible contamination of the stock reagent.

 When use of the burner is to be interrupted, turn the gas off.

 Just before leaving the laboratory, make sure that the gas and water are turned off,
that your desk top is clean, and that reagent bottles and special equipment are
returned to the stockroom.

 Waste solid chemicals, broken glassware, paper, used matches, and other solid
residue should not be dumped into the sink.

 Use appropriate trash cans.

 Try to produce the least amount of waste when performing laboratory activities.

 The laboratory is a place for serious study.

 Start promptly upon entering the laboratory room and remain inside until the end of
the laboratory period.

 The following are not permitted: SMOKING, LOUD TALKING, CHEWING, PLAYING
GAMES, WHISTLING, SINGING, PRACTICAL JIOKING, and OTHER PRACTICES
NOT CONDUCIVE TO EFFICIENT WORK.

 Just like any other class, a laboratory class should be prepared for.

 The student must read and study the experiment from beginning to end IN ADVANCE,
i.e., before coming to class. Planning helps prevent mistakes, loss of time, and leads
to a better understanding of the principles.

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 Report all accidents and injuries to the instructor after any urgent first aid (such as
washing acid out of the eyes).

 The instructor, when necessary, will refer the victim or see to it that he is brought to
the school clinic for medical attention. Ask a fellow student to report the accident or
injury in case you cannot do it yourself.

 Treat spilled acids or bases as follows:

On hands or face – before much damage is done, wash off the chemical with
a large amount of water then with sodium bicarbonate solution. In case of sulfuric
acid, wipe off first then wash off with plenty of water.

On clothing
- Acids  wash with sodium bicarbonate solution or dilute ammonium hydroxide
- Bases pour dilute acetic acid then neutralize with sodium bicarbonate
solution

 If any chemical gets into the eyes or mouth, go to the nearest sink and wash off with
as much water as possible.

 If the eye is involved, hold the eyelids open with your fingers and allow the water to
run freely over the eyeball.

 In case of fire, keep your distance from it, let the instructor handle it. However, you will
probably be asked to assist in extinguishing small fire with fire extinguishers and fire
on burning cloth with a wet cloth or sack.

 Consistent with the aim of training students in professional responsibility, accuracy of

results will enter into consideration in evaluation and grading.

 Standards of accuracy in terms of tolerance of error will be set by the instructor in

keeping with the experience of the student, equipment and time allotted to the
experiment.

 Some of the proceeding rules may be subject to modification by the instructor

according to the judgment of the laboratory teacher.

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 During laboratory activities, it is important for you to read prior concepts and the
laboratory procedures so that you can perform the laboratory activities with minimal errors
and maximum use of laboratory time. Your laboratory notebook is essentially your diary in
the laboratory because you will be using it to record your data, search for definition for
unfamiliar terms and plan your activities to finish your activity on time. Take note that you
only have two hours of laboratory time per meeting. In your actual performance, probably
you will be using about one and a half hour per meeting, considering that there is still a
pre lab discussion and orientation. In using your laboratory notebook, it is recommended
to use one page (usually the front page) for your pre-laboratory concepts, flowchart of
steps, observations and data while the other side (back page) should be used to write your
personal notes about the activity and procedures. Hence you need to use a sturdy
notebook with adequate number of pages to accommodate all activities in the biochemistry
laboratory.

There may be unfamiliar concepts in the laboratory manual that needs to be

clarified. Use your free time to search for additional concepts that can help you
understand your laboratory activity. You may also watch relevant educational videos
in biochemistry online to clarify some steps or concepts. It is also important to simplify
the definition of terms and concepts you have learned, not to copy the technical
definitions in books and journals because you might end up more confused if the
definitions are too technical. Take note, information in the laboratory should not be too
time-consuming since you need to finish data gathering during the allotted time form
laboratory meetings.

The steps in the laboratory manual may be too long for you to understand DURING
the actual laboratory meeting. In other words, do not read you manual on the day of
the performance of the activity – you will end up confused. You cannot expect your
laboratory instructor to accommodate all your questions because you are supposed to
be PREPARED in performing the activities. Try to summarize the main procedures
making a flowchart of steps. You can do this at least two meetings prior to the actual
performance of the laboratory activity. Note also some steps in the procedure which
require prior preparation (i.e. bringing of specimens or materials, warming up of
equipment, preparation of solutions etc.). This will save you time in understanding the
general flow of activities and minimize errors in the lab. This will also ensure that all
members of the team help each other in finishing the laboratory activities on time
during the actual performance of procedures.

The activities in the laboratory are designed for you to observe chemical reactions
or gather data which are relevant to understand the concept being learned through
experiential learning. In other words, you learn concepts by experience, which is the
actual hands-on activity. Take note that the activities in this laboratory will require you

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 to gather data for analysis. In doing so, you will understand the concepts better,
instead of relying on books or journals for understanding the concepts. Hence, it is
important to record your actual data during the lab activity. Try to organize your
laboratory notebook so that your data corresponds to an actual observation as you do
the procedure.

Write your sample computations in your laboratory notebook. Some computations

include solving for mean and standard deviation, concentration of compounds using a
linear regression equation, and recording IC50 or %inhibition. Take note that your
laboratory notebook should show all computations to obtain data which will be
presented in graphical or tabular form in your laboratory report. This will also train you
to trace how the individual data in your graph or table was obtained. As much as
possible, keep your computations organized and logical. Solving for mean and
standard deviation during laboratory activites may be done after the laboratory activity.
Focus on GATHERING OF DATA during laboratory activities. However, be critical if
the deviations of data are too high. Perform another replicate to make sure that your
data is precise when you present them. For quantitative data, you need to perform a
minimum of three replicates in the laboratory.

B. Laboratory Report
Components
Introduction
- Keep this brief. The introduction generally discusses relevance of a topic in
relation to the laboratory activity. Briefly introduce your general aim in this
section.

Infographic
- An infographic is basically a summary of the objectives, results and conclusion
presented in an art form.

Flowchart of Procedures
- The procedures should be in a flowchart form, with all major procedures
emphasized. Flowcharts are concise and straightforward, compared to wordy
procedures in most laboratory manuals.

Results and Findings

- This section should be highly visual, focusing on major findings and data.
Figures and graphs are encouraged.

Discussion of Results
- This section uses theories, laws and other concepts in biochemistry to justify
the validity of inferences. Keep this brief and straightforward as well.

Conclusion
- This section briefly describes the generalization (with limitations) about the
activity which has been completed. The conclusion also addresses the key
concepts which are written in the aims of each activity.

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 References
- The reference section should contain all cited literature in the laboratory report.

 Proposed Format:
Paper size: 8” x 11”
Font: Arial 10, spaced singly
Margin: 1 inch in all sides

What makes a good laboratory report?

 Does it contain the required sections?
 Is it clearly written?
 Does it use scientific terms properly?
 Does it use good grammar?
 Are the words spelled correctly?
 Are the calculations performed correctly?
 Is it unnecessarily long?
 Is the title meaningful?
 Does the title page contain the author’s name and address?
 Does the title page contain the name(s) of the author’s lab partners?

Introduction
 Does it give general background?
 Does it point out poorly understood or unknown factors related to the study?
 Does it discuss the significance of the work?
 Does it flow well? Is it logically written? Is it concise?

Infographic
 Is it simple?
 Is it straightforward?
 Are appropriate icons used?
 Is it scientific?

Flowchart of Procedures
 Is it simple?
 Is it easy to understand?
 Are key procedures included?
 Does it look cluttered?

Results
 Does it explain the rationale and strategy for the experiments performed?
 Does it describe, in words, what was done?
 Does it answer the questions raised in the Introduction?
 Does it flow well? Is it logically written? Is it concise?

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 Discussion
 Does it summarize the findings obtained in the Results section?
 Does it discuss the expected results?
 Does it discuss the unexpected results?
 Does it answer the questions raised in the Introduction?
 Does it reach conclusions?
 Does it explain why the conclusions are important?
 Does it flow well? Is it logically written? Is it concise?

Conclusion
 Is the conclusion unnecessarily long?
 Does it provide an adequate generalization?
 Does it provide an answer to the objectives of the activity?

References
 Are all references cited in the report?
 Is the format followed?
 Are updated references used?
 Are references alphabetically arranged?

C. Answers to Research Questions

- Answers to research questions should be BRIEF, anchored on
normative concepts, and are supported by reliable literature sources.
Limit your answers to ten sentences. Cite all references using APA
format, 6th edition.

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 In studying biochemistry, it is important to note that chemical systems inside cells are
extremely complex. This complexity is due to the diversity of components, structures and many
interactions inside the cell. The interactions are also complex, requiring analysis of metabolic
pathways and their roles to maintain homeostasis. Since cells interact with other cells in tissues,
tissues interact with other tissues, and organs interact with other organs to sustain life, a single
laboratory activity cannot completely capture all the complexity in the chemical environment inside
the cells.

In our biochemistry laboratory class, we will perform laboratory activities to infer possible
mechanisms in living organisms. It is important to note that chemical environments in the
laboratory are controlled based on the design of a scientist. Biochemistry laboratory activities
within this semester will be limited to in vitro and ex vivo activities. But these activities are
important because it gives us important data which can be used to analyse and develop our
inferences to understand living systems. In this activity, we will be performing ex vivo (Latin “out
of the living”) and in vitro (Latin “in glass”) activities to aid us in understanding biomolecules.
Studying the effect of a substance on a tissue culture is an ex vivo activity while color reactions
involving biomolecules is an in vitro activity. These two activities are useful in developing
inferences in in vivo (Latin “within the living”) chemical systems. However, it is also important to
note the limitations of these chemical systems to avoid misleading or inaccurate inferences.

At the end of the activity, students should be able to:

 Describe the characteristics of ex vivo and in vitro chemical systems;
 Discuss the relevance of ex vivo and in vitro data in understanding biochemical
reactions.

 Baker’s yeast  Clean cheesecloth  0.1M NaOH

 Small plastic cups  Hotdog casings solution
 Egg white  Unflavored gelatin  Starch
 Distilled water  Phenolphthalein  Sodium
 Cover slip and slide  Methylene blue/ bicarbonate
 Table sugar congo red  Microscope slide
 Small inflatable  1M NaCl solution  Cover slip
balloon  Sucrose
 Paper towels  0.1M HCl solution

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 Blender
 Electric microscope

1. In a 250 mL Erlenmeyer flask, Prepare a yeast mixture by mixing 25 granules

of baker’s yeast to 15 mL of water. Soaking the yeast cells will activate them.
Label this as “Yeast Suspension A.”

2. Prepare another yeast mixture, similar to what you did in the first step. This
time, transfer the mixture to your smallest beaker. Boil the mixture for 5 to 10
minutes then let it cool at room temperature. Label this as “Yeast Suspension
B.”

Yeast Suspension A Yeast Suspension B

3. Place a drop (25 µL) of “Yeast Suspension A” in a microscope slide. Carefully

mix this drop of yeast mixture on the slide using a clean micropipette tip.

4. Add a drop of congo red or methylene blue stain to the drop of “Yeast Mixture
A” on the slide. Cover the stained suspension drop with a cover slip.

NOTE: To do this, you may introduce the congo red or methylene

blue stain on one side of the cover slip. Then place a tissue on the
opposite side to pull the stain towards the yeast suspension, then
towards the tissue paper. This will also remove excess stain from
the yeast suspension.

Tissue paper

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5. Do the same procedure for “Yeast Suspension B” (Steps 3 to 4). Set aside the
slides for 5 minutes.

6. Take a picture of the resulting cell suspensions on the two slides at low power
objective (40x to 60x magnification).

7. Observe for the following on both slides and document your results:
a. Appearance of the cells,
b. Response of cells to the stain, and
c. Budding process

Record your results and answers in the laboratory worksheet.

8. Add a ½ tablespoon of table sugar to the prepared yeast suspensions. Mix the
contents of the test tubes gently, just to allow the sugar to dissolve completely
in the yeast suspension.

9. At the mouth of the test tube, fasten a small balloon. Secure the balloon using
a rubber band.

10. Store the yeast suspensions inside your lockers overnight.

11. Record all observations in your laboratory worksheet.

Questions:
- Which observation is related to active transport? Why?
- Which observation is related to metabolism? Why?
- Which observation is related to reproduction? Why?

Record your results and answers in the laboratory worksheet.

1. Prepare a 10 mL of 1.0 M NaCl solution. This will be your stock NaCl solution.

2. Transfer 50 mL of the stock solution to a plastic cup. Label this as “Cup A.”

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3. In separate plastic cups, prepare 50mL of the following:
Cup B –0.1 M salt solution
Cup C – Distilled water

Show all pertinent solutions in your laboratory worksheet.

NOTE: To prepare the 0.1M NaCl solution, use the dilution formula.

C1 V1 = C2 V2

(1.0M) x = (0.1M) (50mL)

x = 5mL

This means you need to get 5mL of the stock solution (1.0 M NaCl solution),
then add 45 mL of distilled water to prepare a 50mL of 0.1M NaCl solution.

4. Place a drop of a yeast suspension on a slide. Cover the suspension with a

cover slip. Observe the morphology of the cells at high power objective (400x
to 600x magnicifation).

5. Introduce a drop of 1.0M NaCl solution to the yeast suspension on the prepared
slide in Step 4.

6. Observe the yeast cells for 20 seconds, 40 seconds and 60 seconds. Note any
changes in morphology.

7. Using another slide, prepare another yeast suspension. This time, introduce a
drop of 0.1 M NaCl solution. Observe the yeast cells for 20 seconds, 40
seconds, and 50 seconds.

8. Using another slide, prepare the last yeast suspension. This time, add a drop
of distilled water to the yeast suspension.

Question:
- What is the effect of solute concentration to the morphology of yeast
cells?

Record your results and answers in the laboratory worksheet.

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1. Prepare the following solutions:
Solution 1: 30 mL of 5% NaCl
Solution 2: 30mL of 5% starch solution
Solution 3: 30mL of 40% sucrose solution
Solution 4: 4.5% NaHCO3 solution

2. Prepare four hotdog or sausage casings. Tie one end of the casing tightly using
a string. Carefully pour about 15mL of each of the solutions to each of the
casings.

3. Close the casings by tying the other end with a string.

When tying the end of a casing, leave a small unfilled space to

observe if the casing has increased in weight or not due to solvent
movement across the casing.

4. Weigh each filled casing and record the measurements in your laboratory
worksheets.

5. Immerse the casings in distilled water for 30 minutes.

6. Carefully dab the casings and measure the weight changes (if there is).

7. Test for the presence of each of the solutes in the distilled water inside the
beaker by using the following methods:

a. To test for NaCl, use 2-3 drops of AgNO3 solution. The positive result is
the appearance of a white cloudy precipitate of AgCl.

b. To test for starch, use 2-3 drops of Lugol’s solution. The positive result is
the appearance of a bluish-purple color, indicating the iodine-amylose
complex.

c. To test for sucrose, heat 1 mL of the distilled water inside the beaker.
Caramelization is will result when sucrose is heated. You can observe this
if the distilled water suddenly turns brownish color, with a distinct caramel
odor.

d. To test for NaHCO3, measure the pH of distilled water. If NaHCO3 is

present, the pH of the solution will slightly become more alkaline.

Record your results and answers in the laboratory worksheet.

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 1. In a 1000 mL beaker, mix one pack of an unflavoured gelatin with 237 mL of
distilled water, 2.5mL of 1% phenolphthalein solution and few drops of 0.1M of
NaOH solution. The solution should be bright pink in color. NOTE: This
procedure needs to be performed beforehand.

2. Boil the mixture to completely dissolve the gelatin.

3. After boiling, allow the gelatin to cool slightly (at least 50oC). In this
temperature, you can comfortably hold the beaker with your hands without
scalding.

4. Pour the gelatin in a plastic container. Make sure that the depth of the gelatin
is at least 3 cm. You may want to use a narrower container for preparing your
gelatin sample. Refrigerate the gelatin overnight.

You need to prepare the gelatin overnight, so it is advisable

that the gelatin has already been prepared before doing this
activity. Your laboratory instructor can prepare the gelatin
for the class.

5. After the gelatin has solidified, cut them into cubes with various dimensions: 1
cm3, 2 cm3 and 3 cm3. You should have at least 5 cubes of each dimension to
conduct the activity.

6. In separate plastic containers, sort the gelatin cubes by size. Immerse the
cubes in each container completely in 0.1 M HCl solution for three minutes.

7. Collect the gelatin cubes and dab them immediately dry using a paper towel.

8. Slice the gelatin cubes and note which part of the cube has decolorized.
Compute for the % decolorization by subtracting the dimension of the
decolorized portion from the dimension of the cube using the formula:

𝐕𝐨𝐥𝐮𝐦𝐞 𝐨𝐟 𝐂𝐮𝐛𝐞 − 𝐕𝐨𝐥𝐮𝐦𝐞 𝐨𝐟 𝐝𝐞𝐜𝐨𝐥𝐨𝐫𝐢𝐳𝐞𝐝 𝐩𝐨𝐫𝐭𝐢𝐨𝐧

% 𝐃𝐞𝐜𝐨𝐥𝐨𝐫𝐢𝐳𝐚𝐭𝐢𝐨𝐧 = 𝐱 𝟏𝟎𝟎%
𝐕𝐨𝐥𝐮𝐦𝐞 𝐨𝐟 𝐜𝐮𝐛𝐞

Present your data in bar graph form.

Question:
- What is the relationship of size of cells to their surface area?

Record your results and answers in the laboratory worksheet.

19
1. Obtain a medium-sized egg, preferably hatched within eight days from a
poultry shop. An egg which is laid within one hour is best preferred for this part
of the activity.

2. Separate the egg white from the egg yolk.

You can use an empty plastic bottle (preferably used bottled

water) to suck out the yolk from the raw egg. Of course, do
not break the yolk because we do not want the egg white to
be contaminated with the yolk. Always wear your gloves!

3. Blend the egg white at a reduced speed. If there is no blender, you may mix
the egg white using a fork in a beaker.

4. Strain the egg white using a cheesecloth to remove the unmilled sections and
chalazae cords.

5. Place 20 mL of the blended egg white in a 100-mL or 250-mL beaker.

Preferably, the egg white solution should be stirred constantly during titration.

6. Record the initial pH of the egg white. Plot this in a graph (on the dot, as
illustrated below), with the x-axis referring to the volume of 0.1N NaOH and
0.1N HCl solution used and the y-axis referring to pH.

Initial pH of the egg white

Left upper portion

pH

Right lower portion

mL of 0.1N NaOH solution mL of 0.1N HCl solution

7. Add an increment of 0.20 mL of 0.1N NaOH solution to the egg white solution.
Record the pH of the egg white solution every 0.20 mL of added solution. Your
data corresponds to the pH of the solution for the left upper portion of the
titration curve. Titrate until the pH of the solution reaches 11.0.

20
 8. Obtain another 20 mL of the blended egg white in another 100-mL or 250-mL
beaker.

9. This time, titrate the egg white solution with 0.1N HCl solution. Record the pH
for every 0.20mL increment of the HCl solution. Titrate until the pH of the
solution is 2. Present your data in graphical form.

10. Perform titration of distilled water using the method described in this part of the
activity.

How do the graphs compare?

Question:
- How do egg proteins resist a drastic change in pH, based on what you
have observed?

Record your results and answers in the laboratory notebook.

Why should you be careful in interpreting results of in vitro or ex vivo

activities in studying biochemistry?

Why is there a need for the circulatory system, instead of relying on

diffusion, to distribute nutrients and oxygen to different parts of the
body?

Through an argument, evaluate the validity of the statement below.

Use the Toulmin Argumentation Pattern to present your answer. Cite
all relevant resources you used to support your argument.

“Cellular function is determined greatly by molecular structure.”

Suggested Reading Material:

Findlay, S.D. & Thagard, P. (2012). How parts make up wholes. Frontiers in Physiology
3(455), 1-10.

21
Cotterill, O.J., Gardner, F.A., Cunningham, F.E. & Funk, E.M. (1959). Titration curves and turbidity
of whole egg white. Poultry Science. 38. 836-842. DOI: 10.3382/ps.0380836

Morris, G.J., Winters, L., Coulson, G.E. & Clarke, J. (1983). Effect of osmotic stress on the
ultrastructure and viability of the yeast Saccharomyces cerevisiae. Journal of General
Microbiology 129, 2023-2034.

22
 Quantitative analyses play important roles in understanding biochemistry concepts in the
laboratory. A quantitative analysis presents an advantage over qualitative analysis because the
data can be used to infer relationships of various variables or establish cause-effect inferences.
In fact, a biochemistry laboratory involves a lot of quantitative data, ranging from determination of
concentration of biomacromolecules or simple molecules in sample materials to determining
enzymatic activity in biological samples.

Recently, color reactions in biochemistry have adapted a micro-scale method – small amounts
of reagents and chemicals are used and experiments are done using a 96-well microplate or
microarray. Most methods of quantification in the biochemistry laboratory are expensive and may
require multiple complex steps prior to analysis. These methods, despite having high sensitivity,
may be too impractical when studying biochemistry concepts in a typical academic setting. An
alternative to these quantitative methods is through the use of image analysis, which is presented
in this activity.

Image analysis only requires two hardware: your desktop computer or laptop, and a simple
flatbed scanner (Figure 1). Using this image analysis set-up, you need to (1) scan your results,
(2) adjust the image to enhance contrast and remove interferences and (3) analyze the mean
gray values and convert them into quantifiable data, similar to the absorbance data using a
spectrophotometer.

Flatbed Scanner

96-well plate

Laptop
Clear acetate

23
 In performing image analysis, you will need to download and install ImageJ in your laptop or
desktop computer. ImageJ is an image processing program created by Wayne Rasband of the
Research Services Branch, National Institute of Mental Health in Bethesda, Maryland. This imaga
analysis software has several features which could be used to process and analyze images in
most commonly used image formats.1

The ImageJ software can be used to process images in TIFF, GIF, JPEG, PNG, DICOM,
BMP, PGM and FITS formats.2 In this activity, you will be using the mean integrated density into
red, green and blue channels. A higher mean integrated density means that an image is lighter
(in terms of mean gray value).2 In performing image analysis, you are going to use a 96-well plate,
so all procedures are actually performed using a microscale method. Using a microscale method
produces lesser waste in the laboratory and expensive alternative for analytical methods.

At the end of the activity, students should be able to:

 Perform analysis of sample images using ImageJ software
 Interpret results using Microsoft Excel

 Coomassie Blue Stain

 Bovine serum albumin
 Protein samples with unknown protein concentration

 Desktop computer
 Flatbed Scanner

1. Don your gloves. Be sure that your test tubes are properly rinsed with distilled
water. This assay is sensitive to contaminants and residues or detergents.

2. To make the calibration curve, pipet 0.25 mL of each of the stock solutions and
diluted bovine serum albumin solutions into separate test tubes. Make each up to
1.0 mL with distilled water.

3. Pipet 0.25 mL of distilled water into a further tube to provide the reagent blank.
Use a small test tube for this step. Again, add distilled water to prepare a 1mL
sample.

4. For your unknown protein concentration, pipette 0.25 mL each in three test tubes.
This will be your three replicates.

24
 You can use the following samples:
 Fish paste
 Soy sauce
 Beef or chicken broth
 Other protein samples

Analysis of protein in food using Bradford assay is not as

straightforward as it seems. Food samples are complex matrix of
different compounds which could interfere with the reaction of
Coomassie Blue and the protein found in the protein sample. It is
possible that there is an underestimation of protein in food.4

5. In separate test tubes, add 0.10 mL of the Bradford reagent to each tube with 0.10
mL of each diluted protein sample.

6. Mix well by inversion or gentle vortex mixing. The reaction will proceed for 2 to 5
minutes.

7. Load a volume of 0.10 to 0.15 mL of each solution to 96-well plates. You may use
the guide on the next page to know where to place the samples and the solutions
for constructing the calibration curve.

C2 to C7: 31.3µg/mL to 1000µg/mL BSA solutions

D2 to D7: 31.3µg/mL to 1000µg/mL BSA solutions
F2 to F4: Unknown protein concentration
C10 to C12: Blank (distilled water)

8. Scan your 96-well plate and upload to your desktop. Label your image to make it
easy to access.

25
 When using the flatbed scanner, it is a little tricky to adjust the
conditions to produce an image with an adequate quality for image
analysis. It is suggested that you use a white background for the
96-well plate. Second, you need to use an enclosed box to make
sure that there are no residual light which could illuminate the
sample being scanned. Lastly, you can use a clear acetate to
protect the surface of the flatbed scanner.

Print a copy of the scanned image and submit it together with

your laboratory worksheet.

1. Download the software “Image J” through this link: https://imagej.nih.gov/ij/. You

may do this prior to this activity so that the software is already installed.

2. Upload the scanned image of your 96-well plate. Open this file using Image J. Use this
template as a guide in analysing your results.

26
Figure 3. Edited Image

27
3. Open the Icon for ImageJ to start using the software.

Select “Open” among the main tabs then choose the file name of the image that you
want to analyse.

4. If you want to analyze the image, choose “Image” then “Color” followed by “Split
Channels.” This step will split your image into red, blue and green channels.

The resulting images are the red, blue and green channels of the original image.

28
Red channel

Green channel

Blue channel

29
5. Visually evaluate which channel exhibits a pattern in terms of increasing or decreasing
intensity of color. In this case, the red channel seems to exhibit a pattern, and will be
used subsequently for image analysis.

6. On the ImageJ tab, select “Analyze” then “Set Measurement.” Choose the “Mean Gray
Value” since this will give you data on the mean integrated density of the individual
wells.

30
7. Choose the “Multipoint Tool.” Double click on the icon and select “Circle” among the
options for “Type. This will ensure that a large portion of the target image will be
included in the analysis.

8. Double click on the multipoint icon to choose the specific point tool to be used.

31
9. Select all wells to be analysed using the “Multi-point” tool. After selecting images, click
on “Analyze” then “Measure.”

The results will appear like the one shown on the next page.

The data can be copied and analysed in Microsoft Excel.

32
10. Convert the mean integrated densities to “Absorbance” by dividing each value by
255. Get the negative logarithm of these values.3

For example:

-log (140/255) = 0.260412 (This data will be used for analysis)

-log (143/255) = 0.251204

-log (147/255) = 0.239223

11. Plot the “Concentration” versus “Mean Absorbance” by highlighting all the values on
the identified linear region of the curve. Select “Insert” then click on the icon for scatter

12. Determine the linear regression and linearity of the curve by pressing the right click on
any of the plotted points. Then, select “Add Trendline.”

13. Click on “Display Equation on chart” and “Display R-squared value on chart.”

14. Evaluate the R-squared value (coefficient of determination). The linear regression
equation is reliable (in a simple linear regression) if the R2 value is at least over 0.95.

33
15. You can now quantify a given sample using your constructed linear regression
equation, with the format y = mx + b, where x is the concentration while variable y is
the –log value of the mean gray values of the images.

16. Using the linear regression equation, estimate the protein concentration in your
sample. In this case, the unit of concentration of protein used to make the calibration
curve was expressed as µg/mL. Hence, your answer should also be expressed using
the same unit.

If your linear regression equation is y = 0.0002x + 0.2223, and the data is

0.350, the predicted concentration is:

0.350 = 0.0002x + 0.2223

0.350 – 0.2223 = 0.0002x

0.350 – 0.2223 = x
0.0002

x = 638.50 µg/mL

In you laboratory report, show the calibration curve of the protein

standard, linear regression equation, R2 and graphical presentation
of the protein concentration of your samples. Your data should reflect
mean and standard deviation of a minimum of three replicates.

34
What limitations of image analysis have you observed? What is an
appropriate plan to resolve this?

What is the principle behind Bradford Assay for determining the

concentration of protein in your samples? How do you minimize errors
caused by interference of other compounds in a complex chemical
matrix using Bradford Assay?

Using the Toulmin Argumentation Pattern, create an argument to

affirm or refute the statement below.

“Intemolecular interactions are greatly influenced by molecular

structure.”

35
[1] Abramoff, M.D., Magalhaes, P.J., & Ram, S.J. (2004). Image processing with Image. J.
Biophotonics International, 11(7), 36-42.

[2] Ferreira, T., & Rasband, W. (2012). ImageJ user guide IJ1.46r. Retrieved from
http://rsbweb.nih.gov/ij/docs/guide/user-guide.pdf

[3] Soldat, D.J., Barak, P. & Lepore, B.J. (2009). Microscale colorimetric analysis using a desktop
scanner and automated digital image analysis. Journal of Chemical Education 86(5), 617-620.

[4] Mæhre, H.K., Dalheim, L., Edvinsen, G.K., Elvevoll, E.O. & Jensen, I-J. (2018). Protein
determination – Method matters. Foods 7(5). doi:10.3390/foods7010005

36
 Enzymes are complex protein molecules which speeds up the rate of chemical reactions by
lowering the activation energy of that reaction – hence their reputation as biological catalysts.
Most chemical reactions inside our cells are actually catalysed by enzymes that is why we need
to understand how enzymes work. The catalytic activity is performed by enzymes by holding
substrates in specific orientations in the active site, making a chemical reaction energetically
favourable. In fact, the structure of the active site is dependent on the three-dimensional structure
of the enzyme, which in turn is dependent on correct sequence or and type of amino acids. Note
that the active site is an area in the enzyme where the substrate and the amino side chains of
amino acids interact. Any factor which disrupts the three-dimensional structure of the active site
may cause an enzyme to lose its function.

Enzyme kinetics is an important topic in undergraduate biochemistry because it provides data

on how to characterize enzyme function. In an enzyme assay, you need to measure the initial
rate. A linear trend is observed if the substrate concentration is higher than an enzyme
concentration. In this activity, you do not know the concentration of invertase yet that is why you
need to establish rate conditions.

Yeast invertase is composed of homo-oligomer of eight sub-units (Figure 1). Each sub-unit is
composed of 512 amino acid residues with a molecular mass of 58.5kDa. The enzyme is a
tetramer of protein dimers that oligomerize by intersubunit extension of the two β-sheets. Yeast
invertase forms two classes of dimers, AB/CD and EF/GH, which are located at opposite vertices
of the square. The subunits of these two classes of dimers associate differently with each other,
and, thus, the EF/GH dimers can be described as an “open” assembly, whereas the AB/CD dimers
form a “closed” assembly.2

37
 The active site of invertase is located at the interface within each pair of dimers. Because of
the two different ways subunits can form dimers, the active sites in these dimers may have
different chemical environments. The first two dimers (AB/CD) form a very narrow pocket that
seems unable to accommodate an oligosaccharide with more than three or four sugar units. In
contrast, the wider (20 x 16 Å) entrance cavity observed in the other two dimers (EF/GH) allow
longer substrates.

Most of the residues in yeast invertase are short chain amino acids, such as ser-412, ser-414,
ser-415, thr-379, thr-380, and ser-447. These amino acid residues seem to contribute to the
efficiency of invertase in the hydrolysis of sucrose. Yeast invertase, however, shows no significant
activity when inulin is used as a substrate. Recall that inulin has a β-glycosidic linkage among its
monomers. The mechanism will not be fully discussed in this activity, since your focus is on
determining Km and Vmax of invertase.

In this activity, you will be performing three important skills in biochemistry: (1) partial isolation
of enzyme, (2) quantification of the isolated enzyme and (3) determination of parameters of
enzyme activity such as Km and Vmax. Specifically, you will be studying yeast invertase, also known
as β-fructofuranosidase, an enzyme which breaks down sucrose into glucose and fructose. Yeast
invertase exists in two different forms – the cytoplasmic enzyme is nonglycosylated, while the
external invertase contains about 50% carbohydrate of the high mannose type.1

For quantification of total protein in your protein isolate, you will be performing Bradford assay,
which was previously performed in Laboratory Activity 2. This assay relies on the binding of the
dye Coomassie Blue G250 to protein. The more anionic blue form of the dye, which binds to
protein, has an absorbance maximum at 590 nm. Thus, the quantity of protein can be estimated
by determining the amount of dye in the blue ionic form. The dye appears to bind most readily to
arginyl and lysyl residues of proteins.

At the end of the activity, students should be able to:

 Isolate invertase from Saccharomyces cerevisiae cell suspensions
 Quantify the protein content of the invertase isolate
 Determine the Vmax and Km of invertase
 Construct a Michaelis-Menten plot and Lineweaver-Burke Plot

 Baker’s yeast  Phosphoric acid  Distilled water

 NaHCO3  Bovine serum  Volumetric flasks
 Ice cold ethanol albumin (1000 mL and 10
 Tris-HCl buffer  Dinitrosalicylic acid mL)
 Bradford Reagent solution

 Laptop
 Flatbed scanner
 Refrigerated centrifuge

38
1. In an Erlenmeyer flask, suspend 17.5 g of dried yeast in 40 mL of 0.10M NaHCO3
solution.

2. Cover the suspension with cotton ball then incubate at 35oC to 37oCovernight.

Your laboratory instructor can perform this part of the activity, as incubation
of yeast cells requires at least 15 hours.

3. After incubation, centrifuge the suspension at 7500 rpm for 30 min at 4oC. Use the
refrigerated centrifuge for this procedure.

4. Collect the supernatant then store into aliquots of 1.5 mL. You can use an Eppendorf
tube (5.0 mL capacity) to store your aliquots.

5. Clarify the supernatant in the Eppendorf tube by adding ice-cold ethanol to produce a
29.0 % ethanol solution in the supernatant. (This means adding 0.435 mL of ice-cold
ethanol to the supernatant).

6. Mix the supernatant gently by inverting the tube.

7. Place the supernatant in an ice bath for 5 minutes.

8. Centrifuge the Eppendorf tubes at 10, 000 rpm for 10 min at 4oC.

9. Transfer the supernatant to a new Eppendorf tube for the second precipitation of the
enzyme.

10. Add more ice cold ethanol to yield a solution which is about 40.0 % ethanol in a volume
which is less than 1mL. (This means adding 0about 0.37 mL of 95% ethanol).

11. Mix the supernatant gently by inverting the tube.

DO NOT shake the supernatant vigorously because it may lead to

denaturation of invertase.

12. Place the tube containing the supernatant in ice for 5 minutes.

13. Collect the pellets then re-suspend in 0.60 mL of 5.0 mM Tris-Cl (pH=7.4). Store the
supernatant at -20.0oC if not used. This will be the source of yeast invertase.

39
 An enzyme-catalyzed reaction is started when the enzyme (E) binds to its
substrate (S) to form an enzyme-substrate complex (ES). In the reaction
below, an enzyme E combines with the substrate S to form an enzyme-
substrate complex with a rate constant of k 1. However, the enzyme-
substrate complex could also dissociate back to E and S with a reaction
rate of k-1. But, the formation of product is irreversible, so the reverse
process from E + P to ES does not occur significantly (with a rate constant
of k-2).

Enzyme molecules are generally larger than the substrate molecules

except for proteinases, nucleases and amylases that act on larger
substrates (macromolecules). The binding to the enzyme occurs at the
active site which is small portion of the enzyme molecule. The active site is
usually described as a cleft or a pocket which binds with the substrate.3

Catalytic function is accomplished at the active site because various

chemical structures important in binding with the substrate are brought
together in an organized spatial arrangement. Thus, the unique property of
an enzyme is based on its three-dimensional structure and on an active
site whose groups may be brought near to each other from different regions
of the enzyme, which is a polypeptide chain.3

1. Don your gloves.

 Be sure that your test tubes are properly rinsed with distilled water. This assay
is sensitive to contaminants, even detergents.

2. For the calibration curve, pipet 0.25 mL of each of the stock solution and diluted bovine
serum albumin solutions into test tubes, and make each up to 1.0 mL with distilled
water.

3. Obtain 0.25 mL of distilled water into another tube to provide the reagent blank. Use a
small test tube for this step. Do this procedure in two replicates.

4. For your experimental replicates, pipette 0.25 mL each in three test tubes. This will be
your three replicates.

5. Add 0.25 mL of the Bradford reagent to each tube and mix well by inversion or gentle
vortex mixing. Allow the reaction to proceed.

6. Measure the absorbance of the solutions at 595 nm against the reagent blank between
2 min and 1 h after mixing. An alternative method for this is through the use of image
analysis.

40
 Record the absorbance in your laboratory notebook.

You need to prepare a calibration curve for DNSA assay using glucose as
the standard prior to determining Km and Vmax of invertase. For the
calibration curve, use the following concentration of concentration of
glucose or dextrose:
0.19 µM
0.38 µM
0.75 µM
1.5 µM
3.0 µM
6.0 µM
12.0 µM
24.0 µM

To perform the assay, mix 0.30 mL of DNSA reagent in six separate test
tubes. Add 0.30 mL of the substrate glucose or dextrose monohydrate.
Place in a water bath for 7 to 9 minutes. Add 3.0 mL of distilled water to
each test tube. Measure the absorbance at 540 nm using a blank solution
composed of 0.30mL of glucose or dextrose monohydrate and 3.30 mL of
distilled water. Plot the absorbance versus concentration and determine
the linear regression equation.

Perform the following procedures.

1. Label 11 clean test tubes with “0” to “10.”

2. Using a clean 1-mL syringe, place 0.30 mL of DNSA reagent to each test tube.

3. Using another clean 1-mL syringe, add 0.30 mL of sucrose solution to tube “0.” This is
the blank to which no invertase will be added. It will also be used to “zero” the
spectrophotometer. It can also be used as a “blank” for image analysis.

4. Place 2.0 mL of sucrose solution in a water bath (37oC) for a few minutes so that it
warms to the reaction temperature.

41
 The optimal conditions should be determined prior to performing an
enzyme kinetics procedure. First, the concentration of the enzyme is
determined. After determining the optimal enzyme concentration, the
concentration of the substrate is changed to determine the enzyme
velocity, which corresponds to the slope of a linear regression equation.

You may use the following concentrations of sucrose below. To save time,
your laboratory instructor will assign one concentration per group. All data
need to be shared to all groups to construct the Michaelis-Menten Plot and
Lineweaver-Burke Plot. Use the proposed assignment of group per
concentration.

50.0 mM – Group 1
20.0 mM – Group 2
10.0 mM – Group 3
5.0 mM – Group 4
2.5 mM – Group 5
1.0 mM – Group 6
0.5 mM – Group 7
0.1 mM – Group 8

5. Pipet 2.0 mL of sucrose solution to a clean Erlenmeyer flask or reaction vessel. Add
2.0 mL of invertase solution and mix. Immediately after adding the enzyme to sucrose
solution, simultaneously start the timer. This is your reaction mixture.

6. After 2 minutes, use a syringe to take 0.30 mL sample of the reaction mixture. Add this
to tube “1.” Note that the test tubes contain DNSA reagent already. Immediately place
the tubes in boiling water bath to stop the enzymatic hydrolysis of sucrose. Stand this
tube for 7 to 10 minutes until the DNSA reagent changes color to red/ reddish brown.

7. Every successive 2 minutes, take 0.30 mL of the reaction mixture and add it to one of
the successively numbered tubes.

8. Using a clean glass pipette (or other calibrated glassware), add 3.0 mL of distilled
water to each test tube.

9. Flick the tubes gently with a finger to mix the contents.

10. If the latter samples are too dark, dilute the samples with distilled water.

11. Determine the slope of the reaction progress curves. To do this, simply create linear
regression of your points using Microsoft Excel. The slope of the reaction is actually
the velovity o

42
12. Determine the slope of the best fitting line and fill up the table below. (Ask your
instructor for assistance.)

Concentration of Slope of the linear regression

Substrate (µM) equation (µM*min-1)

50.0
25.0
10.0
5.0
2.5
1.0
0.5
0.1

13. Plot a graph of the results. An example of Michaelis-Menten graph and Lineweaver-
Burke Plot (inset) is shown below (Figure 2).

You can use amylase and starch solution to study enzyme kinetics if invertase is not
available.
[Substrate] (µM/min)

[Substrate] (µM)

Plot your Michaelis-Menten graph to determine the Km of yeast invertase.

Use a graphing paper for your laboratory notebook while plot your graph in
Microsoft Excel for your laboratory report. Briefly explain the graph.

43
Km is also known as the Michaelis-Menten constant. It shows the
concentration of the substrate at half of the Vmax for the reaction. It is a
measure of binding affinity of the substrate to the active site of the
enzyme. A low Km value indicates a large binding affinity. This means that
the reaction will approach Vmax more rapidly. An equation with a high
Km indicates that the enzyme does not bind as efficiently with the substrate.
This means that the Vmax will only be reached if the substrate concentration
is high enough to saturate the enzyme.4
The Michaelis-Menten Equation is:

According to Michaelis-Menten's equation, at low concentrations of

substrate, KM >> [S], so the equation is V0 = Vmax [S]/KM which resembles
a first order reaction. At high substrate concentrations, [S] >> KM, so the
[S]/ ([S] + KM) becomes essentially one and the initial velocity approached
Vmax, which resembles zero order reaction.3

What is an enzyme inhibitor? What are the possible mechanisms on

how inhibitors decrease enzyme activity?

What happens to Km and Vmax when there is an enzyme inhibitor in a

reaction vessel? Why do you say so?

How do you know if an inhibitor is competitive, noncompetitive or

uncompetitive?

Using the Toulmin Argumentation Pattern, present an argument to

the statement below:

“All molecules should share structural similarities to substrate to be

effective in enzyme inhibition.”

44
[1] Sainz-Polo, M.A., Ramirez-Escudero, M., Lafraya, A., Gonzalez, B., Marin-Navarro, J.,
Polaina, J. & Sanz-Aparicio, J. (2013). Three-dimensional structure of Saccharomyces
invertase: Role of non-catalytic domain in oligomerization and substrate specificity. The
Journal of Biological Chemistry 288(14), 9755-9766.

[2] Marques, W.L. Raghavendran, V., Stambuk, B.U. & Gombert, A.K. (2015). Sucrose and
Saccharomyces cerevisiae: A relationship most sweet. FEMS Yeast Research 16(1).

[3] Bhagavan, N. V. (2002). Enzymes I: General Properties, Kinetics, and Inhibition. Medical
Biochemistry, 85–108. doi:10.1016/b978-012095440-7/50008-1

[4] https://en.wikibooks.org/wiki/Structural_Biochemistry/Enzyme/Michaelis_and_Menten_
Equation

45
 Have you ever wondered by fat soluble vitamins such as Vitamin A and Vitamin E are
classified as antioxidants? If you try to analyze the chemical structures of these two fat-solugle
vitamins, they bear similarities to the structure of fatty acids and simple esters. Take a look at the
structures in Figure 1.

3
1

2 4

One of the popular procedures performed in studying the antioxidant property of materials is
through the use of the α,α-diphenyl-β-picrylhydrazyl (DPPH) radical scavenging activity assay.
This assay makes use of the stable free radical DPPH, which has a strong purple color that can
be measured spectrophotometrically. In the presence of compounds that are capable of either
transferring an electron or donating hydrogen, the DPPH will become discolored. In the literature,
the change in DPPH absorbance after the addition of a test material is often used as an index of
the antioxidant capacity of a material.1

In this activity, you will be using quantitative structure-activity relationship activity in

determining whether molecular features are liked to their in vitro activity, or property. The use of
in silico methods and quantitative structure activity relationships (QSAR) has helped in the
determination of compounds to test on biological systems. QSAR is a helpful tool for investigating
the relationship between a molecular structure and its biological activity.2 Fatty acids and esters
bear structural similarities between Vitamin A and E, but will these molecules exhibit in vitro radical
scavenging activity as well?

46
At the end of the laboratory exercise, students should be able to:
 Perform in silico analysis of molecular descriptors of selected fatty acids and simple
esters;
 Perform in vitro DPPH• radical scavenging assay using the fatty acids and fatty acid
esters;
 Construct an approximated regression model of the molecular descriptors of fatty acid and
esters with radical scavenging activity; and,
 Explain the limitations of the approximated multiple linear regression model.

 Materials:
o 96-well plate
o 1mL disposable syringe
o Micropipette
o Cuvette

 Hydrocarbon
o hexane
o hexadecane

 Fatty Acids:
o lauric acid
o oleic acid
o linoleic acid

 Esters:
o methyl laurate
o methyl linoleate
o ethyl linoleate

 Standard radical scavengers

o α-tocopherol
o butylated hydroxytoluene

 Other reagents:
o DPPH reagent
o ethanol
o dimethylsulfoxide (DMSO)

 Desktop computer or laptop

 Spectrophotometer
 Flatbed Scanner

 MolView, ChemDes, Microsoft Excel

47
1. In your desktop computer or laptop, go to http://molview.org/ to obtain the molecular
descriptors of the fatty acids and fatty acid esters.

2. Using the vertical tools, draw the structure of lauric acid, which is one of the fatty acids
that you will study in this activity.

3. Once you have drawn the molecular structure, click on “Tools.” Click on “Information Card”
to determine basic information about the molecule.

48
4. Copy the canonical SMILES of the fatty acid. The abbreviation SMILES mean Simplified
Molecular Input Line Entry System.

5. Do the same procedure for the following fatty acids and simple esters.

oleic acid

linoleic acid

methyl laurate

methyl oleate

ethyl linoleate

α-tocopherol

49
 hexadecane

hexane

butylated hydroxytoluene

6. Go to http://www.scbdd.com/chemdes/ and sign in using the given public user name

and password.

50
7. Choose the molecular descriptors that you will use by clicking on “Web Server.” Click
“Custom Computation”. Under the options, choose “Molecular Properties (6).”

8. Insert the canonical SMILES under on the pointed box (red arrow) then click “Submit.” The
six molecular descriptors will be displayed.

9. Do the same steps for the other fatty acids and fatty acid esters. Copy the properties you
have obtained and save to your desktop.

Fatty Acid Molecular Descriptors

TPSA LogP2 Hy UI LogP MR
lauric acid
oleic acid
linoleic acid
methyl laurate
methyl oleate
ethyl linoleate

51
1. Prepare a 2 mL of 10.0 mg/mL stock solution of each of the fatty acids, esters and other
standards in this activity using dimethylsulfoxide as your solvent. (NOTE: This could
already be prepared prior to this step to save time.)

2. Prepare 1 mL each of the diluted solutions from the stock solution through serial dilution.
You should prepare the following concentrations:
5.0 mg/mL
2.5 mg/mL
1.25 mg/mL
0.63 mg/mL
0.32 mg/mL
0.16 mg/mL
0.08 mg/mL
0.04 mg/mL

3. In separate clean test tubes, mix 0.1 mL of each of the sample solutions with 0.4 mL of
DPPH solution in a test tube. For the control, add 0.1mL of DMSO instead of the samples.

4. Keep the test tubes in the dark for 30 minutes, preferably at room temperature.

5. Dispense 0.1 mL of the mixtures into 96 well plates.

6. Scan your image using a flatbed scanner then analyze the image using ImageJ.

7. Calculate the % DPPH radical scavenging activity using the formula:

𝐀𝐃𝐏𝐏𝐇 − 𝐀𝐬𝐚𝐦𝐩𝐥𝐞
% 𝐃𝐏𝐏𝐇 𝐑𝐒𝐀 = ∗ 𝟏𝟎𝟎
𝐀𝐃𝐏𝐏𝐇

Where ADPPH is the –log (mean gray value/255) of the control while Asample is the –log(mean
gray value/255) of the sample fatty acid, ester and other standards.

8. Perform the DPPH radical scavenging activity assay in three replicates. Record the mean
% inhibition of each of the potential inhibitors.

9. The % inhibition will be used for constructing your linear regression equation in the next
section.

52
1. Open a Microsoft Excel worksheet on your desktop computer or laptop.

2. Perform a correlation analysis between the % inhibition and the molecular descriptors you
have obtained in ChemDes.

To perform correlation analysis of variables, open a Microsoft Excel

worksheet.

In two columns, encode your data. In the example below, I have two
variables: Variable A and Variable B.

In one cell, write “=CORREL, then fill up the data in array 1 and array 2.
In the example below, Variable A is assigned to Array 1 while Variable B
is assigned to Array 2. Usually, the variables in the x-axis corresponds to
Variable A.

53
 Press “Enter” tab after completing the command. The correlation coefficient
(R) will be displayed. In the example below, the correlation coefficient is
0.9985.

3. From these data, choose three to molecular descriptors/ charge descriptors which has the
highest correlation with the % inhibition. Refer to the glossary of terms to be familiarized
with the descriptors.

4. Open a new Excel worksheet. Perform a correlation analysis among the molecular
descriptors. If one of the molecular descriptors shows very weak correlation with the other
molecular descriptors, you choose another molecular descriptor to replace your first
choice.

Activity MD1 MD2 MD3

Activity 1
MD1 - 1
MD2 - - 1
MD = Molecular Descriptor

54
5. Click on “Data” tab. Then click on “Data Analysis.”

6. On the choices of statistical tests in the “Data Analysis” box, choose “Regression.”

7. Once you are in the “Regression” box, fill up all data with the following:
Input Y range: Highlight the data and label under “IC50.” This is the activity we are
studying.

Input X range: Highlight all data and label of the properties you have chosen to include
in the equation.

55
8. Tick on the “labels” to avoid confusing the beta coefficients.

9. Tick on “New Worksheet Ply” and click “OK.” The result of the linear regression will appear
on a new MS Excel worksheet. Evaluate the ρ value (Significance F).

If the Significance F value is <0.05, then your model is adequate. Then, look at the values
of the coefficients. These are the β coefficients of your multiple linear regression.

If the value of Significance F value is not <0.05, you have to revise your model. This will
entail a lot of patience from you so do your best!

56
 The general form of a multiple linear regression is:

Y = β0 + β1X1 + β2X2 …+ βnXn + C

Where:
β0 is the value of Y if when all independent variables are zero
β1 to βn are the beta coefficients
X1 to Xn are the independent variables
C is the constant

The beta coefficients give you an idea how much of variable X contributes
to Y if all other variables are held constant.

Interpretation:
For example,

Activity = 12.56 + 2.55TPSA + 0.68 MR - 0.25Hy + C

Assume that ρ<0.05. (Statistically significant)

If all other factors are zero, Y = 12.56. If TPSA increases by 1, Activity

increases by 2.55 units. If Activity increases by 1, Activity increases by 0.68
unit. Lastly, if Hy increases by 1 unit, Activity decreases by 0.25 unit.

NOTE: You need at least 5 molecules per molecular descriptor to be

included in your equation. It means that you will be using a simple linear
regression if there are 5 to 9 molecules only.

10. Evaluate the statistical significance of the linear regression equation. If the p value is
greater than 0.05, you have to remove or add independent variables (X variables) until the
ρ value is less than 0.05.

Write your final multiple linear regression equation and the p value below.

Multiple Linear Regression Equation: p value:

Interpret your generated multiple linear regression equation. Relate your

linear regression equation to in vitro radical scavenging activity. Present
the limitations of your proposed equation based on your in silico analysis.

57
How does Orlistat inhibit the activity of porcine pancreatic lipase?
Illustrate the inhibitory mechanism of Orlistat on the active site of
pancreatic lipase.

What is the role of lipase inhibition in the management of

cardiovascular diseases?

Create an argument based on the statement below using the Toulmin

Argumentation Pattern format.

Pi bonds are the main cause of radical scavenging activities of

hydrophobic molecules.

58
[1] Karamac, M., R. Amarowicz, S. Weidner, S. Abe, F. Shahidi. Antioxidant activity of rye
caryopses and embryo extracts. Czechoslovakian Journal of Food Science 20(6):209 – 214,
2002.

[2] Razo-Hernandez, Pineda-Urbina, K. Velazco-Medem, M.A., Villanueva-Garcia, M., Sumaya-

Martinez, M.T., Martinez-Martinez, F.J. & Gomez-Sandoval, Z. (2014). QSAR study of the
DPPH radical scavenging activity of coumarin derivatives and xanthine oxidase inhibition by
molecular docking. Central European Journal of Chemistry 12(10), 1067-1080. DOI:
10.2478/s11532-014-0555-x

[3] http://www.dmstat1.com/res/TheCorrelationCoefficientDefined.html

59
Steroid molecules have several functions in the body. In fact, sex hormones such as testosterone,
estrogen and progesterone are all derived from a common steroid precursor. The enzyme 17β-
hydroxysteroid dehydrogenase type 1 (17β-HSD Type 1) is a 34.9 kDa protein which catalyzes
the interconversion of the oxidized and reduced form of estrogenic and androgenic steroids at the
17-position [1]. 17β-HSD1 expression positively correlates to estrone activation and proliferation
of breast cancer cells.[2] In Figure 1, notice that the 17th position in the steroid ring in estrogen (17β-
estradiol) and testosterone is a hydroxyl functional group, while in estrone, the 17th position is a
keto- functional group.

The 17β-hydroxysteroid dehydrogenase type 1 enzyme was thought to be related to sustain

high levels of estrogen [3] since it contributes to the conversion of estrone into biologically active
estradiol. Inhibition of this enzyme is aimed to decrease estradiol levels in postmenopausal
women, thereby inhibiting the growth and development of estrogen-dependent tumors. The active
site of the enzyme is shown in Figure 1, based on the interaction of the substrate to the active
site of the enzyme.[4]

60
 In this laboratory activity, you will be investigating molecular properties which are relevant in
inhibiting the activity of modified steroid molecules using an in silico method. The in silico method
in this activity will be focused on using MolView for you to observe molecular properties of the
selected modified steroids and ChemDes to obtain the molecular descriptors of the selected
molecules. Good luck!

At the end of the activity, students should be able to:

 Construct an approximated multiple linear regression model of the structure-activity
relationship of E-ring modified steroids and their Inhibitory activity against 17β-
Hydroxysteroid Dehydrogenase Type 1;
 Discuss part-whole relationships related to the structureof E-ring modified steroids and
their inhibitory property against 17β-Hydroxysteroid Dehydrogenase Type 1
 Interpret the approximated linear regression equation for the structure-activity relationship
of the selected E-ring modified steroids; and,
 Discuss the limitations of the linear regression model.

 Desktop computer or laptop

 MolView, ChemDes, Microsoft Excel

1. In your desktop computer or laptop, go to http://molview.org/ to obtain the molecular

descriptors of the selected modified steroid molecules.

2. Draw structure of the modified steroid shown below. Assign this molecule as “Modified
Steroid 1.”

61
3. Once you have drawn the molecular structure, click on “Tools.” Click on “Information card”
to determine basic information about the molecule.

4. Copy the canonical Simplified Molecular Input Line Entry System (SMILES) displayed on
the information card.

62
5. Do the same for the following modified steroid molecules.

Modified Steroid 2 Modified Steroid 3

Modified Steroid 4 Modified Steroid 5

Modified Steroid 6 Modified Steroid 7

Modified Steroid 8 Modified Steroid 9

Modified Steroid 10

63
6. List all canonical SMILES of the phenylalanine derivatives here. You will use the canonical
SMILES to determine the molecular descriptors in ChemDes.

Modified Steroid Canonical SMILES

Modified Steroid 1
Modified Steroid 2
Modified Steroid 3
Modified Steroid 4
Modified Steroid 5
Modified Steroid 6
Modified Steroid 7
Modified Steroid 8
Modified Steroid 9
Modified Steroid 10

7. Go to http://www.scbdd.com/chemdes/ and sign in using the given public user name

and password.

64
8. Choose the molecular descriptors that you will use by clicking on “Web Server” then click
“Custom Computation”.

9. Select “Charge (25)” among the options. Insert the canonical SMILES under “Input your
SMILES” then click “Submit.” The result will present 25 data. Choose the following data
pertaining to Tpc, Tnc and LDI.

65
10. Go back to the “Web Server” then click “Custom Computation”.

11. This time, choose “Molecular Properties (6)” among the options. Obtain the following data:
TPSA, logP2, Hy, UI, logP and MR. Refer to the glossary to be familiarized with these
molecular descriptors.

12. Copy the properties you have obtained to the table below.

Data from ChemDes

Molecular Descriptors Charge
Modified Steroid Activity

TPSA

logP2

logP
(IC50 μM)

Tpc

Tnc

LDI
MR
Hy

UI
Modified Steroid 1
Modified Steroid 2
Modified Steroid 3
Modified Steroid 4
Modified Steroid 5
Modified Steroid 6
Modified Steroid 7
Modified Steroid 8
Modified Steroid 9
Modified Steroid 10
LEGEND: TPSA = Topological Polarity Surface Area, logP2 = Square of logP value based on the Crippen method, Hy =
Hydrophilic Index, UI = Unsaturation Index, logP = logP based on Crippen method, MR = Molar Refractivity, Tpc = total of positive
charges, Tnc= total of negative charges, LDI = local dipole index

13. Your laboratory instructor will give the data on the IC50 (µM) of the modified steroid
molecules in this exercise. Obtain the IC50 data after submitting your completed table of
data from ChemDes.

1. Copy the data on the IC50 of the modified steroid molecules in this exercise.

2. Perform a correlation analysis between the inhibiting activities and the chosen ChemDes
molecular descriptors. Choose five of any of the molecular descriptors or charge
descriptors from the data you gathered in ChemDes. Explain your reasons for choosing
these data.

Rationale:

66
3. Open a new Excel worksheet. Perform a correlation analysis among the chosen ChemDes
data using the table format like the one shown below.

Activity P1 P2 P3 P4 P5
Activity 1
P1 - 1
P2 - - 1
P3 - - - 1
P4 - - - - 1
P5 - - - - - 1
P = Property

4. Open a new Excel worksheet and “Data” tab. Then click on “Data Analysis.” On the
choices of statistical tests in the “Data Analysis” box, choose “Regression.”

5. Once you are in the “Regression” box, fill up all data with the following:
Input Y range: Highlight the data and label under “Activity.”
Input X range: Highlight all data and label of the properties you have chosen to include
in the equation.

6. Tick on the labels to avoid confusing the beta coefficients. Tick on New Worksheet Ply
and click “OK.” The result of the linear regression will appear on a new MS Excel
worksheet.

7. Evaluate the statistical significance of the linear regression equation. Write your multiple
linear regression equation and the p value below.

Multiple Linear Regression Equation: p value:

67
Why should the modified steroid molecules be based on the structure
of estradiol instead of cholesterol?

What will happen to the affinity of a steroid molecule to the enzyme if

the hydroxyl group on the 17th position of the molecule is changed to
a carboxylic acid group?

Using Toulmin Argumentation Pattern, create an argument to answer

the statement below:

Benzopyrene, a carcinogen, can also inhibit the enzyme 17β-

hydroxysteroid dehydrogenase type 1.

68
[1] Fischer, D.S., Allan, G.M., Bubert, C., Vicker, N., Smith, A., Tutill, H.J., Purohit, A., Wood,
L., Packham, G., Mahon, M.F., Reed, M.J. & Potter, B.V.L. (2005). E-ring modified steroids
as novel potent inhibitors of 17β-hydroxysteroid dehydrogenase type 1. Journal of
Medicinal Chemistry 48, 5479 – 5770.

[2] He, W., Gauri, M., Li, T., Wang, R. & Lin, S-X. (2015). Current knowledge of the
multifunctional 17β-hydroxysteroid dehydrogenase type 1 (HSD17B1). Gene. DOI:
10.1016/j.gene.2016.04.031

[3] Miyoshi, Y., Ando, Ando, A., Shiba, E., Taguchi, T., Tamaki, Y. & Noguchi, S. (2001).
Involvement of up-regulation of 17β-hydroxysteroid dehydrogenase type 1 in maintenance
of intratumoral high estradiol levels in postmenopausal breast cancers. International
Journal of Cancer 94, 685 – 689.

[3] Sawicki, M.W., Erman, M., Puranen, T., Vihko, P. & Ghosh, D. (1999). Structure of the
ternary complex of human 17β-hydroxysteroid dehydrogenase type 1 with 3-hydroxyestra-
1,3,5,7-tetraen-17-one (equilin) and NADP+. Proceedings of the National Academy of
Science 96, 840-845.

69
 Carbohydrates are naturally occurring molecules which have approximately the general
formula Cn(H2O)n, which implies that they are indeed hydrates of carbon. These molecules serve
as sources of energy, structural support to plants and animals, components of DNA
(deoxyribonucleic acid) and RNA (ribonucleic acid).1 Carbohydrates are also found in natural
products, blood group antigens, antibiotics, and bacterial cell wall1. These polyhydroxy molecules
play several roles in human biology and disease development and are currently applied in the
food and pharmaceutical industry2. Polysaccharides, in particular, also influence the properties
and texture of food which is currently studied to address several health issues.3

In the field of medical technology, sugars are also involved in the diagnosis of diseases and
in the determination of blood type (A, B, O or AB). Glucose is quantitatively determined in blood
or in urine to support the diagnosis of problems related to sugar metabolism, such as Diabetes
mellitus. In this activity, you will be performing quantitative tests for carbohydrates its application
to urinalysis.

At the end of the activity, students should be able to:

 Explain the chemical principles of qualitative tests for carbohydrates;
 Identify the molecular descriptors of selected monosaccharides and disaccharides which
are related to their reducing properties; and,
 Perform quantitative determination of sugar in artificial urine sample.

 Benedict’s reagent
 Monosaccharide solutions: glucose solution, galactose solution, fructose solution
 Disaccharide solutions: sucrose solution, maltose solution, lactose solution
 Glassware: cuvette, Erlenmeyer flask, burette, pipette, test tubes, volumetric flask, pH
meter

Desktop Computer
Flatbed Scanner

Image J, MolView, PubChem, ChemDes and Microsoft Excel

70
1. Go to: https://pubchem.ncbi.nlm.nih.gov/.

2. On the search bar, type the name of the selected monosaccharides and disaccharides in
this activity, but enclose the names in quotation marks.

NOTE: For this activity, you may use either the alpha- or beta- form of the saccharides. The saccharides which
will be used for quantitative Benedict’s test are: galactose, glucose, fructose, lactose, maltose and sucrose.
Since sucrose is non-reducing, you will be performing hydrolysis of the sugar prior to performing Benedict’s
Test.

3. Scroll down along the information provided by PubChem to determine the canonical
Simplified Molecular Input Line Entry System (SMILES).

71
4. List all canonical SMILES of the imino sugars here. You will use the canonical SMILES to
determine the molecular descriptors in ChemDes.

Saccharide Canonical SMILES

galactose
glucose
fructose
maltose
sucrose
lactose

1. Go to: http://www.scbdd.com/chemdes/. Sign in using the given public user name and
password.

72
2. Choose the molecular descriptors that you will use by clicking on “Web Server” then click
“Custom Computation”. For this activity, you will be using the “Charge (25)” descriptors
and “Molecular Property Descriptors (6).”

Insert the canonical SMILES on the pointed box (red arrow) then click “Submit.”

3. There are 25 data which is included in the “Charge 2D descriptors. Choose the following
data: QHmax (most positive charge on H atom), QCmax (most positive charge on C atom),
QOmin (most negative charge on O atom) and LDI (local dipole index).

For “Molecular Property Descriptors,” choose TPSA (total polar surface area), logP2
(square of logP values) and Hy (Hydrophilic Index) only.

4. Copy the properties you have obtained to the table below.

Molecular Descriptors Charge 2D Descriptors
Saccharide TPSA LogP2 Hy QHmax QCmax QOmin LDI

Galactose
Glucose
Fructose
Maltose
Sucrose
Lactose

Observe the trends in the molecular descriptors. Which information seems to have an
increasing or decreasing trend? Which data are similar? What is the possible explanation
why your data appears this way?

73
 NOTE: You will be given sample saccharides to determine whether these are
reducing or non-reducing. The saccharide solutions are: glucose, lactose, sucrose,
fructose, and starch.

1. Prepare a calibration curve of dextrose monohydrate by preparing a 3mL stock

solution with a concentration of 4 mg/mL.

2. Perform serial dilution of the stock solution to prepare 1 mL of the following

concentrations of dextrose monohydrate:

2.0 mg/mL
1.0 mg/mL
0.5 mg/mL
0.25 mg/mL
0.13 mg/mL
0.06 mg/mL
0.03 mg/mL

3. Using a clean 1-mL syringe, place 0.10 mL of DNSA reagent to eight test tubes. The
eighth test tube is assigned as tube “0.”

4. Using another clean 1-mL syringe, add 0.10 mL of each of the diluted solutions to tube
“0.”

5. Stand all 8 test tubes in a large beaker of just-boiled water for 5 to 10 minutes until the
contents have changed color (the contents of tube “10” will be dark red).

6. Using a clean glass pipette, add 3.0 mL of distilled water to each test tube.

7. Flick the tubes gently with a finger to mix the contents.

8. Determine your linear regression equation by using image analysis. Express the
concentration as mg/mL dextrose monohydrate equivalents.

9. Obtain three test tubes and place 0.1 mL of each of your sample sugar solution.

10. Using a clean 1-mL syringe, place 0.30 mL of DNSA reagent.

11. Place the tube in a boiling water bath. The positive result for a reducing sugar is the
appearance of a red/ reddish brown color.

12. Dilute with 1.0 mL of distilled water.

74
13. Dispense 0.1mL of the resulting mixtures to 96-well plates. Again, if the samples are
too dark, you need to dilute them accordingly with distilled water. (TAKE NOTE OF
YOUR DILUTION FACTORS!)

14. Obtain the mean and standard deviation of your data.

Which of the saccharides are reducing? Which are non-reducing? Why is this so?

1. Pipette 10 mL of Benedict’s solution into a 125 mL Erlenmeyer flask.

2. Add 1 gram of anhydrous sodium carbonate and several boiling stones to the flask.

3. Shake the mixture well to suspend the sodium carbonate.

4. Place the flask on a hot plate. Heat the contents in the flask to boiling.

NOTE: Keep the contents of the Erlenmeyer flask boiling steadily while performing the
activity. If the volume of the liquid in the flask is reduced, add distilled water to keep
the volume constant.

5. Add 3 mL of the sugar solution to the flask. If you are using a monosaccharide, allow
the monosaccharide solution to boil for 30 seconds. If you are testing a disaccharide,
allow the disaccharide solution to boil for 1 minute. This step is used for calibration of
the appropriate concentration of sugar solutions to be used in this procedure.

NOTE: If the blue color of the Benedict’s reagent is not removed after boiling for the
prescribed amount of time, add another 3 mL of the sugar solution and boil again for
the prescribed amount of time. Continue this process of adding 3-mL aliquots and
boiling until the blue color disappears.

If the blue colour is completely removed after adding 6 mL of the sugar solution, dilute
a fresh sample of sugar solution in half and repeat steps 1 to 3. If the blue colour still
remains after adding 12 mL of the sugar solution, concentrate a fresh sample of sugar
solution (by heating it and allowing some water to evaporate) to half its original volume
and repeat steps 1 to 3.

When the preliminary titration requires between 6 and 12 mL in removing the

blue colour, the sugar solution is within the proper concentration range to
proceed. Note this concentration since you need a specific concentration to
determine which saccharide is present in a solution. Prepare a 100mL solution
of the saccharide with this optimized concentration, based on initial reaction
with Benedict’s Reagent.

6. Fill a burette with about 15 mL of the correctly diluted (or concentrated) sugar solution
prepared in steps 1–4 above. Clamp the burette so that it is positioned above the hot
plate.

75
7. Pipet 10 mL of Benedict’s Quantitative Solution into a 125mL Erlenmeyer flask.

8. Add 1 gram of anhydrous sodium carbonate and two boiling stones to the flask. Shake
the mixture well to suspend the sodium carbonate.

9. Place the flask on a hot plate situated underneath the buret. Heat the contents in the
flask to boiling.

10. Perform titration of the Benedict’s Quantitative Solution in the flask with the sugar
solution in the burette. The endpoint of this titration occurs when the blue colour
disappears from the solution in the flask.

NOTE: Keep the contents of the flask boiling steadily throughout the experiment. If the
volume of the flask is reduced, add distilled water to keep the volume constant.

11. Calculate the concentration of the unknown sugar solution using the following
procedure and conversion factors then find your sugar in the table below.

Note the mass in grams of sugar required to completely reduce 10 mL of Benedict’s

Quantitative Solution. This is the number of grams of sugar that was titrated into the
10-mL sample of Benedict’s Quantitative Solution before reaching the endpoint.

Sugar No. of grams required to reduce 10mL of

Benedict’s Solution
glucose 0.0200 g
galactose 0.0200 g
fructose 0.0202 g
lactose 0.0271 g
lactose 0.0282 g
sucrose 0.0196 g

12. This time, prepare 2mg/mL solution of all the sugars. Record the time it requires for
the sugars to completely decolorize Benedict’s Quantitative Solution. Record your
results in the table below:
Sugar Time Required to Completely
Decolourize 10mL of Benedict’s
Quantitative Solution (minutes)
glucose
galactose
fructose
lactose
maltose
sucrose

76
1. Open a Microsoft Excel worksheet. Perform a correlation analysis between the
molecular descriptors of the sugars and the time it requires to completely decolourize
Benedicts’s Quantitative Solution.

2. Open a new Excel worksheet. Perform a correlation analysis among the molecular
descriptors. If one of the molecular descriptors shows very weak correlation with the
other molecular descriptors, you choose another molecular descriptor to replace your
first choice.

Activity MD1
Activity 1
MD1 - 1
MD2 - -
MD3 - -
MD = Molecular Descriptor

3. Click on “Data” tab. Then click on “Data Analysis.” On the choices of statistical tests in
the “Data Analysis” box, choose “Regression.”

4. Once you are in the “Regression” box, fill up all data with the following:
Input Y range: Highlight the data and label under “Activity.”
Input X range: Highlight all data and label of the properties you have chosen to include
in the equation.

5. Tick on the “labels” to avoid confusing the beta coefficients.

6. Tick on “New Worksheet Ply” and click “OK.” The result of the linear regression will
appear on a new MS Excel worksheet.

7. Evaluate the statistical significance of the linear regression equation. If the p value is
greater than 0.05, you have to remove or add independent variables (X variables) until
the p value is less than 0.05.

Please do note that you only have six molecules in this activity so your model should
be a simple linear regression only.

Write your final linear regression equation and the p value below. Interpret your
equation.

Linear Regression Equation: p value:

77
Is the quantitative Benedict’s test a reliable method to diagnose
Diabetes mellitus using urine samples? Why or why not?

Is pH an important factor to consider when studying the properties of

saccharides? Why or why not?

The type of glycosidic bond influences the reducing property of

saccharides.

Create an argument about the statement above using the Toulmin

Argumentation Pattern.

78
[1] Lichtenthaler, F.W. (2010). Carbohydrates: Occurrence, structures and chemistry. Wiley-VCH
Verlag GmbH & Co. KGaA, Weinheim: 1-30.

[2] Hayes, M. & Tiwari, B.K. (2015). Bioactive carbohydrates and peptides in foods: An overview
of sources, downstream processing steps and associated bioactivities. International Journal
of Molecular Science 16, 22485-22508.

[3] Lovegrove, A., Edwards, C.H., De Noni, I., Patel, H., El, S.N., Grassby, T., Zielke, C., Ulmius,
M., Nilsson, L., Butterworth, P.J., Ellis, P.R. & Shewry, P.R. (2017). Role of polysaccharides
in food, digestion and health. Critical Reviews in Food Science and Nutrition 57(2), 237-253.

79
 The α-amylase (AAMY) is an enzyme present in microorganisms and in tissues from animals
and plants. This enzyme catalyzes the hydrolysis of a-1,4-glycosidic bonds in glycogen- and
starch-related polysaccharides (such as the mainly linear amylose and the branched
amylopectin), and some oligosaccharides. The enzyme liberates reducing sugars such as α-
maltose, α-glucose, and α-limit dextrins in a stepwise fashion. In mammals, AAMY can be found
in saliva and pancreatic secretions. Ptyalin, the AAMY isoform present in human saliva, is a
metalloenzyme and requires calcium ions for function. The optimum conditions for ptyalin activity
are a pH range of 5.6–6.9, a temperature of 37oC, and the presence of certain anions and
activators, such as chloride, bromide, and iodide.1 the amount of ptyalin differs in people of
different ethnic backgrounds.

Amylase inhibition has currently been recognized as a possible management for diabetes
mellitus, a disorder on sugar metabolism. Diabetic mellitus (DM) is a condition arising due to
abnormal metabolism of carbohydrate which could be caused by insulin deficiency and/or insulin
resistance. This disorder prevails worldwide with its occurrence increasing at an alarming rate
globally and locally. Various complications encompass all the vital organs of the body as a
consequence of the metabolic derangement in diabetes. At present, the treatment of DM is based
on parenteral insulin and oral antidiabetic drugs. Oral hypoglycemic agents include
sulphonylureas, biguanides, and other drugs like acarbose are also used to regulate blood sugar
among diabetic patients.2

The principle of AAMY inhibition is based on decreasing post-prandial (after meal) sugar levels
in the blood. Absorption of glucose can be delayed by reducing the rate of digestion of starch.
Inhibition of the mammalian alpha amylase enzyme in the intestine would delay the degradation
of starch and oligosaccharides to monosaccharides before they can be absorbed. This would
decrease the absorption of glucose and consequently reduce postprandial blood glucose level.
Therefore, screening of alpha-amylase inhibitors in medicinal plants has received much
attention.2 In this activity, you will be investigating the relationship the structural features of
metabolites which are derived from plants, and the inhibition of α-amylase.

At the end of the activity, students should be able to:

 Perform in silico analysis of molecular descriptors of selected metabolites;
 Perform in vitro amylase inhibition assay using the selected metabolites;
 Determine the correlation of molecular descriptors and amylase inhibition of the
metabolites;
 Construct an approximated regression model of the structure of metabolites with amylase
inhibition; and,
 Explain the limitations of the approximated multiple linear regression model.

80
 Gallic acid  Agar  Phosphate
 Quercetin  Starch solution Buffered Solution
 Cinchonine  Potassium iodide  NaOH and HCl for
 Propyl gallate solution pH adjustment
 Acarbose Dinitrosalicylic acid  Alpha amylase
 Ethanol (DNSA) reagent

 MolView, ChemDes and Microsoft Excel

1. In separate test tubes, prepare a 1000 µg/mL stock solution of each of the following
metabolites:
A – gallic acid I = gallic acid + caffeine
B – quercetin J – quercetin + cinchonine
C – cinchocine K – quercetin + propyl gallate
D – propyl gallate L – quercetin + caffeine
E – caffeine M – cinchonine + propyl gallate
F – gallic acid + quercetin N – cinchonine + caffeine
G = gallic acid + cinchonine O – propyl gallate + caffeine
H = gallic acid + propyl gallate P – Acarbose

2. Aliquot your stock solutions into 500 µg/mL, 250µg/mL, 125µg/mL, 62.5µg/mL and
31.25µg/mL into separate test tubes. You will be testing all of these concentrations to
determine the IC50 in the amylase inhibition assay (Session 3).

3. If the stock solutions are prepared beforehand, place them in a refrigerator at least 18 to
24 hours prior to use. However, it is advised that stock solutions need to be prepared
fresh prior to the assay.

1. Prepare a gel (for electrophoresis) consisting of 1% agar in 0.4M phosphate buffer (pH
7.5).

2. Preincubate each of the metabolites (31.25 µg/mL to 1000 µg/mL) with the enzyme for 15
minutes at 36oC.

3. Load the preincubated metabolite-enzyme mixtures in to different wells. Untreated

enzyme served as a positive control in a separate well.

4. Load the buffer for preparing the gel in the electrode compartments.

5. Apply a stabilized current of 100V through the gel for 2 hours at 4◦C.

81
6. For visualization of the amylase bands, immerse the gels in 0.5% soluble starch and
incubated at 37◦C for 30 min.

7. Wash the excess starch using phosphate buffer solution.

8. Flood the washed gel with iodide potassium iodide solution for 1 minute. Colorless bands
against a deep blue background indicated amylase activity.

1. Using 2mL Eppendorf tubes, mix 100 μL of the secondary metabolites in Section 1 with
200 μL of α- amylase enzyme and 100 μL of 2mM of phosphate buffer solution (pH-6.9).

2. Incubate the mixture for 20-minutes at 36oC.

3. After incubation, add 100 μL of 1% starch solution. Do the same for the controls where
200 μL of the enzyme was replaced by buffer.

4. After incubation for 5 minutes, add 500 μL of dinitrosalicylic acid reagent to both control
and test.

5. Immerse the tubes containing the mixtures in a boiling water bath for 5min.

6. Cool the tubes.

7. Record the absorbance at 540nm using spectrophotometer. You may also use image
analysis for this part of the activity. Please refer to Activity 2 for the steps.

8. Compute for percentage inhibition of α-amylase enzyme using the formula:

𝐜𝐨𝐧𝐭𝐫𝐨𝐥 − 𝐭𝐞𝐬𝐭
𝐈𝐧𝐡𝐢𝐛𝐢𝐭𝐢𝐨𝐧 (%) = 𝟏𝟎𝟎 ∗
𝐜𝐨𝐧𝐭𝐫𝐨𝐥

9. Plot the graph and determine the IC50 of each of the metabolites and combinations.

1. In your desktop computer or laptop, go to http://molview.org/ to obtain the molecular

descriptors of the fatty acids and fatty acid esters.

2. Using the vertical tools, draw the structure of each of the metabolites, and Acarbose.

3. Once you have drawn the molecular structure, click on “Tools.” Click on “Information Card”
to determine basic information about the molecule.

4. Copy the canonical SMILES of the fatty acid. The abbreviation SMILES mean Simplified
Molecular Input Line Entry System.

82
5. Go to http://www.scbdd.com/chemdes/ and sign in using the given public user name
and password.

6. Choose the molecular descriptors that you will use by clicking on “Web Server.” Click
“Custom Computation”. Under the options, choose “Molecular Properties (6).”

7. Do the same steps for the other metabolites. Copy the properties you have obtained and
save to your desktop.

Compound Molecular Descriptors

TPSA LogP2 Hy UI LogP MR

Gallic acid

Quercetin

Cinchonine

Propyl gallate

Caffeine

Acarbose

83
1. Open a Microsoft Excel worksheet in your desktop computer or laptop.

2. Perform a correlation analysis between the % inhibition and the molecular descriptors you
have obtained in ChemDes. From these data, choose three to molecular descriptors which
has the highest correlation with the % inhibition.

3. Open a new Excel worksheet. Perform a correlation analysis among the molecular
descriptors. If one of the molecular descriptors shows very weak correlation with the other
molecular descriptors, you choose another molecular descriptor to replace your first
choice.

Activity MD1 MD2 MD3

Activity 1
MD1 - 1
MD2 - - 1
MD3 - - - 1
MD = Molecular Descriptor

4. Click on “Data” tab. Then click on “Data Analysis.” On the choices of statistical tests in the
“Data Analysis” box, choose “Regression.”

5. Once you are in the “Regression” box, fill up all data with the following:
Input Y range: Highlight the data and label under “Activity.”
Input X range: Highlight all data and label of the properties you have chosen to include
in the equation.

6. Tick on the “labels” to avoid confusing the beta coefficients.

7. Tick on “New Worksheet Ply” and click “OK.” The result of the linear regression will appear
on a new MS Excel worksheet.

8. Evaluate the statistical significance of the linear regression equation. If the p value is
greater than 0.05, you have to remove or add independent variables (X variables) until the
p value is less than 0.05.

Write your final multiple linear regression equation and the p value below. Interpret your
equation.

Multiple Linear Regression Equation: p value:

84
 How does Acarbose inhibit the activity of alpha amylase? Illustrate the
inhibitory mechanism of Acarbose on the active site of alpha amylase.

Interpret your proposed linear regression model. What are limitations

of the proposed regression model?

Hydrogen bonding is an important intermolecular interaction to

promote enzyme-substrate interaction among metabolites and the
enzyme’s active site.

Create an argument about the statement above using the Toulmin

Argumentation Pattern.

[1] Valls, C., Rojas, C., Pujadas, G., Garcia-Vallve, & Mulero, M. (2012). Characterization of
the activity and stability of amylase from saliva and detergent. Biochemistry and Molecular
Biology Education 40(4), 254-265.

[2] Sangeetha, R. & Vedasree, N. (2012). In vitro α-amylase inhibitory activity of the leaves of
Thespesia populnea. Internation Scholarly Research Network. DOI:10.5402/2012/515634

85
 The extraction of DNA and gel electrophoresis are among the skills that students need to learn
in biochemistry. DNA extraction allows students to relate an abstract idea to a tangible product
and gain a better understanding of the DNA molecule.1 These laboratory techniques are
widespread, but the equipment and materials needed for their performance are invariably
expensive, often out of the reach of typical laboratory classes. In addition, some of the reagents
are too hazardous for these settings.

The stages involved in extracting DNA from cells can be categorized as: cell harvesting, lysis,
protein digestion, and precipitation. The major variances in previously described protocols are in
the cell harvesting stage. The stages that follow cell harvesting are fairly uniform throughout the
literature. In this activity, you will be perfoming two methods using the conventional method and
using household materials. Along this activity, try to design your own method to modify the
procedures and evaluate if they work or not.1

A gel electrophoresis system that can be constructed and run using only safe household
materials widely available from national hardware, pet supply, pharmacy, and supermarket
chains. Previously reported alternative electrophoretic systems typically use a mixture of
household and laboratory materials. Although the quality of DNA gel electrophoresis and
visualization using safe household materials does not rival that reached with research-quality
materials, the gels are relatively inexpensive, easy to perform, and instructive in simple laboratory
settings.2

At the end of the activity, students should be able to:

 Discuss the principles of DNA extraction and gel electrophoresis
 Perform DNA extraction and gel electrophoresis

 0.2M lithium acetate in 1% SDS  70% ethanol

solution  Eppendorf tubes
 96%-100% ethanol

 1M Tris-Acetate-EDTA buffer
 Agar

86
  6 grams NaCl  Liquid detergent (Breeze or any
 120 g green peas or 150 grams liquid detergent)
chicken liver  Cold 70% ethyl alcohol
 120 mL cold distilled water  Clean wooden splinter
 Enzyme powder or meat  Clean cheesecloth or strainer
tenderizer

 Electrophoresis chamber:  Distilled water

rectangular-shaped plastic  2 alligator clips
container with flat base  Aluminium foils (1 cm wide)
 Electricity source: 5 pieces of 9-  Cast or Petri Dish
volt batteries  Scalpel
 Stainless steel wire – 20 gauge  Double clip
 A piece of art foam, (4 x 4 in)  Green food coloring
 Agarose powder  Eppendorf tube
 Sodium bicarbonate

This method was adapted from the protocol described by Looke et al (2011).

1. Pick one yeast colony from a plate or spin down 100-200 µL of liquid yeast culture
(OD600=0.4).

2. Suspend the cells in 100 µL of 200 mM LiOAc, 1% SDS solution.

3. Incubate for 5 minutes at 70oC.

4. Add 300 µL of 96% ethanol then vortex for 5 minutes.

5. Spin down DNA and cell debris at 15000 rpm for 3 minutes.

6. Wash pellet with 70% ethanol.

7. Dissolve pellet in 100µL of water or TE buffer.

8. Spin down cell debris gor 15 seconds at 15000rpm.

9. Use 1.0 µL of supernatant for agarose gel electrophoresis.

87
Prepare 1L of 1xTAE buffer. You will be using this as your running buffer and in preparing the
agar.

1. Tape the sides of the casting gel so that it will hold the gel.

2. Pour 100mL of 1x TAE buffer into a clean 250mL flask. Add 1 gram of agarose to make
1% agarose gel.

3. Microwave until the solution is clear. As much as possible, microwave in 10 second pulses.

4. If the solution is clear and fully liquefied, bring to your bench top and let it cool to about
60oC. You can already hold the flask in this temperature.

5. When you can already hold the container, pour the agarose solution in a cast, with the
well comb in place. The wells should be deep, with a few millimetres of gel remaining at
the bottom.

6. Let the solution to sit for 20 to 30 minutes or until the agarose gel has solidified.

7. Place the gel into the tank.

8. Pour the TAE buffer into the tank until the gel is covered.

9. Add 1.0 µL of loading dye per 5.0 µL of DNA sample. Gel loading dye is typically made at
6X concentration (0.25% bromphenol blue and 30% glycerol). It helps to track how far the
DNA sample has travelled and allows the sample to sink into the gel.

10. Program the power supply to the desired voltage (1-5V/cm between electrodes).

11. Add enough running buffer to cover the surface of the gel.

12. Attach the leads of the gel box to the power supply. Turn on the power supply and verify
that both gel box and power supply are working.

13. Remove the lid. Slowly and carefully load the DNA samples into the gel. An appropriate
DNA size marker should always be loaded along with the experimental samples.

14. Replace the lid to the gel box. Double check that the electrodes are plugged into the
correct slots in the power supply.

15. Turn on the power. Run the gel until the dye has migrated to an appropriate distance.

16. Dilute DNA-dye methylene blue 200X concentrate down to 1X (250 µL DNA-dye
methylene blue + 50 mL water).

17. Trim down the agarose gel to the smallest area possible.

88
18. Incubate the gel (dimension approximately 80 x 60 mm) with 50 mL methylene blue for
about 20 minutes. The staining solution should cover the gel to a depth of about 5 mm.

19. Destain repeatedly in water for 5 to 10 minutes.

1. In a blender, mix 120 grams of green peas, 6 grams of NaCl and 120mL of cold water.
Blend for at least 15 seconds until the mixture resembles a soup.

2. Pour the soupy mixture through a strainer to an Erlenmeyer flask. This is your aqueous
solution.

3. Add 30mL of liquid detergent to the aqueous solution. Gently mix then leave it for 5 to 10
minutes.

4. Divide the aqueous solutions into three test tubes.

5. Add 0.36 gram of meat tenderizer and stir gently.

6. After 3 minutes, add the cold alcohol solution (70% ethyl alcohol).

7. Look for a white, stringy stuff where the alcohol layer and aqueous layer meet. This is
your DNA sample.

8. Collect the DNA sample using a clean wooden splinter. Transfer the DNA sample in test
tubes with alcohol.

1. Prepare a 1% agarose gel solution by mixing 1 gram of agarose in 100mL of the buffer
solution (may be TAE buffer).

2. Place the mixture in a microwave for 1 to 3 minutes to dissolve the agarose in the buffer.
Microwave the solution in pulses, to avoid losing your buffer solution.

3. When you can already hold the container, pour the agarose solution in a cast, with the
well comb in place. The wells should be deep, with a few millimetres of gel remaining at
the bottom.

4. Let the solution to sit for 20 to 30 minutes or until the agarose gel has solidified.

89
1. Prepare the gel electrophoresis tank by placing the aluminium foils on the bottom of the
plastic container (rectangular plastic container with flat base). Place the solid agarose gel
at the center of the gel electrophoresis tank.

2. Fill up the gel electrophoresis tank with the same buffer you used to prepare your gel until
the gel is completely immersed.

3. Mix 1 drop of glycerol and 2 drops of green food coloring in an Eppendorf test tube. Add
the genomic DNA sample with glycerol-food coloring solution in a 1:1 ratio using a pipetter.
You will need a very small sample for electrophoresis, so you have to estimate the amount.

4. Load 10 µL of the DNA-dye solution to the wells.

5. Attach the aluminium foils to the battery using the alligator clips.

6. Always remember to run your DNA samples towards the positive electrode. The
complete set-up looks like the one shown below (Figure 1).

7. Run the gel electrophoresis for 1 hour to 1.5 hours. However, you have to note if the dye
has already migrated to about 75% of the whole gel. This implies that the DNA sample
has been separated.

8. Remove the gel and allow it to dry. Immerse the gel in 10000x diluted 2% crystal violet
solution (Gentian violet) for 12 to 24 hours.

9. Visualize the DNA under a bright light.

90
 Why is ethidium bromide used to help in the visualization of DNA?

Is the nucleotide in DNA or RNA the same as dietary nucleotides?

Why or why not?

Using the Toulmin Argumentation Pattern format, build an argument

on the statement below.

“An acidic chemical environment is essential for better electrophoretic

mobility of DNA during electrophoresis.”

[1] Hearn, R.P. & Arblaster, K.E. (2010). DNA extraction techniques for use in education.
Biochemistry and Molecular Biology Education 38(3), 161-166.

[2] Ens, S., Olson, A.B., Dudle, C., Ross III, N.D., Siddiqi, A.A., Umoh, K.M. & Schneegurt, M.A.
(2012). Inexpensive and safe DNA electrophoresis using household materials. Biochemistry
and Molecular Biology Education 40(3), 198-203.

91
 Nucleosides are glycosylamines that form nucleotides, which are the building block of nucleic
acids1. Structurally, nucleosides are composed of a pentose sugar (ribose, deoxyribose or D-
arabinose) and a nitrogenous base of cellular origin (adenine, guanosine, cytosine, thymine or
uracil) or from natural products (xanthine, theobromine, or caffeine). Nucleoside analogues exhibit
antiviral1,3 and anticancer2,4,5 activities. Some synthetic nucleoside analogues such as Cytarabine
and Gemcitabine were actually based from the structure of the nucleoside analogue from
Cryptotheca crypta, a marine sponge.4

The nucleoside analogues exhibit cytotoxic properties by interfering with the synthesis of
nucleic acids by cells. They can alter nucleic acids, interfere with enzymes involved in the
synthesis of nucleic acids or modifying the metabolism of physiologic nucleosides. 2 The
anticancer activities of nucleoside analogues were investigated in the laboratory experiments
were performed using cancer cell lines such as murine leukemia cells and human T-lymphocyte
cells5. This in silico activity will focus with identifying some molecular properties which could be
used to explain the cytotoxic and anticancer properties of nucleoside analogues5 which were
selected for this study.

At the end of the activity, students should be able to:

 Identify the molecular descriptors of nucleoside analogues which are related to their
cytotoxic activities;
 Construct an approximated multiple linear regression model of the structure-activity
relationship of nucleoside analogues and their cytotoxic activities; and,

 Microsoft Excel
 MolView
 ChemDes

 Desktop Computer

92
1. Go to: http://molview.org/

2. Draw structure of the nucleoside analogue shown below.

Nucleoside Analogue 1

3. Once you have drawn the molecular structure, click on “Tools.” Click on “Information card”
to determine basic information about the molecule. Please refer to Activity No. 2 for the
details of this step.

4. Copy the canonical Simplified Molecular Input Line Entry System (SMILES) displayed on
the information card.

5. Do the same for the following molecules:

Nucleoside Analogue 2 Nucleoside Analogue 3

Nucleoside Analogue 4 Nucleoside Analogue 5

93
 Nucleoside Analogue Nucleoside Analogue 7
6

Nucleoside Analogue 8 Nucleoside Analogue 9

( )2

Nucleoside Analogue 10

6. List all canonical SMILES of the nucleoside analogues here. You will use the canonical
SMILES to obtain the molecular descriptors of the nucleoside analogues in ChemDes.

Nucleoside Analogue Canonical SMILES

Nucleoside Analogue 1
Nucleoside Analogue 2
Nucleoside Analogue 3
Nucleoside Analogue 4
Nucleoside Analogue 5
Nucleoside Analogue 6
Nucleoside Analogue 7
Nucleoside Analogue 8
Nucleoside Analogue 9
Nucleoside Analogue 10

94
1. Go to: http://www.scbdd.com/chemdes/. Sign in using the given public user name and
password.

2. Choose the molecular descriptors that you will use by clicking on “Web Server.” Then click
on “Chemopy Descriptors.” Click on “Web Server again then click on “Custom
Computation”. Click on “Molecular Properties (6).”

3. Insert the canonical SMILES then click “Submit.” Copy the following molecular descriptors
from the results: TPSA (total polar surface area), Hy (hydrophilicity index) and MR (molar
refractivity).

4. Repeat step 2. This time, select “Charge” among the options. There will be 25 data which
will be given. Choose the following data only: LDI, QHmax, QCmax, QNmin, and QOmin.

5. Copy the properties you have obtained to the table below.

Data from ChemDes
Nucleoside Molecular Charge
Analogue Descriptors Activity
TPSA Hy MR LDI QHmax QCmax QOmin QOmax (log IC50-1)
Nucleoside Analogue 1
Nucleoside Analogue 2
Nucleoside Analogue 3
Nucleoside Analogue 4
Nucleoside Analogue 5
Nucleoside Analogue 6
Nucleoside Analogue 7
Nucleoside Analogue 8

TPSA = Topological Polarity Surface Area, Hy = Hydrophilic Index, MR = Molar Refractivity, LDI = local dipole index, QHmax
= most positive charge on H atom, Cmax = most positive charge on C atom, QNmin = most negative charge on N atom,
QOmin = most negative charge on O atom

95
1. Open a Microsoft Excel worksheet.

2. Perform a correlation analysis between the DPPH radical scavenging activities and the
molecular descriptors you have obtained in Part A. From these data, choose three
molecular descriptors which have the highest correlation with the activity.

3. Open a new Excel worksheet. Perform a correlation analysis among the molecular
descriptors. If one of the molecular descriptors shows very weak correlation with the other
molecular descriptors, choose another molecular descriptor to replace your first choice.
Activity MD1 MD2
Activity 1
MD1 - 1
MD2 - - 1
MD3 - - -
MD = Molecular Descriptor

4. Click on “Data” tab. Then click on “Data Analysis.” On the choices of statistical tests in the “Data
Analysis” box, choose “Regression.”

5. Once you are in the “Regression” box, fill up all data with the following:
Input Y range: Highlight the data and label under “Activity.”
Input X range: Highlight all data and label of the properties you have chosen to include
in the equation.

6. Tick on the “labels” to avoid confusing the beta coefficients.

7. Tick on “New Worksheet Ply” and click “OK.” The result of the linear regression will appear
on a new MS Excel worksheet.

8. Evaluate the statistical significance of the linear regression equation. If the p value is
greater than 0.05, you have to remove or add independent variables (X variables) until the
p value is less than 0.05.

Write your final multiple linear regression equation and the p value below. Interpret your
equation.

Multiple Linear Regression Equation: p value:

96
 What is the role and application of the concept of aromaticity to the
properties of nucleosides and nucleotides?

Using the Toulmin Argumentation Pattern format, build an argument

on the statement below.

“A nucleoside can be isolated from a nucleotide solution by using

simple extraction using hexane.”

[1] Huang, R-M., Chen, Y-N., Zeng, Z., Gao, C-H., Su, X. & Peng, Y. (2014). Marine nucleosides:
Structure, bioactivity, synthesis and biosynthesis. Marine Drugs 12I, 5817 – 5838.

[2] Galmarini, C.M., Mackey, J.R. & Dumontet, C. (2002). Nucleoside analogues and
nucleobases in cancer treatment. The Lancet Oncology 3, 415 – 424.

[3] Krecmerova, M. (2012). Nucleoside and nucleotide analogues for the treatment of herpes
virus infections: Current stage and new prospects in the field of acyclic nucleoside
phosphonates. Herpesviridae – A look into this unique family of viruses, D. George Dimitri
Magel (Ed.).

[4] Schwartsmann, G., da Rocha, A.B., Berlinck, R.G.S., & Jimeno, J. (2001). Marine organisms
as a source of new anticancer agents. The Lancet Oncology 2, 221 – 225.

[5] Sarmah, P. & Deka, R.C. (2010). Anticancer activity of nucleoside analogues: A density
functional theory based QSAR study. Journal of Molecular Modeling

97
 Lipases or triacylglycerol acylhydrolases are hydrolytic enzymes which act on ester bonds
between alcohol and carboxylic acids. Pancreatic lipases, specifically, are important in lipid
absorption through the hydrolysis of dietary fats[1]. Orlistat, a gastric lipase inhibitor, competes
with dietary fats for sites in the lipase molecule to block the absorption of dietary fats [2]. This drug
is being used commercially as an anti-obesity drug. The structure of Orlistat is shown below
(Figure 1). The molecular structure of Orlistat has hydrophobic regions.

The catalytic mechanism of pancreatic lipase happens in the enzymatic pocket of the enzyme.
The active site is located in the upper part of the β-sheet of the enzyme. Figure 2 shows the
lipolytic mechanism involves acylation. The hydroxyl group of serine153 (or ser 152) of the
enzyme is deprotonated by an aspartic acid and a histidine belonging to the catalytic triad. The
activation permits the hydroxyl group of serine to attack the carboxylic group of the
triacylglycerols. The tetrahedral intermediate is further stabilized by gln and thr residues. Finally,
the de-acylation occurs on ser 153 by the introduction of a water molecule or alcohol (R2-OH),
regenerating the enzyme [3].

98
 A dipeptide is composed of two amino acids which are linked together by a peptide bond.
Many protein-rich food such as milk, fish, maize and gelatin were reported in some reports to
contain Angiotensin Converting Enzyme-inhibitory peptides.1 Recent research has also isolated
active peptides from snake venom2 and marine organisms.3 The commercially used drugs such
as Captopril, Lisinopril and Enalapril were actually based from the peptides in snake venom.2 The
peptides are usually extracted from the protein sources and studied in the laboratory. The enzyme
involved in this study is porcine lipase, similar to what you investigated in Laboratory Activity 04.
This time, you will analyze the molecular descriptors of selected dipeptides4 which exhibited
similar structural features with Orlistat.

However, notice that the active site of pancreatic lipase has several hydrophobic amino acid
residues such as phenylalanine, alanine and leucine (Figure 3). In this activity, you will be
investigating the inhibitory properties of selected fatty acids and their esters using the in vitro
lipase inhibition assay. The lipase inhibition assay focuses on determining the ability of an inhibitor
which has similar property to Orlistat. Fatty acids have hydrophobic hydrocarbon “tails,” but is this
structure in fatty acids and fatty acid esters important in inhibiting the catalytic activity of porcine
pancreatic lipase?

99
At the end of the activity, students should be able to:
 Determine the molecular descriptors of selected amino acids and dipeptides which are
related to lipase-inhibiting activity;
 Construct an approximated multiple linear regression model of the structure-activity
relationship selected dipeptides; and,
 Predict the lipase-inhibiting activity of a dipeptide.

 Amino acids: tyrosine, glycine, histidine, tryptophane

 Dipeptides: gly-phe, ala-gly, gly-glu, ala-ala, ala-tyr, gly-his
 Orlistat
 Porcine lipase
 Cuvette

 Spectrophotometer
 Desktop Computer

 MolView, ChemDes and Microsoft Excel

1. Go to: http://molview.org/

2. Draw structure of the dipeptide shown below.

Glycyl-phenylalanine

3. Once you have drawn the molecular structure of the dipeptide, click on “Tools” then click
on “Information card.”

4. Copy the canonical Simplified Molecular Input Line Entry System (SMILES) displayed on the
information card. Label this molecule as “Dipeptide 1.”

100
5. Do the same for the following dipeptides:

Dipeptide 2 Dipeptide 3 Dipeptide 4

Ala-gly
Gly-glu Ala-ala

Dipeptide 5 Dipeptide 6

Ala-tyr Gly-his

6. List all canonical SMILES of the dipeptides here. You will use the canonical SMILES to
determine the molecular descriptors in ChemDes.

Dipeptides Canonical SMILES

Dipeptide 1
Dipeptide 2
Dipeptide 3
Dipeptide 4
Dipeptide 5
Dipeptide 6

1. Go to: http://www.scbdd.com/chemdes/. Sign in using the given public user name and
password.

2. Choose the molecular descriptors that you will use by clicking on “Web Server” then click
“Custom Computation”. For this activity, you will be using the “Charge (25)” 2D descriptors
and “Molecular Property Descriptors (6).”

3. Insert the canonical SMILES then click “Submit.”

101
4. There will be 25 data which will be presented for “Charge 2D descriptors. Choose the
following data only: QHmax (most positive charge on H atom), QCmax (most positive
charge on C atom), QOmin (most negative charge on O atom) and LDI (local dipole index).
For “Molecular Property Descriptors,” choose TPSA (total polar surface area), logP2
(square of logP values) and Hy (Hydrophilic Index) only.

5. Copy the properties you have obtained to the table below.

Molecular Descriptors Charge 2D Descriptors

Dipeptide TPSA LogP2 Hy QHmax QCmax QOmin LDI
Dipeptide 1
Dipeptide 2
Dipeptide 3
Dipeptide 4
Dipeptide 5
Dipeptide 6

Observe the trends in the molecular descriptors. Which information seems to have an
increasing or decreasing trend? What “part” of the dipeptides contributes to these
molecular descriptors?

1. In separate 10mL volumetric flasks, prepare 1000 µg/mL stock solutions of the dipeptides
and Orlistat in dimethylsulfoxide (DMSO). You may dispense the solutions in 5-mL test
tubes during the assay. Prepare 500 µg/mL, 250 µg/mL, 125 µg/mL, 62.5 µg/mL and 31.3
µg/mL working solutions of the fatty acid and fatty acid esters using serial dilution.

2. Prepare a 1000 µg/mL porcine lipase solution in Tris-HCl buffer (pH = 8.0). Centrifuge the
mixture at 2000 rpm for 5 minutes, then collect the supernatant. This will be the source of
your enzyme. If there is no available centrifuge, you may filter the enzyme using a 0.2 µm
syringe filter. Make sure that you use a glass syringe for this step.

3. Prepare a stock solution of PNPB (p-nitrophenyl butyrate) by dissolving 20.9 mg of PNPB

in 2.0 mL of acetonitrile.

4. In separate Eppendorf tubes, combine 50 µL of the dipeptide and 50µL of PNPB. Agitate
or vortex the solution.

5. In separate plastic cuvettes, dispense 100 µL of porcine pancreatic lipase. Add Tris-HCl
buffer solution to bring the volume to 1000 µL.

6. Incubate the tubes at 36oC for 15 minutes.

7. Add 100 µL of PNPB-dipeptide mixture to the porcine lipase.

8. Incubate the plate again at 36oC for 30 minutes. Measure the absorbance after 30 minutes.
Plot your graphs.

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9. Determine the pancreatic lipase activity by measuring the hydrolysis of para-nitrophenyl
butyrate to para-nitrophenol at 405 nm using a UV-visible spectrophotometer.

10. Calculate the % inhibition using the formula:

𝑨−𝑪
% 𝐈𝐧𝐡𝐢𝐛𝐢𝐭𝐢𝐨𝐧 = 𝟏 − [ ] 𝐱 𝟏𝟎𝟎
𝑩−𝑫
Where B is the activity of the enzyme without inhibitor, and D is the negative control without inhibitor; A is
the activity of the enzyme with inhibitor, and C is the negative control with inhibitor.

11. Perform the lipase inhibition assay in triplicate. Record the mean % inhibition of each of
the potential inhibitors. The mean % inhibition will be used for developing a linear
regression model.

1. Open a Microsoft Excel worksheet in your desktop computer or laptop.

2. Perform a correlation analysis between the % inhibition and the molecular descriptors you
have obtained in ChemDes. From these data, choose three to molecular descriptors which
has the highest correlation with the % inhibition.

3. Open a new Excel worksheet. Perform a correlation analysis among the molecular
descriptors. If one of the molecular descriptors shows very weak correlation with the other
molecular descriptors, you choose another molecular descriptor to replace your first
choice.

Activity MD1
Activity 1
MD1 - 1
MD2 - -
MD3 - -
MD = Molecular Descriptor

4. Click on “Data” tab. Then click on “Data Analysis.” On the choices of statistical tests in the
“Data Analysis” box, choose “Regression.”

5. Once you are in the “Regression” box, fill up all data with the following:
Input Y range: Highlight the data and label under “Activity.”
Input X range: Highlight all data and label of the properties you have chosen to include
in the equation.

6. Tick on the “labels” to avoid confusing the beta coefficients.

7. Tick on “New Worksheet Ply” and click “OK.” The result of the linear regression will appear
on a new MS Excel worksheet.

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 8. Evaluate the statistical significance of the linear regression equation. If the p value is
greater than 0.05, you have to remove or add independent variables (X variables) until the
p value is less than 0.05.

Write your final multiple linear regression equation and the p value below. Interpret your
equation.

Multiple Linear Regression Equation: p value:

Is lipase inhibition an effective method to address health concerns

related to all cardiovascular diseases? Why or why not?

The property of the peptide bond is responsible for promoting

increased affinity of inhibitors to the active site of lipase.

Using Toulmin Argumentation Pattern, create an argument about the

statement above.

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 The enzyme tryptophan hydroxylase is responsible for the hydroxylation of tryptophan, which
in turn, is important in the synthesis of serotonin.1 This enzyme can be found in the brain and in
the gut. The enzyme in the gut, tryptophan hydroxylase-1 (TPH-1), is involved in the synthesis of
gut-derived serotonin. The inhibitors of TPH1 are utilized in treating chemotherapy-induced
vomiting as well as diarrhoea in cancer patients1. In the several studies, tryptophan hydroxylase-
1 inhibitors are also used to treat osteoporosis1,2. Serotonin in the gut regulates intestinal mobility
and mucosal secretion3.

Gut-derived serotonin inhibits bone formation while brain-derived serotonin promotes bone
formation and decreases bone resorption1. The enzyme tryptophan hydroxylase-1 and tryptophan
hydroxylase-2 (TPH2) are responsible for the production of serotonin in the gut1,3 although THH1
accounts for about 95% of total serotonin produced in the body. Hence, the TPH1 are the main
target of inhibitors to treat osteoporosis. In this in silico activity, you will be focusing with the
identification of some molecular descriptors and properties which could be used to explain the
inhibitory activity of the phenylalanine derivatives against the enzyme tryptophan hydroxylase-1.

At the end of the activity, students should be able to:

 Identify physicochemical features of phenylalanine derivatives which contribute to their in
vitro inhibiting activity;

 Construct an approximated multiple linear regression model of the structure-activity

relationship of nucleoside analogues and their anti-cancer activities; and,

 Predict the anti-cancer activity of a nucleoside analogue based on the approximated linear
regression model.

 Microsoft Excel, MolView and ChemDes

105
1. Go to: http://molview.org/

2. Draw structure of the phenylalanine derivative shown below.

Assign this molecule as “Phenylalanine Derivative 1

3. Once you have drawn the molecular structure, click on “Tools.” Click on “Information card”
to determine basic information about the molecule. Please refer to the previous activities
for the details of this step.

4. Copy the canonical Simplified Molecular Input Line Entry System (SMILES) displayed on
the information card.

5. Do the same for the following molecules:

Phenylalanine Derivative 2 Phenylalanine Derivative 3

Phenylalanine Derivative 4 Phenylalanine Derivative 5

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 Phenylalanine Derivative 6 Phenylalanine Derivative 7

Phenylalanine Derivative 8 Phenylalanine Derivative 9

Phenylalanine Derivative 10

6. List all canonical SMILES of the phenylalanine derivatives here. You will use the canonical
SMILES to determine the molecular descriptors in ChemDes.

Phenylalanine Derivative Canonical SMILES

Phenylalanine Derivative 1
Phenylalanine Derivative 2
Phenylalanine Derivative 3
Phenylalanine Derivative 4
Phenylalanine Derivative 5
Phenylalanine Derivative 6
Phenylalanine Derivative 7
Phenylalanine Derivative 8
Phenylalanine Derivative 9
Phenylalanine Derivative 10

107
 1. Go to: http://www.scbdd.com/chemdes/. Sign in using the given public user name and
password.

2. Choose the molecular descriptors that you will use by clicking on “Web Server.” Then click
on “Chemopy Descriptors.” Click on “Web Server again then click “Custom Computation”.

3. Insert the canonical SMILES then click “Submit.” Copy the six molecular descriptors.

4. Perform step 2. This time, select “Charge” among the options. There will be 25 data which
will be given. Choose the following data only: Tpc, Tnc, Mnc, Mpc, LDI, QHmax, QCmax,
QNmin, and QOmin.

5. Copy the properties you have obtained to the table below.

Data from ChemDes

Molecular Charge
Phenylalanine Descriptors Activity
Derivative (pIC50-1)

QOmi
TPSA
logP2

QOm
QHm
QCm
logP

Mpc
Mnc
Tpc
Tnc

LDI
MR
Hy

ax
ax

ax
UI

n
Phenylalanine Derivative 1
Phenylalanine Derivative 2
Phenylalanine Derivative 3
Phenylalanine Derivative 4
Phenylalanine Derivative 5
Phenylalanine Derivative 6
Phenylalanine Derivative 7
Phenylalanine Derivative 8
Phenylalanine Derivative 9
Phenylalanine Derivative 10

TPSA = Topological Polarity Surface Area, logP2 = Square of logP value based on the Crippen method, Hy = Hydrophilic Index, UI =
Unsaturation Index, logP = logP based on Crippen method, MR = Molar Refractivity, Tpc = total of positive charges, Tnc= total of
negative charges, Mnc = Mean of negative charges, Mpc= meant of positive charges, LDI = local dipole index, QHmax = most positive
charge on H atom, Cmax = most positive charge on C atom, QNmin = most negative charge on N atom, QOmin = most negative
charge on O atom

108
1. Open a Microsoft Excel worksheet in your desktop computer or laptop.

2. Perform a correlation analysis between the % inhibition and the molecular descriptors you
have obtained in ChemDes. From these data, choose three to molecular descriptors which
has the highest correlation with the % inhibition.

3. Open a new Excel worksheet. Perform a correlation analysis among the molecular
descriptors. If one of the molecular descriptors shows very weak correlation with the other
molecular descriptors, you choose another molecular descriptor to replace your first
choice. Choose your final two molecular descriptors to be used for your linear regression
equation.

Activity MD1 MD2

Activity 1
MD1 - 1
MD2 - - 1
MD = Molecular Descriptor

4. Click on “Data” tab. Then click on “Data Analysis.” On the choices of statistical tests in the
“Data Analysis” box, choose “Regression.”

5. Once you are in the “Regression” box, fill up all data with the following:
Input Y range: Highlight the data and label under “Activity.”
Input X range: Highlight all data and label of the properties you have chosen to include
in the equation.

6. Tick on the “labels” to avoid confusing the beta coefficients.

7. Tick on “New Worksheet Ply” and click “OK.” The result of the linear regression will appear
on a new MS Excel worksheet.

8. Evaluate the statistical significance of the linear regression equation. If the p value is
greater than 0.05, you have to remove or add independent variables (X variables) until the
p value is less than 0.05.

Write your final multiple linear regression equation and the p value below. Interpret your
equation.

Multiple Linear Regression Equation: p value:

109
 Tryptophan hydroxylase 2 (TPH2) is an isoform of tryptophan
hydroxylase 1 (TPH1) which is produced by the myenteric neurons.
This means that the neurons are found in the muscles of the small
intestines. Will the phenylalanine derivatives inhibit the activity of
Tryptophan hydroxylase 2?

Using the Toulmin Argumentation Format, present an argument about

the statement below.

“All aromatic rings can elicit the same inhibitory activity of

phenylalanine derivatives.”

[1] Ouyang, L., He, G., Huang, W., Song, X., Wu, F. & Xiang, M. (2012).Combined structure-
based pharmacophore and 3D QSAR Studies on phenylalanine series compounds as TPH1
inhibitors. International Journal of Molecular Sciences 13, 5348 – 5363.

[2] Michalowska, M., Znorko, B., Kaminski, T., Oksztulska-Kolanek, E. & Pawlak, D. (2015). New
insights into tryptophan and its metabolites in the regulation of bone metabolism. Journal of
Physiology and Pharmacology 66(6), 779 – 791.

[3] De Vernejoul, M-C., Collet, C. & Chabbi-Achengli, Y. (2012). Serotonin: good or bad for bone.
BoneKey Reports 1, 1 – 6.

110
 – a public database which provides access to an integrated suite of analysis and information retrieval tools
for compound searching, structure-based clustering, descriptor generation (chemical properties), and retrieval of
published bioactivity and target protein information.

– an empirical index related to hydrophilicity of compounds:

Where NHy is the number of hydrophilic groups (-OH, -SH, -NH), Nc the number of carbon atoms and A the number of
atoms (hydrogen excluded). Hy index is between -1 and 3.64.

– A molecular descriptor calculated as the average of the charge differences over all i-j bonded
atom pairs:

Where B is the number of bonds

– partition coefficient of a molecule between an aqueous and lipophilic phases, usually octanol and
water. The partition coefficients are usually transformed into a loganthmic form. Below is the formula for
determining logP.
P = [C] octanol / [C]aqueous

The relative affinity of a drug molecule for an aqueous or lipid medium is important for the drug’s activity
because absorption, transport, and excretion depend on partitioning phenomena. [C]octanol is the
concentration of a solute in the lipid phase (n-octanol) and [C]aqueous is the concentration of the solute in
the aqueous phase. Compounds for which P>1 are lipophilic or hydrophobic, and compounds for which
P<1 are hydrophilic. Lipophilicity represents the affinity of a molecule or a moiety for a lipophilic
environment.

– square of LogP value based on the Crippen Method

- Molecular descriptor of a liquid which contains both information about molecular volume and
polarizability usually defined by Lorenz-Lorenz Equation:

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Where MW is the molecular weight, Q the liquid density (the ratio MWIQ corresponds to the molar volume V), and n the
refractive index of the liquid. The refractive index is defined as a ratio of the velocity of light in vacuum to the velocity
of light in the substance of interest. Used as an indicator of the purity of organic compounds, it is related to several
electric and magnetic properties such as polarizability as well as to critical temperature, surface tension, density, and
boiling point.

– Molecular descriptors numerical values associated with the chemical constitution for
correlation of chemical structure with various physical properties, chemical reactivity, or biological activity. The
descriptors may be physicochemical (hydrophobic, steric, or electronic), structural (based on frequency of occurrence
ofa substructure), topological, electronic (based on molecular orbital calculations), geometric (based on a molecular
surface area calculation), or simple indicator parameters (dummy variables).

A biological activity = f(molecular descriptors) …

– Multiple Linear Regression( MLR) method helps in establishing correlation between the
independent and dependent variables. Here, the dependent variables are the biological activity or physiochemical
property of the system that is being studied and the independent variables are molecular descriptors obtained from
different representations. In linear regression models, the dependent variable is predicted using only one descriptor or
feature. Multiple linear regression models consider more than one descriptor for the prediction of property/ activity in
question.

- prediction depends on the structure of molecules and atoms

present in the compound. Biological activity is understood in terms of numerical values (example bioavailability,
inhibitory concentration) and presence/absence of a condition (example infected/not infected, mutagenic/non-
mutagenic).

WORKFLOW of a QSAR Model Development

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 - the part of the surface area of the molecule associated with oxygens, nitrogens,
sulfurs and the hydrogens bonded to any of these atoms. This surface descriptor is related to the hydrogen-bonding
ability of compounds.

– The sum of all negative charges of the atoms in a molecule.

– The sum of all positive charges of the atoms in a molecule.

- The unsaturation index (UI) is defined as:

UI = log2 (1+ nDB + nTB + nAB)

Where nDB = the number of double bonds, nTB = number of triple bonds, and nAB = number of aromatic bonds.

Unsaturation Index is also calculated using the formula:

UI = log2 (1 + b)

Where b is calculated as

b = [2Nc + 2 – NH – NX + NN + NP + 2 (NO-S – NSO3)] / 2 – C

Nc, NH, Nx, NN, NA and C are the number of carbon atoms, hydrogen, halogen, nitrogen, phosphorus, and
independent cycles, respectively. NO-S and NSO3 are the number of oxygen atoms bonded to sulfur and the number
of SO3 groups, respectively.

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115
A. Validity of Data in Argument
Actual data is used; valid assumption- or inference-based, sophisticated – 2 points
Assumption- or inference-based, but simplistic – 1 point
Invalid/ Misinterpretation of data – No point

B. Quality of Links in Argument

Correct, chemistry-based or mechanistic reasoning – 2 points
Correct but non-mechanistic chemistry reasoning – 1 point
Incorrect reasoning/ simplistic or naïve reasoning – 1 point

C. Validity of Data in Rebuttal

Actual data is used; valid assumption- or inference-based, sophisticated – 2 points
Assumption- or inference-based, but simplistic – 1 point
Invalid/ Misinterpretation of data – No point

D. Quality of Links in Rebuttal

Correct, chemistry-based or mechanistic reasoning – 2 points
Correct but non-mechanistic chemistry reasoning – 1 point
Incorrect reasoning/ simplistic or naïve reasoning – 1 point

116