Download Science Course Outline Template

FACULTY OF SCIENCE
SCHOOL OF BIOTECHNOLOGY AND
BIOMOLECULAR SCIENCES
BIOC2181
FUNDAMENTALS OF BIOCHEMISTRY
Course Manual
Session 1, 2017
UNIVERSITY OF NEW SOUTH WALES
SCHOOL OF BIOTECHNOLOGY AND BIOMOLECULAR SCIENCES
BIOC2181 FUNDAMENTALS OF BIOCHEMISTRY
COURSE MANUAL
2017
TABLE OF CONTENTS
Page No.
Course Outline:
Information about the Course……………………...…………. 2
Staff Involved in the Course…..…………..………….…..
3
Course Details…….…………………………………….…
3
Course Schedule……………………………………….…..
4
Assessment Tasks and Feedback……………………….
5
Course Topics and Additional Class Information……….
6
Additional Resources and Support…..……………..……
8
Required Equipment, Training and Enabling Skills.…….…. 8
Administration Matters.………………………………………
9
UNSW Academic Honesty and Plagiarism ….…………..…. 13
Lecture Summaries………………..…………………..…..
14
Study Guide………….………………………………….…..
24
Practicals – General Information…………………………..…..29
Laboratories:
Appendix:
Academic Misconduct and Class Attendance…………….
32
Laboratory Safety………………………………………….
33
Safety declaration……………………………………….…..
37
Spectrophotometry…..………………………………….…..
38
Enzymes……………….……………………………….……
54
Oxygen Electrode Simulation.……………………………..
71
Glycolysis.………………………………………………….
72
Separation Techniques……………………………………
82
Glucose Tolerance Test……..……………………………
90
Instrumentation…………………………..………………….
97
1
BIOC2181 Fundamentals of Biochemistry - Course Outline
1. Information about the Course
NB: Some of this information is available on the UNSW Handbook1
Year of Delivery
2017
Course Code
BIOC2181
Course Name
Fundamentals of Biochemistry
Academic Unit
School of Biotechnology and Biomolecular Sciences
Level of Course
Level 2
Units of Credit
6UOC
Session(s) Offered
Session 1
Assumed Knowledge,
Prerequisites or Co-requisites
BABS1201 Molecules, Cells and Genes and CHEM1011 Chemistry A
or CHEM1031 Higher Chemistry A or CHEM1831 Chemistry for
Health, Exercise and Medical Science
Hours per Week
6 HPW
Number of Weeks
12 weeks
Commencement Date
Monday 27 February, 2017
th
Summary of Course Structure (for details see 'Course Schedule')
Component
HPW
LECTURES
2-3
Lecture 1
1
5 pm
Monday
Mathews B
Lecture 2
1
12 pm
Thursday
Mathews B
Lecture 3
1
3 pm
Friday
Mathews B
LABORATORIES
Time
Day
Location
3
Wallace Wurth 122
Lab – Option 1
3
10 am - 1 pm
Tuesday
Lab – Option 2
3
2 pm - 5 pm
Tuesday
Wallace Wurth 123
Wallace Wurth 122
Wallace Wurth 123
TUTORIALS
Large Group Tutorials
1
5pm
weeks 6 & 12
Mathews B
Small Group Tutorials*
1
10 am or 2 pm*
Tuesday
Weeks 2, 5, 8 and 12*
Wallace Wurth 122 / 123
TOTAL
Special Details
1
1-2
6
* Small Group Tutorials are held during weeks in which no wet laboratory
practicals are scheduled. The tutorials will be located in the teaching
laboratories and will be held during the first hour of the allotted laboratory times.
UNSW Online Handbook: http://www.handbook.unsw.edu.au
2
2. Staff Involved in the Course
Name
Contact Details
Consultation
Times
Dr Nirmani Wijenayake
[email protected]
By
appointment
Dr Richard Edwards
Dr Vladimir Sytnyk
Dr Lucy Jo
Dr Rebecca LeBard
Dr Kyle Hoehn
[email protected]
[email protected]
[email protected]
[email protected]
[email protected]
Demonstrators
& Tutors
See Moodle for
demonstrator lists
Moodle Discussion Boards
Technical &
Laboratory
Staff
Ms Shamima Shirin
Ms Angela Guider
[email protected]
[email protected]
Staff
Role
Course Convenor
Lecturers
Additional
Teaching
Staff
By
appointment
Scheduled
laboratory and
tutorial times
Scheduled
laboratory
times
3. Course Details
BIOC2181 Fundamentals of Biochemistry introduces modern biochemistry,
fundamental aspects of the structure-function relationships of proteins and an overall
coverage of intermediary metabolism. Major topics covered include: the nature and
functions of enzymes; the metabolic working of cells, tissues and organs; the
interrelationships between pathways of carbohydrate, lipid and amino acid metabolism;
the vital roles of enzymes and hormones in catalysis and metabolic regulation; the
energy-trapping mechanisms of animals and plants; and interesting variations on the
central metabolic pathways in various life forms. The practical coursework
complements the lectures and introduces the principles of biochemical analysis.
Course
2
Description
Course Aims
Student
Learning
4
Outcomes
3
 This course aims to introduce students to modern biochemistry with a particular
emphasis on how we, as humans, convert foods to useful energy.
 This course also aims to provide a solid context for new learning material by
providing clinical, medical and everyday applications that correspond to the central
themes and topics.
 Practicals are designed to reinforce the core biochemical concepts covered in
lectures and introduce students to current laboratory techniques and biochemical
assays.
By the end of this course, you will be able to:
 Describe and contrast the major metabolic pathways in humans.
 Explain the various mechanisms that control and regulate anabolic and catabolic
processes simultaneously in the cells of living tissues.
 Discuss the integration of major metabolic pathways in the context of various
human conditions, such as fasting, starvation, obesity and exercise.
 Follow the correct procedures for working safely and effectively in a modern
biochemical laboratory.
 Perform a range of biochemical assays, analytical techniques and biochemical
calculations through the application of current scientific methods in an experimental
environment.
2
UNSW Handbook: http://www.handbook.unsw.edu.au
Learning and Teaching Unit: Course Outlines
4
Learning and Teaching Unit: Learning Outcomes
3
3
4. Course Schedule
Week
No.
Week
Begins
LECTURE/TUTORIAL
LECTURE
LECTURE
PRACTICAL/TUTORIAL
Monday 5pm Mathews B
Thursday 12pm Mathews B
Friday 3 pm Mathews B
Wallace Wurth 122/123
1
27 Feb
Introduction – NWG
Amino Acids – RE
Proteins - RE
Online Safety Quiz
2
6 March
Enzymes – RE
Enzyme Kinetics – RE
Lecture Review 1: Online Activity
Tutorial 1: Biochemical
calculations
3
13 March
Carbohydrates – VS
Glycolysis – VS
Regulation – VS
Practical 1. Spectrophotometry
Lecture Review 2: Online Activity
Practical 2. Enzymes
4
20 March
5
27 April
Oxidative Phosphorylation (1) - NWG
Oxidative Phosphorylation (2) - NWG
Oxidative Phosphorylation (3) – NWG
6
3 April
Large Group Tutorial 1:
Scientific Writing - NWG
Glycogen Metabolism – NWG
Review Lecture 3: Online Activity
Practical 3. Oxygen Electrode
Simulation (Computer prac)
7
10 April
TEST 2
Gluconeogenesis – NWG
Review Lecture 4: Online Activity
GOOD FRIDAY
Practical 4. Glycolysis
TEST 1
TCA Cycle – VS
17 April
8
24 April
9
1 May
10
8 May
11
15 May
12
22 May
MID-SESSION BREAK
Introduction to Fats – LJ
Lipoproteins – LJ
Fat oxidation and synthesis – NWG
Ketone bodies – NWG
Protein Catabolism – RLB
Review Lecture 5: Online Activity
The Urea Cycle – RLB
Hormonal Control of Metabolism –
RLB
Fuel Supply in Exercise – RLB
Metabolic Specialisation of Tissues –
KLH
Fuel Supply in Fasting – KLH
Large Group Tutorial 2:
Concluding Lecture – NWG
Review Lecture 6: Online Activity
TEST 3
Integration of Metabolism - KLH
13
PRAC QUIZ (5%)
Tutorial 2: Test 1 Review
PRAC QUIZ (5%)
Tutorial 3: Test 2 Review
Practical 5. Separation
Technique - TLC
NO LAB
Practical 6. Glucose Tolerance
PRAC QUIZ (5%)
Tutorial 4: Test 3 Review
29 May
NWG - Nirmani Wijenayake; RE - Rich Edwards; VS - Vladimir Sytnyk; LJ - Lucy Jo; RLB – Rebecca LeBard; KLH – Kyle Hoehn
4
5. Assessment Tasks and Feedback
Task
Mid-session Test 1*
Mid-session Test 2*
Mid-session Test 3*
Knowledge & abilities
assessed
Theory presented in
All RE lectures
Theory presented in
All VS lectures and NWG
OX-PHOS lectures
Theory presented in
All of LJ lectures and NWG
GM, GNG, and FATS lectures
% of total
mark
Feedback
Date of Assessment
Task
10 %
Week 4, Monday 20th
March, 5-6pm
10 %
Week 7, Monday 10th
April, 5-6pm
10 %
Week 10, Monday 8th
May, 5-6pm
WHO
Tutor and
Course
Coordinator
Tutor and
Course
Coordinator
Tutor and
Course
Coordinator
WHEN
HOW
Week 5
Review Tutorial &
Moodle
Week 8
Review Tutorial &
Moodle
Week 12
Review Tutorial &
Moodle
Pre-lab and other
assigned online quizzes
Practical theory, ability to
perform biochemical
calculations and lab safety
10 %
Pre-lab quizzes must be
completed prior to each
lab
Course
Coordinator
During the
quiz
Adaptive Feedback
Final Theory
Examination*
(2 hours)
Theory presented in
Weeks 1 - 12 lectures
45 %
June examination period
(date to be announced)
Course
Coordinator
-
-
Practical Quizzes
Practical work conducted
throughout Weeks 1 - 12 and
reviewed in lectures and
tutorials
15 %
Weeks 5, 8, & 12
Tutor and
Course
Coordinator
Week
following
the quiz
During the lab
-
-
-
TOTAL:
-
100 %
-
* Please note that the format of all three mid-session tests and the final theory examination will consist of a combination of multiple choice, short answer and
extended (short essay) answer questions. Further details of each assessment task will be released on Moodle and/or during lectures prior to each test.
5
6. Course Topics and Additional Class Information
Major Topics
 Introduction to key biochemical themes and concepts
(Lecturer: Dr Nirmani Wijenayake)
 Amino acids, protein structure, enzymes and enzyme kinetics
(Lecturer: Dr Rich Edwards)
 Carbohydrates, glycolysis and the TCA cycle (Lecturer: Dr Vladimir Sytnyk)
 Oxidative phosphorylation (ATP generation) (Lecturer: Dr Nirmani Wijenayake)
 Glycogen metabolism and gluconeogenesis (Lecturer: Dr Nirmani Wijenayake)
 Fats: digestion, transport, breakdown & synthesis (Lecturer: Dr Lucy Jo and
Dr Nirmani Wijenayake)
 Protein catabolism and the urea cycle (Lecturer: Dr Rebecca LeBard)
 Integration of metabolic pathways, hormones and whole body metabolism
(Lecturers: Dr Kyle Hoehn and Dr Rebecca LeBard)
Large Group
Tutorials
Two large group tutorials will be held on Monday 5-6pm on weeks 6 and 12. (See
course schedule on page 4). In preparation for these tutorials, you will be given small
study tasks that MUST be completed PRIOR to each class in order to ensure that you
gain the maximum learning experience from these exercises. The structure of the large
group learning activities will facilitate optimal student-tutor and student-student
interactions that provide you with the opportunity to question and clarify various
aspects of the course content. Tutorials also aim to take you beyond the lecture
material, assisting you to improve your general and scientific communication skills, as
well as your examination techniques.
Small Group
Tutorials
There are 4 small group tutorials that will take place during your assigned laboratory
time in weeks when you do not have a wet laboratory class scheduled. Each tutorial
will be conducted in the first hour of your assigned lab time and will take place in your
allocated teaching laboratory. In most cases, your lab demonstrator will also be your
tutor and you will work with your assigned lab group of students. Tutorials will include a
biochemical calculations workshop and reviewing the answers for the three midsession tests. Details of each tutorial will be provided on Moodle and/or during the
tutorial itself.
Review
Lectures
A total of 6 Review Lectures are scheduled for designated BIOC2181 lecture slots
throughout the session (see course schedule on page 4). During these classes,
previous lecture topics will be revised and no new conceptual material will be covered.
This will provide students with the opportunity to revise course content and reflect upon
their own level of comprehension of the material presented in lectures and integrated
with laboratory classes. For these Reviews, you will work on an online tutorial
independently. The tutorial will provide you with specific feedback based on your
answers.
Mid-Session
Tests
A total of 3 ‘mid-session’ tests will be held during the semester. Each test is worth
10% of your overall assessment. Tests 1, 2 and 3 will be conducted during the
Monday lecture slot of Weeks 4, 7 and 10, respectively, and will be held under strict
examination conditions in the designated lecture theatre (see course schedule on page
4).
NOTE: Students who experience any difficulty in writing English for academic purposes
such as reports, exam short answer or written questions, or problems comprehending
multiple choice questions should consult an advisor at “The Learning Centre” located in
the foyer of the main library entrance to obtain relevant information or up to one hour a
week of private consultation with a peer writing assistant.
6
Practical
Program
Students will be enrolled in one of the following laboratory times:
 Tuesday 10am – 1pm
 Tuesday 2pm – 5pm
BIOC2181 laboratory classes will be scheduled as outlined below. There will not be a
lab class in Week 1; instead, all students are required to complete an online safety
quiz. No laboratory work can be performed until this activity is successfully completed.
Wet lab classes will be conducted in Weeks 3, 4, 7, 9 and 11 only. You will complete
an online virtual lab in week 6.
Students are also required to do a pre-lab quiz prior to each lab. The pre-lab quiz will
be released a week before the lab and will close at 9am on the day of the lab
irrespective of your lab time. If you do not complete the pre-lab quiz and achieve a
grade of 100% prior to the lab, you will not be allowed to participate in the lab
and will be marked absent. More details about these quizzes can be found on page
9.
Small group tutorials are scheduled for Weeks 2, 5, 8 and 12; in the first hour of your
lab time (self-directed study and course revision are highly recommended for the
remaining 2 hours). There will be no lab or tutorial classes held in Week 10.
BIOC2181 Laboratory Class Schedule:
 Week 1 – Online Safety Quiz
 Week 3 – Spectrophotometry
 Week 4 – Enzymes
 Week 6 – Online Oxygen Electrode Virtual practical
 Week 7 – Glycolysis
 Week 9 – Separation Techniques – Thin Layer Chromatography
 Week 11 – Glucose Tolerance Test
NOTE: Final laboratory groups will be announced by Monday of Week 2. A list will be
displayed on the BIOC2181 Moodle site.
Relationship
to Other
Courses
within the
Program
This course essentially covers the same material as BIOC2101 Principles of
Biochemistry (Advanced), but in less detail and with more emphasis on the function of
organisms and less emphasis on some of the underlying chemical mechanisms.
As an alternative to BIOC2101, BIOC2181 Fundamentals of Biochemistry provides a
comprehensive introduction to biochemistry for students who do not intend to proceed
to Level III Biochemistry. It does not fulfill the prerequisite requirements for Level III
Biochemistry, but the Head of School may give approval for students with a grade of
credit to enroll in Level III courses.
7
7. Additional Resources and Support
Text Books
Recommended Texts:

rd
Biochemistry - A Short Course (3 edition), by Tymoczko J.L., Berg J.M.
& Stryer L. (W H Freeman and Company), 2015.
OR

th
Biochemistry and Molecular Biology (4 Edition), by Elliot W.H. & Elliot
D.C. (Oxford University Press), 2009.
Additional Biochemistry Reference Texts:

Essential Biochemistry, by Pratt, C.W. & Cornely, K., 2004.

Concepts in Biochemistry (3 Edition), by Boyer, R., 2006.

Biochemistry (7 Edition), by Berg J.M., Tymoczko J.L. & Stryer L., 2011.

Fundamentals of Biochemistry (4 Edition) Voet, Voet and Pratt, 2013.
rd
th
th
Course Manual
The BIOC2181 Course Manual is available for purchase through the UNSW
Bookshop and can be downloaded via the BIOC2181 Moodle site.
Required and
Additional
Readings
Details of recommended readings and reference materials will be provided by
individual lecturers during lectures and online via Moodle.
Recommended
Internet Sites
Details of recommended internet sites will be provided by individual lecturers
during lectures and online via Moodle.
Societies
ASBMB – Australian Society for Biochemistry and Molecular Biology
www.asbmb.org.au
Computer
Laboratories
Computer laboratory G08, located on the ground floor of the Biological
Sciences Building, is a student laboratory used for course classes and
independent research/studies (when not booked for classes).
8. Required Equipment, Training and Enabling Skills
Equipment Required
Practical Requirements: Laboratory coat and closed shoes (no thongs,
sandals, or open-toed shoes), and safety glasses.
Enabling Skills
Training Required to
Complete this Course
ELISE, Online OHS Quiz conducted via Moodle in Week 1 of Session.
8
9. Administration Matters
Expectations
of Students
PRACTICALS AND TUTORIALS:
A pass in BIOC2181 is conditional upon a satisfactory performance in the
practical and tutorial programs. A satisfactory performance means that:
(i)
You have completed and achieved a mark of 100% in the online
Laboratory H&S Quiz PRIOR to your first lab in Week 3 (see page 29 of
this manual for details); and
(ii)
You have completed all pre-lab quizzes and achieved a mark of 100%
PRIOR to each lab. Each quiz will be worth 1% of your final marks. You
will be allowed multiple attempts for each quiz until you achieve a mark
of 100%. However, each attempt will result in the deduction of marks
from the 1% allocated for each quiz (further information will be provided
during the introductory lecture in week 1)
(iii)
You have attended ALL of the practical and tutorial classes
(iv)
You have kept an up-to-date and accurate record of experimental data
in your laboratory manual. This includes the recording of all data into
the appropriate tables of your manual at the time they are obtained, as
well as the recording of subsequent calculations and answers to the
questions. At the end of each laboratory class, your demonstrator will
check to see that you have completed ALL of your work and that you
have tidied and cleaned your equipment and workspace as instructed
by the demonstrators and technical staff.
(v)
And you have achieved above 50% in all 3 practical quizzes.
Students will be performing a laboratory-based exercise only every second week or
so. In most of the remaining weeks, a one hour structured small group tutorial will
be held in the laboratory. The one hour tutorials will provide an opportune time to
review both lecture and practical material with your tutor, and the remaining 2 hours
would be well-spent further revising core material within smaller study groups or
independently. In order to avoid ‘cramming’ material during the study period at the
end of the semester, we strongly recommend that students keep up to date with
their work and prepare ahead for lectures and practicals to come.
LECTURES:
Attend ALL lectures and try to take comprehensive lecture notes. DO NOT rely
solely on iLectures, lecture hand-outs, lecture notes from other students and textbooks. The lecturer who presents the lectures will set the examination questions
and will also be responsible for marking the relevant examinations/tests. Each
lecturer will take you through the intricacies of the various topics in biochemistry in a
way that you may find difficult to reproduce by simply reading through the syllabus,
lecture hand-outs and the prescribed texts. The most efficient way of ensuring that
you have covered all aspects of the syllabus is by attending ALL the lectures and
participating in ALL tutorials and lab classes.
General
Enquiries
Health and
5
Safety
All general administrative enquiries can be directed to the BSB Student Office,
G27, Ground Floor, Biological Sciences Building, opening hours: Mon-Fri 9am12:30pm and 1:30pm-4:30pm.
Covered shoes, safety glasses, and lab coats must be worn whenever you are
working in the laboratory. Eating, drinking, smoking and running are not permitted
in the lab. Anyone who violates these regulations will not be allowed to proceed with
the practical class.
UNSW H&S policies and procedures (2001) stipulate that everyone attending a
UNSW workplace must ensure their actions do not adversely affect the health and
5
UNSW OHS Home page
9
safety of others. This outcome is achieved through a chain of responsibility and
accountability for all persons in the workplace.
Health and
Safety
(continued)
As part of this, the School has undertaken detailed risk assessments of all course
activities and identified all associated potential hazards. These hazards have been
minimised and appropriate steps taken to ensure your health and safety. For each
activity, clear written instructions are given and appropriate hazard warnings or risk
minimisation procedures included for your protection. Please refer to the Risk
Assessment sections at the beginning of each practical outline in this manual for
specific risks and hazards associated with the laboratory component of this course.
It is your responsibility to prepare for all practical work. You should be familiar with
the procedures scheduled for the practical class and identify all personal protection
requirements needed to complete the exercise in a safe manner. Material Safety
Data Sheets (MSDS) are available from your demonstrator for any hazardous
chemicals. At the commencement of each new practical your demonstrator will
review any risks with you. It is essential that you are present at the beginning of
each class to ensure that you understand any risks and can review the safety
procedures. If you are not present you may be excluded from the class.
You must comply with all safety instructions and observe all safety notices. Failure
to comply with safety instructions may be considered a form of academic
misconduct and may be investigated by WorkCover as a breach of the NSW OH&S
Act (2000).
Following are some simple rules which will ensure good laboratory practice and
minimise the consequences of risks: Wear adequate protective clothing including, when appropriate, gloves and
safety glasses.
 Acquaint yourself with the safety equipment in the lab.
 Do not eat, drink, smoke, or apply make-up in the lab. Do not bring food, drink
etc. into the lab. Do not sit on laboratory benches.
 Do not invite anyone into the lab.
 In the event of an accident with a microbial culture, or hazardous chemical, ask
a fellow student to call someone in authority immediately. Do not move and
risk the spread of contamination. If there is a fire or you are at risk from a
chemical spill, remove yourself from immediate danger and call someone in
authority immediately.
 Dispose of all waste correctly.
 Label all materials correctly and place in the relevant containers provided.
 Operate all equipment carefully and correctly. If in doubt regarding the correct
method of operation consult a demonstrator before proceeding.
 Keep your bench tidy during experimental work and clean up and disinfect your
bench before leaving the laboratory. Ensure that you wash your hands before
leaving.
 If you feel physical discomfort from your work or have an allergic reaction,
consult your demonstrator or another person in authority.
 If you get any biological or chemical substance your eye, immediately go to a
tap and wash your eye. While washing your eye, alert someone to your
situation so that they can assist you and gain the attention of someone in
authority. Continue to wash your eye until someone in authority indicates for
you to do otherwise. Note that you should always wear safety glasses when
handling hazardous substances.
 Information on relevant H&S policies and expectations at UNSW:
http://www.ohs.unsw.edu.au/
The complete “School of Biotechnology and Biomolecular Sciences Undergraduate
Risk Assessment Guide” can be found in the “OHS” content area of the BIOC2181
Moodle site. Additional School of BABS OHS information can be found on the
School
website:
http://www.babs.unsw.edu.au/ohs/school-babs-occupationalhealth-and-safety
10
Assessment
Procedures
Missed Practical Classes or Small Group Tutorials:
If you miss a practical class or a small group tutorial due to illness or some other
unavoidable circumstance that can be verified via professional documentation,
email your course coordinator within three days of the absence. Separate “CatchUp” labs/tutorials are not conducted but if you are able to attend an alternative lab
or tutorial during the week of your absence, you may contact the course coordinator
to ask for permission to do so. If you cannot attend an alternative lab/tutorial, then
you will need to catch up on missed work by speaking to your demonstrator/tutor or
class colleagues.
Missed Large Group Tutorials:
If you miss a large group tutorial you do not need to do anything. Since these
classes contain interactive activities, you are strongly encouraged to attend them in
order to gain the full learning benefit from their design. In order to catch up on
missed large group tutorial activities, you should complete any pre-work, listen to
iLectures and access any supplementary materials via Moodle.
Missed Mid-session Tests:
If you miss a mid-session test due to illness or some other unavoidable
circumstance that can be verified via professional documentation, you must apply
for special consideration according to the UNSW Special Consideration and Further
Assessment Policy outlined below. Depending on their overall performance at the
end of the course, students with compliant applications will either receive an
average mark for their missed test or will be invited to sit further assessment on the
supplementary exam date (see next page).
SPECIAL CONSIDERATION AND FURTHER ASSESSMENT, SESSION 1, 2017:
UNSW
Assessment
6
Policy
Students who believe that their performance, either during the session or in the end
of session final exams, may have been affected by illness or other circumstances
may apply for special consideration. Applications can be made for compulsory class
absences such as (laboratories and tutorials), in-session assessments tasks, and
final examinations. Students must make a formal application for Special
Consideration for the course/s affected as soon as practicable after the problem
occurs and within three working days of the assessment to which it refers.
Students should consult the A-Z section of the “Student Guide 2017”, particularly
the section on “Special Consideration”, for further information about general rules
covering examinations, assessment, special consideration and other related
matters. This is information is published free in your UNSW Student Diary and is
also available on the web at:
https://student.unsw.edu.au/special-consideration
HOW TO APPLY FOR SPECIAL CONSIDERATION:
Applications must be made via Online Services in myUNSW. You must obtain and
attach Third Party documentation before submitting the application. Failure to
do so will result in the application being rejected. Log into myUNSW and go to
My Student Profile tab > My Student Services channel > Online Services >
Special Consideration. After applying online, students must also verify supporting
their documentation by submitting to UNSW Student Central:
 Originals or certified copies of your supporting documentation (Student
Central can certify your original documents), and
 A completed Professional Authority form (pdf available to download).
The supporting documentation must be submitted to Student Central for verification
within three working days of the assessment or the period covered by the
supporting documentation. Applications which are not verified will be rejected.
6
UNSW Assessment Policy
11
UNSW
Assessment
Policy
(continued)
Students will be contacted via the online special consideration system as to
the outcome of their application. Students will be notified via their official
university email once an outcome has been recorded. This could take from a
week up to a month.
SUPPLEMENTARY EXAMINATIONS:
The University does not give deferred examinations. However, further assessment
exams may be given to those students who were absent from the final exams
through illness or misadventure. Special Consideration applications for final
examinations and in-session tests will only be considered after the final examination
period when lists of students sitting supplementary exams/tests for each course are
determined at School Assessment Review Group Meetings. Students will be notified
via the online special consideration system as to the outcome of their application. It
is the responsibility of all students to regularly consult their official student
email accounts and myUNSW in order to ascertain whether or not they have
been granted further assessment.
For Session 1 2017, The BIOC2181 Supplementary Exams will be scheduled on:
th
Thursday 13 July, 2017
Further assessment exams will be offered on this day ONLY and failure to sit for the
appropriate exam may result in an overall failure for the course. Further assessment
will NOT be offered on any alternative dates.
Equity and
Diversity
Those students who have a disability that requires some adjustment in their
teaching or learning environment are encouraged to discuss their study needs with
the course Convenor prior to, or at the commencement of, their course, or with the
Equity Officer (Disability) in the Equity and Diversity Unit (9385 4734 or
http://www.studentequity.unsw.edu.au/).
Issues to be discussed may include access to materials, signers or note-takers, the
provision of services and additional exam and assessment arrangements. Early
notification is essential to enable any necessary adjustments to be made.
Student
Complaint
7
Procedure
School Contact
Faculty Contact
University Contact
Prof Marc Wilkins
Grievance Officer
School of Biotechnology
and Biomolecular
Sciences
[email protected]
Tel: 9385 53633
Dr Gavin Edwards
Associate Dean (Academic
Programs)
[email protected]
Tel: 9385 4652
Student Conduct and
Appeals Officer (SCAO)
within the Office of the
Pro-Vice-Chancellor
(Students) and Registrar.
Tel: 02 9385 8515, email:
studentcomplaints@unsw.
edu.au
University Counselling
and Psychological
8
Services
Tel: 9385 5418
7
8
UNSW Student Complaint Procedure
University Counselling and Psychological Services
12
UNSW Academic Honesty and Plagiarism
What is Plagiarism?
Plagiarism is the presentation of the thoughts or work of another as one’s own.
*Examples include:
 direct duplication of the thoughts or work of another, including by copying material, ideas or
concepts from a book, article, report or other written document (whether published or unpublished),
composition, artwork, design, drawing, circuitry, computer program or software, web site, Internet,
other electronic resource, or another person’s assignment without appropriate acknowledgement;
 paraphrasing another person’s work with very minor changes keeping the meaning, form and/or
progression of ideas of the original;
 piecing together sections of the work of others into a new whole;
 presenting an assessment item as independent work when it has been produced in whole or part in
collusion with other people, for example, another student or a tutor; and
 claiming credit for a proportion a work contributed to a group assessment item that is greater than
that actually contributed.†
For the purposes of this policy, submitting an assessment item that has already been submitted for
academic credit elsewhere may be considered plagiarism.
Knowingly permitting your work to be copied by another student may also be considered to be
plagiarism.
Note that an assessment item produced in oral, not written, form, or involving live presentation, may
similarly contain plagiarised material.
The inclusion of the thoughts or work of another with attribution appropriate to the academic discipline
does not amount to plagiarism.
The Learning Centre website is main repository for resources for staff and students on plagiarism and
academic honesty. These resources can be located via:
www.lc.unsw.edu.au/plagiarism
The Learning Centre also provides substantial educational written materials, workshops, and tutorials
to aid students, for example, in:
 correct referencing practices;
 paraphrasing, summarising, essay writing, and time management;
 appropriate use of, and attribution for, a range of materials including text, images, formulae and
concepts.
Individual assistance is available on request from The Learning Centre.
Students are also reminded that careful time management is an important part of study and one of the
identified causes of plagiarism is poor time management. Students should allow sufficient time for
research, drafting, and the proper referencing of sources in preparing all assessment items.
* Based on that proposed to the University of Newcastle by the St James Ethics Centre. Used with
kind permission from the University of Newcastle
† Adapted with kind permission from the University of Melbourne
13
BIOC2181
Lecture Summaries and Study Guide
2017
LECTURE SUMMARIES
INTRODUCTORY LECTURE
This lecture provides an introduction to the structure and topics of the BIOC2181 course.
AMINO ACIDS, PROTEIN STRUCTURE AND ENZYMES
Lecturer: Dr Rich Edwards (RE)
Introduction: Proteins are responsible for most specific functions of cells. They include the
enzymes that control and regulate the whole of the cell's metabolism, as well as the structural
material in cell membranes and connective tissue, the contractile elements, hormones and
protective agents. The human body contains about 100,000 different proteins. Fundamental
questions are: “What are they made of?”, “How do they differ?” and “How do they work?”
Proteins are very sensitive to changes in the physicochemical properties of their environment
and the maintenance of these properties at constant levels is essential to the structural and
catalytic integrity of living cells.
Amino Acids
 All proteins yield, upon hydrolysis, the same twenty amino acids and in the intact protein,
these are linked by peptide bonds.
 The sequence of amino acids determines the structure, properties and functions of peptides
and proteins.
 Structure of the common amino acids. Classification of side chains as non-polar, polar noncharged, polar charged (acidic or basic), sulfur-containing, aromatic, etc. (see below).
 Optical isomers (D and L forms).
 Properties of amino acid side chains.



acidic and basic groups.
thiol groups and their role in protein aggregation by disulfide bond formation.
aromatic rings and other hydrophobic side chains.
Proteins
 Peptide bond formation and polypeptide chain synthesis.
 Polypeptide chain polarity.
 Consequences of the stereochemistry of the peptide bond.
 The four hierarchical levels of protein structure: primary, secondary, tertiary and quaternary.
 Stabilising effects of hydrogen bonds, van der Waal’s forces, electrostatic forces,
hydrophobic interactions and disulfide bonds.
 Protein folding and denaturation.
14
BIOC2181
Lecture Summaries and Study Guide
2017
Enzymes
 Enzymes as biological catalysts.
 Enzyme specificity and catalytic power.
 Catalytic mechanisms and enzyme classification.
 Enzymes, reaction equilibrium and activation energy.
 Substrate specificity, the active site and formation of an enzyme-substrate complex.
Enzyme Kinetics
 The kinetic properties of enzyme-catalysed reactions.
 The relationship between reaction velocity and substrate concentration.
 The Michaelis-Menten Model and Equation.
 The Lineweaver-Burke plot and significance of KM and Vmax values.
 The effects of a cell’s physical environment on enzyme activity.
 Enzyme inhibition: competitive, non-competitive, reversible and non-reversible.
 The properties of allosteric enzymes.
15
BIOC2181
Lecture Summaries and Study Guide
2017
CARBOHYDRATE STRUCTURE AND CATABOLISM:
Lecturer: Dr Vladimir Sytnyk (VS)
Introduction to Metabolism
Bioenergetics
Living organisms create and maintain their essential orderliness at the expense of their
environment, which they cause to become more disordered in consequence. They are
essentially an ‘open’ chemical system existing in a steady-state condition and must therefore
extract energy, generally as chemical fuel, from their surroundings. Viewed as a machine, they
must obey the same thermodynamic laws applicable to purely physical phenomena. The study
of bioenergetics considers these energy relationships, without which the system of complex
chemical reactions unique to life processes cannot be appreciated. All life processes on this
planet have utilized a single specific molecule, adenosine triphosphate (ATP), as a
concentrated form of chemical energy to which outside energy sources (as food) are converted
and which is then used for biosynthetic purposes to maintain low entropy, i.e. highly ordered
system. ATP will be used as a typical example to illustrate energy relationships applicable to
biochemical reactions in general.
Metabolism
The term ‘metabolism’ encompasses all the chemical processes which occur within living
organisms. ‘Anabolism’ is the sum of those processes by which structural and functional
components of a cell are synthesized from simpler units. ‘Catabolism’ covers the processes
whereby complex compounds are degraded to release energy and to provide the smaller units
for the cell's synthetic processes. All living organisms break down food materials and synthesize
cell components by ordered sequences of chemical reactions called metabolic pathways.
These pathways are frequently common to all cells, thus both man and bacteria break down
glucose to CO2 and H2O by essentially the same pathway. Each chemical reaction in the cell is
catalysed by an enzyme. The operation of a metabolic pathway therefore depends on the
properties of the individual enzymes catalysing the sequence of chemical reactions.
Catabolism: The Oxidation of Fuels
Food As Fuel
Patterns of diet in terms of major fuels (carbohydrate, fat and protein) Overall energy yields and
energy requirements.
Catabolism As A Three-Phase Process
(1)
Digestion
(2)
Breakdown to three main products
(3)
Final oxidation of these common products to CO2 and H2O.
Carbohydrate Structure and Catabolism
 Carbohydrates are polyhydroxy aldehydes or ketones or yield such compounds on acid
hydrolysis.
16
BIOC2181
Lecture Summaries and Study Guide
2017
Glucose: A Model Carbohydrate
 Chemical nature of glucose. The glycosidic bond in disaccharides.
 Complex carbohydrates and their biological function.
 Digestion of important dietary carbohydrates (starch and sucrose).
Glycolysis
 Glycolysis is the universal pathway by which the six-carbon sugar, glucose, is broken down
(“oxidised”) to two molecules of the three-carbon compound, pyruvate. Although this releases
only about 5% of the “biochemical” energy of the glucose molecule, glycolysis provides the
only path for energy supply when oxygen is limiting or temporarily absent from tissues. This
limited (5%) amount of energy can be liberated very quickly in muscle tissue.
 Description of the glycolytic pathway as ‘Phase 2’ in the metabolism of carbohydrate. The
production of NADH and ATP. Glycolysis as a source of intermediates for other metabolic
processes. The importance of glycolysis in various tissues of the body.
 Stoichiometry of glycolysis.
 Metabolism of dietary fructose and galactose.
Fate of Pyruvate
 Lactate production and anaerobic metabolism.
 Ethanol production in some organisms.
 Acetyl CoA formation as a prelude to complete oxidation or for biosynthetic reactions.
Tricarboxylic Acid Cycle
 The TCA cycle is a sequence of enzyme-catalysed reactions that are common to the
catabolism of all organic fuels in aerobic tissues. Complex organic compounds polysaccharides, fats and proteins - are broken down by separate pathways to a few simple
organic compounds which then enter the TCA cycle. In the cycle the carbon atoms from the
organic fuels are finally oxidized to CO2. The energy released in the TCA cycle is used by the
associated processes of the respiratory chain and oxidative phosphorylation to make ATP.
The reactions of the TCA cycle, respiratory chain and oxidative phosphorylation provide most
of the energy in the form of nucleoside triphosphates (i.e. ATP) for most tissues.
 Description of the TCA cycle (Phase 3 of catabolism) with emphasis on its role as a producer
of intra-mitochondrial NADH, FADH2 and CO2 as well as for synthesis of metabolic
intermediates for other related (integrated) metabolic processes.
 Stoichiometry of the TCA cycle. Overall ATP yield from the breakdown of glucose via
glycolysis and the TCA cycle.
17
BIOC2181
Lecture Summaries and Study Guide
2017
OXIDATIVE PHOSPHORYLATION AND THE GENERATION OF ATP
Lecturer: Dr Nirmani Wijenayake (NWG)
Introduction
 ATP as the “energy currency” of the cell.
 Central role of mitochondria in energy transduction.
 Definition of oxidative phosphorylation.
Membrane Structure
 Brief review of membrane composition and structure.
 Fluid mosaic model.
 Integral versus peripheral membrane proteins.
 Role of fluidity in membrane function.
 Transport of metabolites across membranes.
 Membrane structure of the mitochondrion.
The Respiratory Chain of the Mitochondrion
 Concept of electron transport.
 Components of the respiratory chain - properties and locations.
 Fate of hydrogen ions formed and utilised during electron transport.
 Role of the inner membrane of mitochondria.
ATP Formation in the Mitochondrion
 Entry of ADP and Pi into, and exit of ATP from the mitochondrion.
 Control of oxygen consumption by levels of ADP and/or Pi.
 ATP synthase complex (F1 plus F0) and its ATP synthesising and hydrolysing properties.
 P/O ratios and role of transmembrane proton gradient in driving ATP synthesising activity.
 Efficiency of oxidative phosphorylation.
Shuttle Mechanisms of the Mitochondrion
 Transport into the mitochondrion of the reducing equivalents (NADH) produced in the
cytoplasm (glycolysis).
 Effect on overall ATP yield from oxidative phosphorylation.
18
BIOC2181
Lecture Summaries and Study Guide
2017
GLYCOGEN AND GLUCONEOGENESIS: MAINTAINING THE SUPPLY OF GLUCOSE
Lecturer: Dr Nirmani Wijenayake (NWG)
Introduction
In mammals, glucose must be available at all times as it is an important energy source for
rapidly contracting skeletal muscle and an essential energy source for brain and a number of
other tissues. Therefore it must be provided in the diet or synthesised by some tissues from
non-carbohydrate compounds. Furthermore the ability of mammals to consume food at
intervals depends on a capacity to store an excess of absorbed food materials for later use.
Glycogen Metabolism
 An important storage material that fulfils the function of ‘glucose storage’ is the
polysaccharide, glycogen. It is stored mainly in the liver and skeletal muscle, but provides
only a short term store of glucose. Primarily it is used to supply a mammal’s more immediate
energy requirements.
 Structure/Function of glycogen. Sites of glycogen storage.
synthesis and degradation of glycogen.
Enzymic reactions for the
 Disorders of glycogen metabolism: glycogen storage diseases.
Gluconeogenesis
 After glycogen stores are exhausted, and no glucose is available from the diet (e.g.,
starvation), glucose must be synthesised for continued functioning of glucose-dependent
tissues such as the brain and red blood cells. This is accomplished by the process of
gluconeogenesis which is the synthesis of glucose from non-carbohydrate precursors (e.g.,
lactate, certain amino acids).
 Synthesis of glucose from compounds containing five or less carbon atoms.
 Tissue sites for synthesis.
 Pathways and function of gluconeogenesis.
 Mitochondrial and cytoplasmic reactions.
 Overall stoichiometry of gluconeogenesis.
 The Cori cycle.
19
BIOC2181
Lecture Summaries and Study Guide
2017
FATS: DIGESTION, TRANSPORT, BREAKDOWN AND SYNTHESIS
Lecturers: Dr Lucy Jo (LJ) and Dr Nirmani Wijenayake (NWG)
Introduction: Fat Structure, Catabolism, and Anabolism
The term ‘lipids’ encompasses a wide range of compounds such as fats, fatty acids, steroids
and phospholipids. The roles of lipids are related to their various structures, but their two main
functions are as energy reserves, and as structural components for the maintenance of the
integrity of cells.
The main emphasis of this series of lectures will be on the synthesis of fatty acids and their
conversion to triacylglycerols (TAG) which are the stored form of ‘fat’. When a decrease in blood
glucose occurs there is hormone controlled response. This leads to the hydrolysis of stored ‘fat’
in adipose tissue to fatty acids which provide the fuel to all cells, except erythrocytes and brain.
The body has only a limited capacity to store excess glucose as glycogen, it has an almost
unlimited capacity to store fatty acids in the form of TAG (triacylglycerol). This, combined with
the high caloric value of triacylglycerol (approx. 38kJ/g) allows an average human to survive
starvation for 30 to 40 days (β-oxidation of fatty acid to acetyl-CoA provides fuel for most cells).
 Fatty acid structure. Saturated
glycerol.Glycerides. Phospholipids.
and
unsaturated
fatty
acids.
Lipids
containing
 Triacylglycerols from food. Digestion and absorption. Role of pancreatic lipases and bile
salts. Concepts of surface active agents, soaps and micelles in emulsions. Fat digestion is
essential for the absorption of fat-soluble vitamins A, D, E, K and their precursors like
carotene and dihydrocholesterol. Transport of TAG in the blood as lipoproteins. Lipoproteins
are specific types of lipid-protein complexes. The role of lipoprotein lipase (LPL) in the uptake
of fatty acids into cells. Adipose tissue as the main site for triacylglycerol storage and
mobilisation.
 Synthesis of fatty acids in the cytoplasm of liver and adipose cells. Sources of carbon, the
excess acetyl-CoA from the oxidation of glucose and amino acids. Transport of acetyl-CoA
as citrate from the mitochondria to the cytoplasm. This citrate/pyruvate ‘shuttle’ mechanism
also provides ~ half of the NADPH required for synthesis.
 Stoichiometry of fatty acid synthesis. Control of fatty acid synthesis. Synthesis of
triacylglycerols (TAG). Transport of liver TAG as VLDL to adipocytes.
 A fall in blood glucose signals the activation of the hormone-sensitive lipase in adipocytes
which hydrolyses stored TAG to glycerol and fatty acids. The fatty acids are carried in the
blood bound to serum albumin.
 Entry of long chain fatty acids into the cytoplasm. Formation of long chain fatty acylCoA
thioesters. Role of carnitine in the transport of fatty acyl groups across the inner membrane
to the mitochondrial matrix. β-Oxidation in the mitochondrial matrix. Control of fatty acid βoxidation. Energy (ATP) yields from the complete oxidation of fatty acids to CO2 & H2O.
 Excessive β-oxidation in the liver leads to the synthesis and secretion of ketone bodies
(acetoacetate and β-hydroxybutyrate) into the circulation. Ketone bodies are readily
metabolised in peripheral tissues to yield acetyl-CoA. Ketonemia, i.e. high levels of
circulating ketone bodies in the human body, can result from physiological events like
starvation or pathological situations like untreated diabetes. Ketonuria and glucosuria occur
in untreated diabetics.
 Relationships between fatty acid synthesis and oxidation. Integration of fat metabolism at the
level of enzymes, cells and organs.
20
BIOC2181
Lecture Summaries and Study Guide
2017
PROTEIN CATABOLISM AND THE UREA CYCLE
Lecturer: Dr Rebecca LeBard (RLB)
 The requirement of protein as food.
 The distinction between ‘essential’ and ‘non-essential’ amino acids.
 The ‘quality’ of various food proteins.
 Concept of nitrogen balance.
 Digestion and absorption of proteins and amino acids in the diet.
 The role of the digestive proteases (especially pepsin, trypsin, chymotrypsin, elastase and
carboxypeptidase).
 A brief coverage of transamination (aminotransferase) reactions and the formation and
disposal of the carbon skeletons.
 The central role of glutamate dehydrogenase in amino acid metabolism.
 Removal and disposal of excess nitrogenous material.
 Ammonia, uric acid and urea as the major excretory forms of nitrogen.
 The synthesis of urea by the reactions of the urea cycle will be described.
21
BIOC2181
INTEGRATION
METABOLISM
Lecture Summaries and Study Guide
OF
METABOLIC
PATHWAYS,
HORMONES
2017
AND
WHOLE
BODY
Lecturers: Dr Rebecca LeBard (RLB) & Dr Kyle Hoehn (KH)
Introduction
The basic strategy of metabolism is to form ATP, reducing power (NADPH) and the building
blocks for a number of biosyntheses. To this end cell metabolism is an economical, tightly
regulated process. Cells consume just enough nutrients to meet the rate of energy utilization at
any given time. Furthermore they produce just the right balance and quantity of building blocks
for cellular repair and expansion. An understanding of metabolism is not achieved simply by the
rote learning of pathways. It is important to consider how metabolic pathways such as
glycolysis, the TCA cycle, β-oxidation, fatty acid synthesis, the urea cycle, glycogen
metabolism, are functionally related in both single cells and multicellular organisms.
The integration and control of metabolism is modulated by a variety of factors at various levels
within a cell. These include allosteric interactions and covalent modifications of enzymes,
variation in amounts of enzymes and intracellular compartmentation. Carbohydrate and fat are
the two major energy sources available to various tissues which exhibit distinctive metabolic
profiles with respect to utilization of these two different fuel types.
In multicellular organisms the regulation of the metabolism of an individual cell must, in addition,
be integrated with the metabolic state of all the other cells. This control is mediated by two
systems; (a) the central nervous system and (b) the endocrine system. In the latter, organic
compounds called hormones are released by one type of cell and have an effect on the
metabolism of a different type of ‘target’ cell after transport in the body fluids. In humans the
hormones are released by highly specialised cells, the endocrine cells. Different types of
endocrine cells secrete different hormones and one particular endocrine tissue may secrete
different hormones under different conditions. Each hormone has a clearly defined effect on its
target cells. The hormone may be specific for the cells of one particular tissue or may produce a
spectrum of effects in different tissues. In this way these chemical messengers integrate the
function of all the various organs and tissues of the body. The lectures will present a general
survey from the following topics:
 Overview of the strategies used in controlling and coordinating metabolism in higher
organisms and revision of the major metabolic pathways.
 Control at the cellular level by allosteric enzymes, illustrated by the regulation of glycolysis,
pyruvate dehydrogenase, gluconeogenesis, the TCA cycle, β-oxidation and fatty acid
synthesis.
 The endocrine system, secretory cells and the chemical nature of hormones.
 The receptor model of hormone action; cell membrane receptors, second messengers and
the cyclic-AMP cascade, G-proteins; cytosol receptors and gene expression.
 Control of fuel metabolism by insulin, glucagon and adrenaline. Hormonal control of blood
glucose, glycogen synthesis and breakdown, and fat mobilisation.
 Carbohydrate, protein and fat composition of the western diet. Organ utilization of oxygen
and fuels - brain, blood cells, cardiac muscle, skeletal muscle, liver and adipose tissue.
 Relationship between carbohydrate and fat catabolism. The fed and the starved state.
Integration of the control of whole body metabolism as illustrated by the response to an
abnormal metabolic state, e.g. starvation, marathon running, and diabetes mellitus.
22
BIOC2181
Lecture Summaries and Study Guide
2017
Metabolic Pathway Summary
23
BIOC2181
Lecture Summaries and Study Guide
2017
STUDY GUIDE
The following questions are based on important factual material and concepts derived from the
course syllabus. These objectives do not provide a comprehensive coverage of examinable
material, but it is suggested that you use them as a guide to the basic standard necessary for
tutorial preparation and revision.
Amino Acids and Protein Structure
1.
How many different amino acids occur as constituents of proteins?
2.
How many amino acids carry side chains that ionise and what are their structures at pH
7.0?
3.
What is the primary structure of a protein?
4.
What are the differences between secondary and tertiary structure in proteins?
5.
What dictates the tertiary structure of proteins?
6.
What is meant by ‘denaturation’, and what are the chemical and physical conditions that
cause denaturation?
7.
Is denaturation a permanent state, or can a denatured protein be converted to its native
state?
8.
What covalent bonds are involved in the maintenance of tertiary structure?
9.
Which amino acids would contribute to hydrophobic interactions in a protein molecule?
10. What is meant by a ‘polypeptide subunit’, and how many subunits occur in a native
haemoglobin molecule?
Enzymes and Enzyme Kinetics
1.
How does an enzyme decrease the activation energy for a reaction?
2.
What is the active site of an enzyme? Can you list some general features of a “typical”
active site?
3.
What is the difference between coenzymes/cofactors and prosthetic groups?
4.
How is enzyme activity measured, and in what units is it expressed?
5.
Can you calculate enzyme activities, specific activities and molecular activities (turnover
numbers)?
6.
What is the Michaelis-Menten equation?
7.
What are the definitions of the terms KM and Vmax? Why can Vmax not be measured directly?
8.
Can you derive the Lineweaver-Burk equation from the Michaelis-Menten equation?
9.
How does a reversible, competitive inhibitor interact with an enzyme?
10. What does the term “allosteric” mean? How does an “effector” interact with an allosteric
enzyme? What is the significance of allosteric enzymes in the metabolism of cells?
Introduction to Metabolism
1.
What is meant by the terms catabolism and anabolism?
2.
What is the purpose of catabolism?
24
BIOC2181
Lecture Summaries and Study Guide
3.
What is the overall function of ATP in cellular metabolism?
4.
What are the functions of NAD , FAD and NADP in catabolism?
2017
+
Carbohydrates, Glycolysis and the TCA Cycle
1.
What is meant by the terms: hexose, pentose, aldose, ketose, D- and L-sugars, chiral
centre, mutarotation, anomeric carbon atom, reducing and non-reducing sugar?
2.
Give examples (using Haworth formulae) of epimers and anomers.
3.
Draw Haworth formulae for glucose, galactose, fructose, sucrose, maltose and lactose.
4.
Describe the structures of glycogen and starch. What is the nature of the major glycosidic
bonds in these two polysaccharides?
5.
How are the dietary disaccharides lactose and sucrose degraded?
6.
Can you write down the sequence of enzyme-catalysed reactions in glycolysis, complete
with names of enzymes, and cofactors?
7.
In man, which tissues are normally dependent on glycolysis for their ATP production?
8.
How is the pyruvate from glycolysis utilised in the following tissues/organisms: in most
animal tissues; in exercising, anaerobic skeletal muscle?
9.
What is the overall stoichiometry for the conversion of glucose to pyruvate?
10. How is the cytoplasmic reducing power produced in glycolysis normally utilised?
11. What is the main function of the pyruvate dehydrogenase complex in the liver of an over-fed
adult human?
12. Can you write down the sequence of reactions in the TCA cycle, including the names of the
enzymes and cofactors?
13. Can you work out how many ATPs can be formed as a result of the complete breakdown of
a glucose molecule to CO2 and H2O?
Oxidative Phosphorylation and ATP Generation
1.
What are the physico-chemical characteristics of membranes?
2.
What are the key features of lipid bilayer membranes in relation to energy transduction in
mitochondria?
3.
Can you draw a fully labelled diagram of a mitochondrion?
4.
What is meant by the term “high potential” with respect to the molecules NADH and
FADH2?
5.
Why is ATP described as a “high energy” compound?
6.
How is oxidative phosphorylation defined?
7.
What role does the inner mitochondrial membrane play in oxidative phosphorylation?
8.
Can you write down the overall sequence of reactions in the electron transport chain, and
the detailed sequence within one of the complexes?
9.
Why do electrons pass from one complex to another?
10. What is the final electron acceptor in the respiratory chain?
11. How have inhibitors of electron transport helped in the study of oxidative phosphorylation?
12. Can you give examples of electron transport inhibitors that act at Complexes I, III and IV?
25
BIOC2181
Lecture Summaries and Study Guide
2017
13. How is the oxidation of NADH or FADH2 coupled to the phosphorylation of ADP?
14. What is the name of this process and what role does the inner mitochondrial membrane
play in this process?
15. At what points in the electron transport chain are protons pumped, and how have these
points been identified? How many protons are pumped per ATP generated?
16. How does an uncoupler of oxidative phosphorylation work?
17. What is an energy transfer inhibitor? Give an example.
18. How do ADP and Pi enter the mitochondria, and how does ATP exit?
19. What are the roles of the glycerol phosphate and malate-aspartate shuttles in relation to
oxidative phosphorylation?
20. Write the overall reaction for the complete oxidation of glucose and account for the ATP
produced.
Glycogen Metabolism and Gluconeogenesis: Maintaining the Supply of Glucose
1.
What is the function of glycogen?
2.
How is the structure of glycogen related to its function?
3.
Write down the sequence of reactions for the degradation of glycogen, including the names
of enzymes and cofactors.
4.
How are the 1,6-glycosidic linkages cleaved?
5.
Write down the sequence of reactions for the synthesis of glycogen from a glucose
molecule, including the names of the enzymes and cofactors.
6.
Give an example of a glycogen storage disease, the metabolic defect and major clinical
symptoms.
7.
Define gluconeogenesis. Why is the pathway necessary?
8.
What are the major precursors for gluconeogenesis?
9.
Write down the sequence of reactions in gluconeogenesis, including structures of
intermediates and the names of enzymes and cofactors.
10. What are the essential differences between glycolysis and gluconeogenesis, and why do
these differences occur?
11. What are the major tissues responsible for gluconeogenesis?
12. Which compartments within the cell are important in gluconeogenesis?
13. What is the Cori cycle?
14. What is the significance of the term “futile cycle” in relation to gluconeogenesis?
Fat Structure, Catabolism and Anabolism
1.
2.
What are the basic chemical and physico-chemical characteristics of fatty acids and
triacylglycerols (triglycerides)?
What is a “saturated” fatty acid, and how do “unsaturated” fatty acids differ from them?
3.
What nomenclature is used to define saturated and unsaturated fatty acids?
4.
What is the basic structure of a phospholipid?
5.
Why are triacylglycerols the favoured form of energy storage in mammals?
6.
What are chylomicrons? What is the role of lipoprotein lipase?
26
BIOC2181
Lecture Summaries and Study Guide
7.
What is β-oxidation and why is it considered an important pathway?
8.
Why is carnitine important in fatty acid oxidation?
9.
What are ketone bodies? How and when (and in which tissue) are they formed?
2017
10. How and by which tissues are ketone bodies utilised?
11. What is the difference between physiological and pathological ketosis?
12. How does fatty acid synthesis differ from fatty acid degradation?
13. Why is acetyl-CoA carboxylase considered an important enzyme for fatty acid synthesis?
14. What is the significance of malonyl-CoA in fatty acid biosynthesis?
15. What cofactors are used in fatty acid synthesis and oxidation?
16. What are the sources of cytoplasmic NADPH that is needed for fatty acid synthesis?
17. How is citrate involved in lipogenesis?
18. Why are linoleic and linolenic acids essential fatty acids?
19. Which are the major tissues involved in triacylglycerol synthesis?
Protein Catabolism and the Urea Cycle
1.
What are the major digestive proteases that hydrolyse dietary protein?
2.
Why is the pancreas not digested by its own proteolytic enzymes?
3.
Describe the cascade effect of enteropeptidase on the digestive pro-enzymes.
4.
Why are some amino acids “essential” for human life (i.e. they must be provided by the
diet)?
5.
Why do enzymes and other proteins undergo turnover?
6.
Why are some proteins considered to have poor nutritional value while others have good
nutritional value?
7.
Describe the role of aminotransferases (transaminases) in amino acid metabolism.
8.
What is the prosthetic group and what is its function in aminotransferase activity?
9.
Why is glutamate dehydrogenase considered to play a central role in amino acid
metabolism?
10. Describe how the amino nitrogen of any amino acid can be used ultimately to synthesise
urea although the immediate nitrogenous precursors of urea are ammonium ion and
aspartate?
11. What is meant by “glucogenic” amino acid? Name some of them.
12. What is a ketogenic amino acid? Name at least two of them. What are the steps involved in
the biosynthesis of urea in the mammalian mitochondrion?
13. Which is the major organ of urea synthesis in the mammal?
14. How is the urea cycle related to the Krebs (tricarboxylic acid) cycle?
15. What are the roles of glutamic acid and glutamine in the detoxification and formation of
ammonia?
16. Apart from urea, what other compounds are used by organisms to dispose of excess
nitrogen?
27
BIOC2181
Lecture Summaries and Study Guide
2017
Hormones and Co-ordination of Metabolism
1.
What are the two systems that co-ordinate the activities of different organs within an
individual?
2.
What do you understand by the terms “endocrine gland”, “hormone” and “target tissue”?
3.
What are the chemical characteristics of the hormones which can and those which cannot
enter cells?
4.
How does a hormone which binds to a receptor on the external surface of the plasma
membrane of a cell affect the metabolism inside the cell?
5.
What determines whether or not a tissue responds to a particular hormone?
How may the concentrations of circulating hormones be regulated?
6.
How do steroid hormones exert their effects?
7.
What, approximately, is the time span between hormone reaching its target tissue and its
effects being seen, in the case of insulin, glucagon, and steroid hormones?
8.
What are the two major pancreatic polypeptide hormones, and what are their overall roles
in whole body metabolism?
9.
Describe how insulin is synthesized and stored prior to its release from the pancreas.
10. How does glucagon increase the breakdown and decrease the synthesis of liver glycogen?
11. What is the difference between glucagon and glycogen? (After you have answered this
question, promise yourself that you will never confuse these two words.)
12. Insulin antagonizes the effects of glucagon and epinephrine (adrenaline), primarily by
affecting which two enzymes?
Integration, Control and Whole Body Metabolism
1.
What, in metabolic terms, is meant by a “cascade”?
2.
What is the overall role of cAMP-dependent phosphorylation of enzymes in fuel
metabolism?
3.
What is the importance of hormone-sensitive lipase in fat mobilisation?
4.
During short-term fasting (i.e. 1-2 days) which hormone(s) predominate in the circulation,
and which tissue(s) provide the bulk of the metabolic fuel?
5.
During prolonged fasting, which tissues are still predominantly glucose dependent, and
how is their glucose supply maintained?
6.
Which tissue is the major site of gluconeogenesis and ketogenesis, and what factors
accelerate and inhibit these processes?
7.
During prolonged starvation, how does the metabolism of ketone bodies and fatty acids (in
most tissues) result in pyruvate being spared from oxidation?
8.
When pyruvate is being “spared from oxidation”, what is its major metabolic fate?
9.
What is meant by a “futile cycle”?
10. Outline the overall pathways which convert excess dietary carbohydrate to lipid.
11. In the “fed” and “fasted” states, what biochemical changes occur in your outline pathway?
12. How are they brought about and what is the metabolic purpose?
13. What are the major fuel molecules circulating in the blood:
(a) after a normal “western” meal?
(b) after a 48-hour fast?
28
Practicals – General Information
BIOC2181
2017
PRACTICALS
The practical work is an integral and compulsory part of BIOC2181 Fundamentals of
Biochemistry. The practicals are designed to introduce you to basic experimental techniques
and methods. Practical classes will also reinforce and extend certain aspects of the lecture
course. Therefore, you will find that if you make a serious attempt to understand the practicals,
your understanding of the course as a whole will be helped considerably.
PRACTICAL TIMES
You will be required to attend a 3-hour practical class in Weeks 3, 4, 6, 9 and 11 of Session 1.
The times set aside for practical classes are:
TUESDAY
10 am -1 pm OR 2 pm – 5 pm
Students are required to assemble in Laboratory WW122 or WW123, 1st Floor, Wallace Wurth
Building, at the beginning of their appropriate class.
GENERAL INFORMATION
(i) BEFORE you can start your practical classes in Week 3 of session, you MUST complete
an online Laboratory Occupational Health and Safety Quiz that is accessed through
the BIOC2181 Moodle site. Your final quiz mark will be checked prior to your lab in Week
3. If you have not scored 100% in the quiz by 9am on the day of your Week 3 practical
class you will NOT be permitted to attend that lab class or any subsequent lab class until
you have satisfied this requirement.
(ii) You MUST complete a pre lab quiz for each of the wet labs PRIOR to each lab class
(by 9am on the day of your lab). You MUST achieve a mark of 100% for each quiz in
order to participate in the lab class. Students who have not completed the quiz prior to
lab class will not be allowed to participate in the lab and will be marked absent. Each
quiz will be worth 1% of your final marks. You may attempt the quiz as many times as
necessary to achieve 100%. However each attempt will result in the reduction of your
final marks for that quiz (1%). Therefore it is essential you read all the lab material before
attempting the pre-lab quiz.
(iii) At the beginning of each practical class there will be a short talk on the day's experiment
(held in the laboratory). This talk will include IMPORTANT SAFETY instructions and
therefore it is essential that you arrive on time. Students who arrive late and miss the
introductory talk will not be permitted to attend the remainder of the laboratory
class because they have missed important safety information.
(iv) Students must wear a laboratory coat, safety glasses and appropriate foot protection.
Students without footwear or wearing thongs, sandals or other open shoes will
not be permitted in the laboratory.
(v) A medical certificate is required from students who are absent from the practical class
due to illness. Medical certificates are to be submitted via email to the course coordinator
within three days of the absence.
29
BIOC2181
Practicals – General Information
2017
PREPARATION
To derive the full benefit from the practical work, it is necessary to study the notes and relevant
material before the class and not just blindly follow a “recipe”. Students who adopt a “recipe
approach” generally fail to understand the practical and obtain inferior results. This, in turn,
usually means that they are unable to provide satisfactory answers for the related practical
questions and thus obtain a low mark for their laboratory assessment.
CONTINUOUS ASSESSMENT OF LABORATORY COMPONENT
Within the laboratory section of this manual, each experiment is followed by one or two question
pages in which data, associated calculations and answers to specific questions are to be
written. Each student must complete these sheets in full before the end of lab class. Your
Demonstrator will check and assess your work as being either ‘Satisfactory’ or ‘Unsatisfactory’.
If an ‘Unsatisfactory’ mark is awarded, it will be your responsibility to find out why and you will
be given an opportunity to rectify any problems. Failure to do so will result in the deduction of
marks from your final assessment mark in BIOC2181 (see below).
REMEMBER: A pass in BIOC2181 is conditional upon a satisfactory performance in the
practical program.
PRACTICAL EXAMINATION
Written examinations based on the practical course will be held during the semester in the form
of 3 practical quizzes. You will be given further information regarding the style of questions in
these quizzes well in advance of the time for which they have been scheduled. It will be
designed to test your understanding of some of the principles underlying your practical work,
and it may also test your ability to carry out quantitative calculations. This examination will
contribute 15% to your final mark in BIOC2181.
DOCUMENTING YOUR LABORATORY WORK AND ANSWERING RELATED QUESTIONS
The observations from your laboratory work must be recorded neatly at the time the
observations are made. For most experiments, there is ample space provided for this recording
of data in the practical notes themselves. ALL data, graphs and diagrams must be included in
your manual where indicated. These recorded data therefore form the bulk of the information
you will need to complete the questions at the end of each practical. These questions are
designed to make you think about your experimental results, make observations and hopefully
allow you to draw some conclusions from them. They will also help you relate your practical
work to the theory presented in BIOC2181 lectures. Since you are being assessed by your
Demonstrator on your ability to record and discuss your experimental results in a proper
scientific manner, a few things to consider when documenting your lab work include:

Write your results, observations and answers neatly and legibly in pen (pencil can be
used for rough data and graphs).

Graphs should be drawn properly on graph paper, titled and labelled correctly on both
axes (with appropriate units) with the axes ruled in clearly.

The spaces on the question sheets usually indicate the length of answers required.

Where calculations are required, include the steps in your calculations so that your
demonstrator can follow the method by which you attempted them. If the data are provided and your calculations are clearly set out and legible, your demonstrator may be
able to trace any mistakes you might make, and explain them to you. This may be
important in your attempts to rectify the mistakes and thus allow your practical work to
30
BIOC2181
Practicals – General Information
2017
be assessed as ‘Satisfactory’.
LABORATORY EQUIPMENT
All the necessary laboratory equipment required for each practical exercise will be provided for
you on the day of your class.
Instructions concerning the collection, correct use and storage of equipment will be delivered
during the introductory talk at the beginning of each lab class. Therefore, it is ESSENTIAL that
you arrive at your laboratory class on time in order to hear the full equipment and safety
discussion.
During each lab class, various items of equipment and apparatus will only be available from the
technical staff. Other pieces of equipment will be given to you by your Demonstrator or available
at the front of the laboratory. Failure to carry out all laboratory instructions and maintain a tidy
work space may result in the deduction of marks from the practical assessable component of
BIOC2181.
NOTE: You are liable for any damage to any equipment whilst it is in your care. Ensure
that you follow all instructions closely and carefully so that any such damage is easily
avoided. It is also your responsibility to ensure that all equipment is returned or
disposed of correctly, as instructed by demonstrators and technical staff.
31
BIOC2181
General Course Information
2017
ACADEMIC MISCONDUCT
Information concerning the University Regulations concerning Academic Misconduct can be
found on the UNSW website:
https://my.unsw.edu.au/student/academiclife/assessment/AcademicMisconduct.html .
It is essential that all students read this information.
Academic Misconduct may apply to any work or document related to assessment that is
submitted to the School; this includes the laboratory work you document/discuss within this
manual, the three mid-session tests and the final examinations in June.
All work submitted for assessment must represent a student's own individual efforts. Copying
or paraphrasing another person's work and using another student's experimental results are
all examples of academic misconduct (see Academic Honesty and Plagiarism).
ATTENDANCE AT CLASSES
IMPORTANT NOTE: IF STUDENTS ATTEND LESS THAN EIGHTY PERCENT OF THEIR
POSSIBLE CLASSES, THEY MAY BE REFUSED FINAL ASSESSMENT.
32
BIOC2181
Laboratory Safety
2017
LABORATORY SAFETY
Biochemical laboratories contain chemicals and equipment that are potentially dangerous when
misused or handled carelessly. Consequently, safe experimental procedures and responsible
conduct in the laboratory are essential at all times. The regulations governing conduct in the
laboratory are complex and are administered by many different government regulators. The
legislation, codes of practice, guides, standards, UNSW policy or procedures may be found on
the UNSW Safety website https://safety.unsw.edu.au/legislation.
Guidance should be sought from the NSW WHS Act & Regulation 2011, UNSW HS667
CHEMICAL Legislation, Standards and Related Codes of Practice, Environmentally Hazardous
Chemicals Regulation 2008, Guide: Guidance of the Classification of Hazardous Chemicals
under the WHS Regulations, AS/NZS 2243.2: Safety in Laboratories - Chemical Aspects, and
relevant chemical safety data sheets (SDS) to name a few! These policies, procedures,
standards and legislation applies to all university staff and students.
Section 4.11 Students are responsible for:
•
Complying with the requirements of this policy, legislation and Australian Standards
•
Following directions given to them by the person supervising their work
•
Co-operating in the performance of risk assessments
•
Participating in induction and training programs
•
Reading MSDS’s for substances to be handled prior to doing experiments
Failure to comply will result in expulsion from the laboratory class.
PPE1 REQUIREMENTS IN THE LABORATORY

Students must purchase a laboratory coat and wear it when in the laboratory. It
should be removed when leaving the lab e.g. on visits to the computer lab or toilets. Lab
coats should not be left on benches or stools but hung on the coat hooks that are provided
at the back of the laboratory.

Safety glasses MUST be worn at all times.

Disposable plastic gloves will be provided for certain manipulations. These should
be discarded after use or if torn. All gloves should be removed from your hands by first
holding the gloves at the wrist and pulling to turn them inside out before they are discarded
into one of the ‘solids waste’ containers on top of bench.

Never throw gloves or any other laboratory material into the domestic bins.

Never use gloved hands to open doors etc. Either ask someone to open the door for you or
remove one glove temporarily. Always remove gloves before leaving the lab.

Suitable foot protection must be worn. Students with bare feet, thongs, exposed shoes
or strappy sandals will not be allowed into the working area.
__________________________
1
PPE – Personal Protection Equipment
33
BIOC2181
Laboratory Safety
2017
SAFETY RULES IN THE LABORATORY
 Eating, drinking and smoking are forbidden in the laboratory.
 Students with long hair must tie it back.
 Laboratory coats and appropriate footwear (NO thongs or open-toed shoes) must be
worn at ALL times.
 All work with toxic, corrosive or flammable (etc.) chemicals must be conducted in a
fume cupboard where possible.
ALL INJURIES OR ACCIDENTS WITH CHEMICALS MUST BE REPORTED
IMMEDIATELY…Either to your demonstrator or to a member of the technical staff.
RISK ASSESSMENTS
For your own protection and that of those with whom you will be working, you should read,
before each week's experiment is started, the notes and instructions on the Risk Assessment
Sheet preceding each experiment and take note of any hazards in the procedures to be used
for that laboratory session.
Risk Assessments have been carried out on all practicals to highlight the potential for possible
risks to the users. These cover chemical, biological and physical hazards. This is to ensure that
the proper precautions are taken during all laboratory procedures.
The chemical risks have been assessed using MSDSs (Material Data Safety Sheets). These
are available on file at the front of the lab. A copy of the Hazardous Substances Policy is also
on file in the Prep Room.
As strong acids, alkalis and other toxic substances have to be used in some procedures,
the relevant safety instructions will be included at the appropriate places in the manual. Such
dangerous materials must never be pipetted by mouth, they should be manipulated with
great care and, if any come into contact with skin or clothing, wash the affected areas with water
immediately, seek assistance and any antidote that may be applied.
Poisonous solutions will be provided in automatic dispensers; these should be operated gently
and carefully because careless use can cause breakage or a spray of the reagent. Automatic
pipettes will be provided where possible.
34
BIOC2181
Laboratory Safety
2017
EMERGENCY PROCEDURES
 In the event of a fire or other serious emergency, the building may be evacuated. When the
alarm has been activated, a “get ready to evacuate” siren will sound. You should
immediately cease work and secure your workplace (e.g. cap solutions, turn off Bunsen
burners). The second stage is the “evacuate the building” call. You should immediately make
your way to the nearest exit unless another exit is designated by staff. Follow directions
from the staff and evacuation wardens and gather at the Michael Birt Gardens in
front of the Chancellery Building (near Gate 9 on High Street). You should wait there
until you have been checked off by your demonstrator.
 Emergency eye wash stations and Safety showers are installed at the back of the lab. Seek
staff help immediately. If you get something in your eye, you must wash your eyes for at least
20 minutes.
 For procedures to clean up spills, seek staff help immediately.
 Special antidotes (if using cyanide) are located near the Prep Room windows. Seek staff
help immediately.
 If you are in doubt about any safety matter, please consult a member of staff.
Internet sites/references:
National Occupational Health & Safety Commission:
http://www.nohsc.gov.au./hazsubs/index.htm
NSW Work-cover site:
http://www.workcover.nsw.gov.au/links.html
35
BIOC2181
Laboratory Safety
2017
SAFETY IN HANDLING LABORATORY CHEMICALS
PIPETTING
Essentially all hazardous solutions (acids, alkalis, toxic solutions etc.) that are needed in the
practical class will be provided in dispensers which will be set to deliver the correct volume. See
Appendix for proper handling.
For all other pipetting, pipetting aids such as Gilson Pipetmans or Eppendorfs will be provided
for use during classes. See Appendix for proper handling instructions. These should be returned
to the prep. room at the end of the class.
BROKEN GLASSWARE AND OTHER SHARP OBJECTS
Should any breakage of glassware occur, the fragments must be swept up immediately and
placed in the special bins provided for glass. These bins are located at the front of each
laboratory and are clearly marked "BROKEN GLASS ONLY". Other sharp objects e.g. needles
or razor blades should be placed in the yellow “Sharps” Bins located on each bench-top.
Broken glass or other sharp objects MUST NOT be placed in the waste paper bins or in
any other bins, UNDER ANY CIRCUMSTANCES.
DISPOSAL OF “CLINICAL” WASTE
Special labeled enamel or plastic containers are available on each laboratory bench for the
disposal of gloves, gels, tips, microcentrifuge tubes, and any other used disposable plastic ware
or Glad-wrap. Never, ever put this material in the normal domestic waste bins.
DISPOSAL OF CHEMICAL (LIQUID) WASTE
According to the Environmental Policy of the University no chemical waste may be
disposed of down the laboratory sinks.
All chemical residues must be placed in the appropriate waste containers which will be provided
in the laboratory. Solvent, aqueous, biological wastes and some chemicals may have separate
waste containers which are usually located in the fume cupboards. For disposal details, always
check your practical manual, the instructions written on the waste disposal containers in the lab,
or ask your demonstrator.
36
BIOC2181
Laboratory Safety
2017
STUDENT SAFETY DECLARATION
I,………………………………………………………………………………. (Print Full Name Please)
declare that I have completed the safety quiz for BIOC2181, that I have read the Safety in
Laboratories and Appendix Instrumentation sections in my BIOC2181 course manual, and
attended the Pre-lab safety discussion in the first practical.
I am aware of my responsibilities in the laboratory and I agree to co-operate with these
regulations.
Signature:……………………………………
Date:………………….
Countersigned by Demonstrator:………………………………………
Date:……………………………..
37
BIOC2181
Spectrophotometry
2017
SPECTROPHOTOMETRY PRACTICAL:
Introductory Lab Talk Instructions and Notes
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
……………………………………………………………………………………………………………..
38
BIOC2181
Spectrophotometry
20172
SPECTROPHOTOMETRY
THE THEORY OF SPECTROPHOTOMETRIC ANALYSIS
Spectrophotometric analysis is widely used in biochemistry for the quantitative estimation and
identification of compounds. It is usually simple, rapid and sensitive. Spectrophotometry
depends on the fact that there is a fundamental relationship between the chemical structure of
an atom or molecule and its ability to absorb (and/or emit) electromagnetic radiation. Many
compounds of biochemical interest absorb electromagnetic radiation in the ultra-violet, visible or
near infra-red light regions. These compounds give rise to characteristic absorption spectra.
Quantification is possible because the attenuation of radiant energy by absorption is described
by the same laws and equations throughout the electromagnetic spectrum.
For the quantitative estimation of compounds, use can be made of either the compound's
intrinsic ability to absorb light, or a reagent may be added which forms a coloured complex with
the compound to be estimated. Measurements are most commonly made by using light from the
visible or ultra-violet (UV) portions of the spectrum by placing the solution of interest in a
spectrophotometer or a microtiter plate reader. (see Appendix)
As well as being useful for quantitative assays, the spectrophotometer is an invaluable tool in
the identification of unknown compounds.
THEORY
1.
Lambert's Law
When a parallel beam of light traverses a homogeneous medium, its intensity is
reduced by the same relative amount throughout equal intervals of its path. Thus, if a
solution absorbs 10% of the light within the first centimetre of solution, then the second
centimetre of solution absorbs 10% of the remainder and so on.
-dI / I = µ dl
Expressed mathematically,
where dl, is an element of the light path, I is the intensity of the light and µ is the
proportion of the incident light absorbed by the medium at that particular wavelength of
the light.
-µ l
I = Ioe
Integration gives:
where Io is the intensity of the incident light, or, in the case of a solution, the intensity of
the light transmitted by the solvent.
The equation can be transformed to:
µl
Therefore
Io / I = e
log Io / I = µ l log e = αl
10
10
where α is called the absorption coefficient and l = length of the light path.
Rearranging:
α
=
=
µ log10e
or
µ
l g -1 cm -1
2.303
39
BIOC2181
Spectrophotometry
20172
The term log Io / I is called the extinction, the absorbance or optical density. The
10
recommended term is absorbance, denoted by the symbol A.
Thus, according to the derivation above
Absorbance (A) = log Io / I = αl
10
That is, absorbance depends on the absorption coefficient and path length that the light
traverses. As derived, this relationship holds for a solid with uniform absorption. For
solutions however, the effect of solute concentration must also be taken into account.
Hence in such cases, Beer’s Law must be taken into consideration.
2.
Beer's Law
The absorbance of a solution is proportional to the concentration of solute, (i.e. the
number of absorbing molecules in a non-absorbing medium) through which the light
passes.
Beer's Law and Lambert's Law can be combined to give:
A = αcl
where c is the concentration of solute and α, the absorption coefficient, is set equal to
the absorbance of a solution of unit concentration and of unit length of light path .
When citing absorption coefficients the units of concentration employed must be stated,
e.g.
1%
1cm
is the absorption coefficient for a 1% (1 g/100ml or 10 mg/ml)
solution and 1 cm light path
However, the molar absorption coefficient (ε) is written in a different way; ε is the
absorption coefficient for a molar solution in a cell of 1 cm light path. Thus units are:

-1 -1
Absorbance
litres mol. cm
“1” = length of light path
Conc.(molar) x “1” cm
through the cuvette
or microtiter plate.
If Beer's Law is obeyed by a solution, then a plot of absorbance against concentration
will give a straight line. It must be noted that this law does not hold true for all solutions
over all solute concentrations. To measure the concentration of a compound it is
necessary to construct a concentration curve to determine over which concentration
range Beer's Law is obeyed.
40
Spectrophotometry
20172
Absorbance @ λ nm
BIOC2181
Concentration (g/l)
In the Figure above, Beer's Law holds over the linear section of the graph and it is
advisable to measure the concentration of the substance up to (but not beyond) the
dotted line on the concentration axis. Nevertheless, even where Beer's law is not
obeyed, construction of a suitable curve up to a limiting concentration will still relate the
absorbance as a function of concentration.
SPECTROPHOTOMETERS
During the Session students will use two types of spectrophotometers; the LKB Novaspec and
the Multiskan MS microtiter plate reader (see Appendix: Instrumentation).
The LKB Novaspec spectrophotometer is a relatively simple spectrophotometer for use in the
visible region of the spectrum (λ = 330-900 nm). A diagrammatic representation of the light path
is shown over the page. The light source is a tungsten lamp and wavelength selection is
obtained by placing a diffraction grating between the light source and sample. The minimum
sample volume is 1 ml. The light detector is a photomultiplier. When the photomultiplier is
illuminated, electrons are released and the current generated is amplified by a cascade effect in
the photomultiplier. The signal is measured on an ammeter, the scale being calibrated either as
absorbance or transmission. The read-out is via a digital display.
41
BIOC2181
Spectrophotometry
20172
Sample of Solution
in Cuvette
Light Path ( 1 cm )
Figure: Above is a diagrammatic representation of the light path in an LKB Novaspec
Spectrophotometer.
The Multiskan MS microtiter plate reader is an 8-channel vertical light path filter photometer.
The wavelength (λ = 340-750 nm) is selected using interference filters that are held in a filter
wheel. The light source is a quartz tungsten halogen lamp and the 8 equal parallel light beams
are deflected through the bottoms of the 96 wells of the polystyrene microtiter plate to solidstate detectors that measure the intensity of the transmitted light. This is electronically
converted to an absorbance value that can be printed out. The maximum volume that can be
placed in each well is 300 µl. However, as the light path passes through the solution vertically
rather than horizontally across the well, the length of the light path is dependent on the volume
of solution in the well because the volume will determine the depth of the solution, e.g. 100 µl =
0.3 cm and 300 µl = 0.9 cm. A diagrammatic representation of the light path is shown below.
Light Source
Light Detector
Figure: Above is a diagrammatic representation of the light path in a microtiter plate reader.
Note that due to the optical arrangement, the length of the light path will depend on the volume
of solution contained within each well of the plate.
42
BIOC2181
Spectrophotometry
20172
THE TECHNIQUE OF SPECTROPHOTOMETRIC ANALYSIS
Many biological molecules do not absorb light in the visible or ultraviolet regions of the spectrum. These molecules therefore cannot be assayed directly by spectrophotometric analysis. In
many cases, however, this difficulty can be overcome by reacting such molecules with reagents
to form coloured complexes (i.e. complexes which absorb light in the visible region of the
spectrum). The amount of coloured complex that is formed may then be determined by
spectrophotometric analysis, and a value can be obtained for the absorbance (A) of the
coloured complex. According to the Beer-Lambert Law,
A = α cl
thus, the concentration (or amount per unit volume) of the coloured complex may be calculated
if "α" (the absorption coefficient at a particular wavelength) and "l" (the length of the light path)
are known.
Unfortunately, there are many cases where the α (absorption coefficient) of a particular complex
is unknown and the above equation cannot be used. In such cases, the analysis can be
quantified by constructing a STANDARD CURVE as described in the following example.
Example:
Assume that we wish to determine the amount of inorganic orthophosphate (Pi) in a sample of
biological fluid. Solutions of Pi do not inherently absorb light in the visible or ultraviolet region of
the spectrum, but Pi can be converted to a blue-coloured molybdate complex by a reaction with
ammonium molybdate under reducing conditions. To quantify the amount of the now coloured Pi
in a particular sample, it is necessary to construct a STANDARD CURVE.
A standard curve is a graph of the relationship between the absorbance (A) of the blue complex
and known amounts of Pi present in the assay. Hence, to construct the standard curve, it is
necessary to react VARIOUS KNOWN AMOUNTS of Pi with ammonium molybdate, to measure
the absorbance of each known amount and to plot a graph of each of the absorbance (y-axis)
values as a function of each known amount of the Pi complex (x axis). Thus, we can directly
relate the absorbance obtained to the EXACT amount of Pi which was originally present.
A diagram of the resultant STANDARD CURVE is shown on the next page.
43
BIOC2181
Spectrophotometry
20172
Standard Curve of Absorbance versus Amount in µmoles of Inorganic Phosphate (Pi)
Amount of Pi (µmoles )
The Standard Curve can then be used to determine the amount of Pi in a particular sample. In
the example shown in the Standard Curve above, a sample containing an unknown amount of
Pi gave an absorbance of 0.58 when reacted with ammonium molybdate. From the graph the
unknown sample must have contained 0.7 µmol Pi.
In other situations where the solution of a compound or molecule is inherently coloured (e.g. a
solution of the protein haemoglobin) the absorbance may be determined without addition of
further reagents. However, a standard curve of absorbance versus known amounts of
haemoglobin is still required to estimate the amount of haemoglobin in any given sample of
unknown amount or concentration.
44
BIOC2181
Spectrophotometry
20172
PRACTICAL 1A: BIOCHEMISTRY SKILLS
SAFETY ISSUES
Safe practices for laboratory work must be maintained at all times. This includes no eating, no
drinking, no smoking, no mobile phones in the laboratory, laboratory coats and suitable covered
shoes must be worn.
RISK ASSESSMENT
Chemical Risks
1. Acetic acid – corrosive and flammable - Avoid contact with eyes and skin. If contact
occurs wash immediately and seek medical attention.
Procedural Risks
1. Electrical Equipment (microplate reader) - Avoid water/spillages when working with
electrical items.
2. Spills and splashes must be wiped up immediately
3. Disposal of wastes into their suitable containers as directed by demonstrators and lab
staff.
AIMS
By the end of this practical you should be able to:




Confidently perform basic biochemical calculations;
Accurately pipette volumes between 10 and 100 µL;
Use the spectrophotometer;
Construct a standard curve;
INTRODUCTION
In this laboratory class you must confidently master some biochemistry skills required for the
subsequent practical classes. These are outlines in the above aims. Before you progress on to
the next section you must have each section signed off by your demonstrator.
45
BIOC2181
Spectrophotometry
20172
A. BIOCHEMICAL CALCULATIONS
1. Write 100 nM in µM:
2. A 1 molar solution (1M) refers to a concentration. What is another way to express this?
3. What is the concentration of a solution that contains 12.5 mg protein in 500 µL?
How confident were you in converting these units? 1 2 3 4 5
1 = Completely confident, 3 = unsure, 5 = No confidence
A solution of dye, Amido Black 10B dye (10 mM, 6.165 g/L), provided absorbs light much too
strongly for measurement of an absorbance value. It is therefore necessary to dilute it before
proceeding with an experiment to construct a standard curve of absorbance vs. concentration or
amount of dye. The stock 10 mM Amido Black solution needs to be diluted x200 using purified (RO)
water. To achieve the dilution, it is recommended that serial dilutions be used.
4. How would you dilute the Amido Black 10B 1 in 200 to a total volume of 3 mL?
NB: Assume you only have pipettors that cannot dispense less than 20µL.
How confident were you in carrying out this calculation? 1 2 3 4 5
1 = Completely confident, 3 = unsure, 5 = No confidence
5. What is the concentration of the diluted Amido Black 10B dye?
How confident were you in carrying out this calculation? 1 2 3 4 5
1 = Completely confident, 3 = unsure, 5 = No confidence
6. What is the amount (nmol) of Amido Black 10B dye in the diluted sample?
How confident were you in carrying out this calculation? 1 2 3 4 5
1 = Completely confident, 3 = unsure, 5 = No confidence
46
BIOC2181
Spectrophotometry
20172
B. PREPARATION OF A STANDARD CURVE
Standard curves are to be carried out in a microtiter plate like the one shown:
Create a table showing how you would set up your microplate ie. the volumes of RO water and
Amido Black 10B dye in each well. Also show the amount and concentration of Amido Black
10B dye in each well.
For example:
Wells
A1 B1 C1 D1
Volume Amido
Black 10B dye
(µL)
0
Volume water
(µL)
200
Amount
(nmol)
0
Concentration
(mM)
0
Add volumes of your diluted Amido Black 10B dye in the range 0-200 µL to wells in a
microtiter plate and make the total volume in each well up to 200 µL using RO water.
What is a suitable number of replicates for each volume? _________________________
Remember that the pipettes are not accurate for volumes less than one tenth of the maximum
volume e.g. a 200 µL pipette should not be used for volumes of less than 20 µL.
Workings:
How confident were you in carrying out this design? 1 2 3 4 5
1 = Completely confident, 3 = unsure, 5 = No confidence
47
BIOC2181
Spectrophotometry
20172
Mastery of basic biochemistry calculations:
Signed:
Demonstrated ability to use scientific units for volume,
molar concentration and weight.
Demonstrated ability to plan an experiment using
appropriate volumes and concentrations of reagents.
☐
☐
C. PIPETTING
Your demonstrator will show you how to use the pipettes in the laboratory. Following this, you will
individually set up a microtiter plate containing the amounts of acid and base tabulated below, along
with 30µL of indicator to each well. Once you have completed this task, have your demonstrator
check your work.
Column
1
2
3
4
5
6
7
8
Universal indicator (µL)
30
30
30
30
30
30
30
30
0.1 M acetic acid (µL)
120
105
90
75
62
58
45
0
0.1 M sodium carbonate (µL)
0
15
30
45
58
62
75
120
Mastery of pipetting:
Signed:
Demonstrated ability to pipette accurate volumes using a
P20 and P100/200, and to do so consistently.
Demonstrated ability to pipette into a microtiter plate.
Demonstrated ability to put on and eject pipette tips
aseptically.
☐
☐
☐
48
BIOC2181
Spectrophotometry
20172
PRCATICAL 1B: AN APPLICATION OF SPECTROPHOTOMETRIC ANALYSIS
THE MEASUREMENT OF HAEMOGLOBIN
RISK ASSESSMENT
Chemical Risks
1. 0.4% ammonia solution – mild irritant
2. 0.5% potassium ferricyanide/cyanide solution – toxic
Biological Risks
1. Ox blood – an animal product, considered to be a biological hazardous substance in
the laboratory. Wear gloves when handling.
Procedural Risks
1. Electrical Equipment (microplate reader) - Avoid water/spillages when working with
electrical items.
2. Spills and splashes must be wiped up immediately
3. Disposal of wastes into their suitable containers as directed by demonstrators and
lab staff.
AIMS
 To provide an understanding of how the haemoglobin content of blood can be determined
and the clinical significance of blood haemoglobin concentrations.
 To provide an understanding of the use of spectrophotometry to measure (assay) specific
molecules in biological samples.
 To consider issues of experimental design and limitations in quantifying biological
parameters.
INTRODUCTION
The protein haemoglobin is intensely coloured as a result of the absorption of light by the
attached heme group that binds oxygen for oxygen transport. The concentration of “coloured”
molecules can be measured using spectrophotometry. In brief, an instrument (a
spectrophotometer) generates a beam of light at a particular wavelength (the wavelength
chosen is normally that at which absorption is maximal) and measures how much light is
absorbed when the beam passes through a solution. The absorbance (A) of the solution is
directly proportional to the concentration of the light-absorbing molecule.
A= εcl
Where ε = a constant (the absorption coefficient), c = the concentration, and l = the path length
of the light of beam through the solution.
An absorbance of 1.0 means that only 10% of the light passes through the solution (90% is
absorbed) and an absorbance of 2.0 means that only 1% of the light passes through the
solution. It is therefore difficult to measure absorbance values >2 accurately, and most
spectrophotometers can only measure absorbance values reliably in the range 0-2. As a result,
it is often necessary to dilute samples before absorbance measurements are made.
49
BIOC2181
Spectrophotometry
20172
EXPERIMENTAL PROTOCOL
Construct the standards in pairs but each student should work on their own unknown on
the same plate.
Reagents:
Standard haemoglobin solution (5g/100mL). The haemoglobin has been
converted to the stable methemoglobin cyanide by addition of potassium
ferricyanide/cyanide.
“Unknown” haemoglobin solution Unknown (A to F) = __________
Aqueous ammonia (4g NH3/litre)
Standard: Dilute the standard 1 in 10 by pipetting 100 µL of the haemoglobin standard into a
microfuge tube, then making the volume up to 1000 µL with aqueous ammonia. Mix the
contents of the tube thoroughly by gently inverted and label it “diluted standard”.
Unknown: Independently make 1 in 10 dilution of an unknown (A-F) by pipetting 100 µL sample
of an “unknown” blood sample to 1000 µL with aqueous ammonia in another sample tube and
label “unknown (A-F).
Procedure: Transfer samples of the “diluted standard” and diluted “unknown” samples into your
microtiter plate according to the volumes (µL) indicated in the diagram below. Then make each
well up to a final volume of 300 µL with aqueous ammonia.
Standards
1
2
3
4
5
6
7
Unknown
___
Student 1
Unknown
___
Student 2
8
10
9
11
A
0
20
30
40
60
80
100
50
100
50
100
B
0
20
30
40
60
80
100
50
100
50
100
12
C
D
E
F
G
H
Measure the absorbance of the wells on the microtiter plate reader (a type of
spectrophotometer) at 540 nm. The microtiter plate reader will be set to use well A1 as the
blank.
50
BIOC2181
Spectrophotometry
20172
RESULTS
1. Complete the following results table.
Table 1: Haemoglobin Standards and Unknowns
Sample
Column No.
Standards
1
2
3
4
5
6
7
0
0.1
0.15
0.2
0.3
0.4
0.5
Your
unknown
Your
unknown
(50)
(100)
8/10
9/11
Absorbance at
540nm
Mean
Absorbance
Mean – blank
mg of protein
per well
2. Plot a standard curve of mean absorbance (A) against amount (mg) of haemoglobin
added to each well. Show your graph to your demonstrator to check that you have used
the correct method for presenting graphical data.
3. Where the absorbance values fall on your standard curve, determine the amount of
haemoglobin (mg) in each volume of your “diluted unknown” (50 or 100 µL). Then take
into account the volume used and the dilution to calculate values for the original
“unknown” haemoglobin sample (in g Hb/100mL).
4. The “unknown” blood samples were diluted x4 from whole blood as part of the process
of lysing the cells and sample preparation for the assay. Calculate the haemoglobin
concentration (in g per 100 mL) in the whole blood sample from which your “unknown”
was obtained.
Concentration of haemoglobin in Unknown (A-F) __________ was __________ g/100 mL.
51
BIOC2181
Spectrophotometry
20172
Glue a copy of your labelled standard curve
onto this space
(about 10 cm high x 12 cm wide would be a good size)
52
BIOC2181
Spectrophotometry
20172
COMMENTS AND QUESTIONS
1. Comment on the shape of your standard curve. (HINT: Is it linear?)
2. In this experiment, what is the purpose of the ‘blank’ wells (Column 1) in the microtiter plate?
3. Why were two different volumes of unknown solution tested? (HINT: Look at your standard
curve – what might happen if only one volume was tested?)
4. How similar (or different!) were the replicates in your experiment and how could the
accuracy of the determination of your “unknown” haemoglobin sample be improved?
5. Comment on the haemoglobin concentration you have calculated for the unknown whole
blood compared with the normal ranges for male and female adult humans provided below.
Normal Adult Blood [Haemoglobin]
Males
14-18 g/100 mL
Females
12-16 g/100 mL
Mastery of spectrophotometry:
Signed:
Demonstrated ability to use the spectrophotometer; to
insert plate, select blank well and run protocol.
Demonstrated ability to construct a standard curve;
Demonstrated ability to clearly answer questions.
☐
☐
☐
53
BIOC2181
Enzymes
20172
ENZYMES PRACTICAL:
Introductory Lab Talk Instructions and Notes
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
54
BIOC2181
Enzymes
20172
PRACTICAL 2: ENZYMES
AIM
 To study the general characteristics of enzyme-catalysed reactions.
 To investigate the effect of a factor, such as pH, temperature or enzyme concentration, on
the initial rate of an acid phosphatase catalysed reaction, using a stopped assay.
BACKGROUND
All biological organisms depend on enzyme-catalyzed reactions for their functioning, their
biological integrity, their ability to adjust to changes in the environment, in other words, for their
existence. Thus, the study of enzyme-catalyzed reactions is of great importance in acquiring an
understanding of the functioning of organisms. This is best done by examining, in the
laboratory, those factors which affect the rate of enzyme-catalyzed reactions. To be suitable for
use in a class experiment an enzyme must have certain properties:
(i)
(ii)
(iii)
Stability over a long period following extraction from its tissue of origin;
The measurement of activity must be simple, reliable and precise i.e. can be repeated,
giving reproducible results;
Obtainable in sufficient quantity for large numbers of students to be able to use.
Phosphatases are a class of hydrolytic enzymes occurring in the human body and found in a
wide variety of organs, e.g. bone, kidney, red blood cells, liver and prostate gland as well as
serum and intestinal mucosa. They also occur widely in other organisms. The pH optima of
these enzymes range from acid (approx. pH 5) to alkaline (approx. pH 9).
The action of an acid phosphatase is:
OR
O
P
O
O-
acid phosphatase
OH
+ H2O
R
OH
+
HO
P
OH
O
----------------------------------------------------------------------------------------------------------------Question: In what way would the above equation be different for a phosphatase with an
alkaline pH optimum?
----------------------------------------------------------------------------------------------------------------Phosphatases represent a suitable class of enzyme for study in the laboratory and a
phosphatase of plant origin, wheat-germ acid phosphatase, has been chosen. The enzyme is
so called because it gives optimal catalysis at acidic pH values. Also, it is stable in the
laboratory, estimation of its activity is simple and reliable, and its properties are typical in a
qualitative manner of many enzymes.
The rate of an enzyme-catalyzed reaction is altered by many factors such as pH, temperature,
concentrations of substrates and products. For this reason the only meaningful and reproducible
measurement of enzyme activity is given by the initial rate of reaction. In general terms there
are two approaches to the measurement of initial rates of reaction, continuous assays and
stopped assays.
Continuous assays continuously monitor the formation of products or disappearance of
substrates. These allow easy extrapolation to obtain the initial rate, but involve relatively
complex equipment.
55
BIOC2181
Enzymes
20172
In comparison, stopped assays allow the reaction to proceed for a fixed time, for example
10 minutes, before the enzyme is denatured to stop the reaction and the products formed, or
substrates utilized, are measured. This type of method has the advantage of simplicity, but
assumes that the rate of reaction is linear over the time course of the assay. A stopped assay
will be used in today's practical to investigate wheat germ acid phosphatase and an artificial
substrate, p-nitrophenylphosphate (p-NPP). In the reaction the substrate is converted to an
inorganic phosphate and p-nitrophenol (p-NP). The reaction will be stopped by the addition of
an alkali (NaOH) to inactivate the enzyme. In addition, this rise in pH causes the ionisation of
the product p-nitrophenol, resulting in the formation of an intensely yellow-coloured anion, which
can be estimated by its absorbance at 405 nm. This reaction is summarized in Figure 1.
NO2
PO4
-
pH 5
NO2
OH + H2PO4
-
acid
phosphatase
acid phosphatase
alkali (OH )
NO2
O
-
+ H 2O
Figure 1. The reaction catalysed by wheat germ acid phosphatase.
INTRODUCTION
In pairs, you will carry out two experiments. All pairs will perform Experiment 1 - the
construction of a standard curve. In addition, each pair will perform either Experiment 2 (a time
course of the reaction), Experiment 3 (the effect of pH on reaction rate) OR Experiment 4 (the
effect of enzyme concentration on reaction rate). Each of the four experiments are outlined
below.
1. Standard curve – This will be constructed using p-nitrophenol and measuring its
absorbance at 405 nm, so that the enzymatic activity can be quantified.
2. Time course of reaction - The formation of p-NP from p-NPP will be followed over an
extended period of time to establish how long the rate of reaction remains linear. This
will allow the selection of a suitable time period for the stopped assay.
3. The effect of pH on the initial rate of reaction will be determined.
4. The effect of enzyme concentration on the reaction rate will be determined.
All experiments will be carried out in a microtiter plate at room temperature. The absorbance of
the wells for each experiment will be simultaneously read at 405 nm. Raw absorbance values
will be printed out, because the blanks for each experiment are different. You will need to
manually subtract the appropriate blank values, as indicated in the text.
56
BIOC2181
Enzymes
20172
NOTE: Start the section “Setting up of the microtiter plate” before beginning any of the
experiments 1 to 4.
RISK ASSESSMENT
Chemical Hazards
1.
0.4 M NaOH - corrosive
2.
p-Nitrophenol – toxic
Procedural Hazards
1.
Use of Pi pumps for larger volumes.
2.
Spills and splashes.
Waste Disposal
1.
Microtiter plate – empty into the labelled buckets provided next to the sinks
2.
Other solutions – check with your demonstrator.
3.
Used tips – into the yellow sharps bin.
Cleaning
1.
Microtiter plate – once the liquid is discarded, throw into a biological waste bin.
2.
Wipe down your bench area with CaviCide.
Take care with pipetting and the mixing solutions in the small volumes of a 96-well microtiter
plate as the success of this and subsequent assays is mainly dependent on pipetting
accurately, thorough mixing of samples and precise timing.
SETTING UP OF THE MICROTITER PLATE
To take best advantage of the microtiter plate format and minimise pipette swapping for the
different experiments, the microtiter plate should be set out as described below. take care with
the pipetting and mixing of the solutions in the small volumes of a 96-well microtiter plate, as the
success of this and subsequent assays is mainly dependent on:
 Pipetting accurately;
 Thorough mixing of samples;
 Accurate timing.
57
BIOC2181
Enzymes
20172
Method
1. Add 50 µl of 0.1 M citrate buffer (pH 5) into the wells of your microtiter plate, as indicated by
the shaded cells below in Table 1. REMEMBER that you only need to follow the set-up
instructions (below) for the experiments that you and your partner have been allocated.
Table 1: The addition of citrate buffer (pH 5.0).
Experiment 1
Standard Curve
1
2
Experiment 2
Time Course
Experiment 3
Effect of pH
3
4
Experiment 4
Effect of
Enzyme Conc.
5
A
B
C
D
E
F
G
H
2. For experiment 3, pipette 50 µl of each of the 0.1 M citrate buffers with different pH values
into wells B4, C4, E4 and F4, and 50 µl of ach of the 0.1 M Tris buffers into wells G4 and H4,
as indicated in Table 2, below.
Table 2: The addition of buffers with varying pH values.
Experiment 1
Standard Curve
1
2
Experiment 2
Time Course
Experiment 3
Effect of pH
3
4
Experiment 4
Effect of
Enzyme Conc.
5
A
B
C
pH 3.0
(citrate)
pH 4.5
(citrate)
D
E
F
G
H
pH 5.5
(citrate)
pH 6.0
(citrate)
pH 7.0
(Tris)
pH 9.0
(Tris)
58
BIOC2181
Enzymes
20172
3. Pipette 40 µl of 2.5 mM p-NPP (enzyme substrate) in the shaded microtiter plate wells
indicated below in Table 3. NOTE: DO NOT confuse the substrate (p-NPP) with the product
(p-NP).
Table 3: The addition of substrate (p-NPP).
Experiment 1
Standard Curve
1
2
Experiment 2
Time Course
Experiment 3
Effect of pH
3
4
Experiment 4
Effect of
Enzyme Conc.
5
A
B
C
D
E
F
G
H
59
BIOC2181
Enzymes
20172
1. PREPARATION OF A STANDARD CURVE FOR p-NITROPHENOL
Reagents
 0.25 mM p-NP
 0.4 M NaOH
Method
1.
Write out how you will construct a standard curve for p-NP containing five different amounts
of p-NP (up to a maximum of 40 nmol p-NP) using Table 4 to assist you. (Note that an
example series of five different p-NP amounts are shown in the first row of Table 4 to
guide you). Determine the volumes of p-NP and water required so that each well contains
the appropriate quantity of p–NP in a final volume of 160 µL. Show your protocol to
your demonstrator before proceeding.
NOTE: Wells A1 and A2 contain no p-NP and are used as the blank i.e. they contain 160 µL of
water only. Also, each well is to be prepared in duplicate.
Table 4: Setup for standard curve
A1
A2
B1
B2
C1
C2
D1
D2
E1
E2
F1
F2
nmol p-NP per well
0
8
16
24
32
40
Volume 0.25 mM p-NP (µL)
0
Microtiter Plate Wells
Distilled water (µl)
160
DO NOT confuse the product (p-NP) with the substrate (p-NPP) used in later experiments.
2. To each well add 40 µL 0.4 M NaOH.
3. Read the absorbance at 405 nm (after all experiments have been completed) and record
your results in Table 5.
60
BIOC2181
Enzymes
20172
Results
Table 5: Absorbance readings for standard curve.
Microtiter Plate Wells
nmol p-NP per well
A1
A2
B1
B2
C1
C2
D1
D2
E1
E2
F1
F2
0
8
16
24
32
40
0
Absorbance
0
Mean absorbance*
0
* Note that the mean absorbance value for wells A1 and A2 has automatically been subtracted
from the raw mean absorbance values for wells B1/B2 to F1/F2 via the spectrophotometer
program settings.
4. Plot a standard curve of corrected absorbance against nmol of p-NP per well. This standard
curve should be used for the remaining parts of this experiment.
61
BIOC2181
Enzymes
20172
2. VARIATION OF REACTION RATE WITH TIME
The rate of an enzyme-catalysed reaction changes with time as the substrate is utilized and
product levels increase. You will be studying this phenomenon by preparing seven incubation
mixtures of citrate buffer (pH 5.0) and p-nitrophenyl phosphate (substrate), each in a different
well. At specific times, enzyme will be added to a particular well to begin the reaction. The
reaction will be stopped, and the p-NP (product) colour developed by addition of alkali, at the
times indicated.
Reagents




2.5 mM p-NPP (Mr = 371.15; 2.5 mM = 0.928 g/L)
Acid phosphatase ___________ mg/mL (Record the concentration from the blackboard)
0.1 M Citrate buffer pH 5.0
0.4 M NaOH
Method
1.
Ensure that you have already followed the instructions in the section “Setting up of the
microtiter plate” to add citrate buffer and substrate in preparation for this experiment.
2.
Using Table 6, add enzyme (70 µl) and 0.4 M NaOH (40 µL) at the times shown.
REMEMBER:

Times are calculated from 0 min, ie. the time at which enzyme was added to well B3.

NaOH is added in the reverse order to that of the enzyme.

Well A3 will be used as the blank - do not add enzyme to this well.
Table 6: Summary of the times that reaction components are to be added.
Well
Enzyme addition time
(70 µL)
NaOH addition time
(40 µL)
Total incubation
time of the well
B3
C3
D3
E3
F3
G3
H3
0 min
1 min
2 min
3 min
4 min
5 min
6 min
30 min
26 min
22 min
18 min
16 min
13 min
10 min
30 min
25 min
20 min
15 min
12 min
8 min
4 min
3.
When you have finished the incubation, add 70 µL of distilled water and 40 µL of
0.4 M NaOH to well A3 (reaction blank).
4.
Read the absorbance at 405 nm (after all experiments have been completed).
Results
1.
Record your results for this section in the space provided in Table 3. The amounts of p–NP
liberated can be obtained from your standard curve.
62
BIOC2181
Enzymes
20172
2.
Plot a curve of nmol p-NP released against time.
3.
From this curve, determine the initial reaction velocity (place your answer on p.65).
4.
Calculate the specific activity of the enzyme (place your answer on p.65).
Table 7: Variation of reaction rates with time
Incubation
Well
Time
A3
B3
30 min
C3
25 min
D3
20 min
E3
15 min
F3
12 min
G3
8 min
H3
4 min
Absorbance at
405 nm *
p-NP
(nmol per well)
0.00
0
63
BIOC2181
Enzymes
20172
3. THE EFFECT OF pH ON THE ACTIVITY OF ACID PHOSPHATASE
Reagents





2.5 mM p-NPP
Acid phosphatase ___________ mg/mL (Record the concentration from the blackboard)
0.1 M Citrate buffers at pH 3.0, 4.5, 5.0, 5.5 and 6.0
0.1 M Tris buffers at pH 7.0 and 9.0
0.4 M NaOH
Method
1. Ensure that you have already followed the instructions in the section “Setting up of the
microtiter plate” to add buffer and substrate in preparation for this experiment.
2. Using Table 8, add enzyme (70 µl) and 0.4 M NaOH (40 µL) at the times shown.
NOTE: Take care that you add the correct solution the correct well.
Table 8: The times and volumes of enzyme and NaOH to be added.
Time
Procedure
Well
pH
0 min
Add 70 µl enzyme
B4
3.0
1 min
Add 70 µl enzyme
C4
4.5
2 min
Add 70 µl enzyme
D4
5.0
3 min
Add 70 µl enzyme
E4
5.5
4 min
Add 70 µl enzyme
F4
6.0
5 min
Add 70 µl enzyme
G4
7.0
6 min
Add 70 µl enzyme
H4
9.0
10 min
Add 40 µl 0.4 M NaOH
B4
3.0
11 min
Add 40 µl 0.4 M NaOH
C4
4.5
12 min
Add 40 µl 0.4 M NaOH
D4
5.0
13 min
Add 40 µl 0.4 M NaOH
E4
5.5
14 min
Add 40 µl 0.4 M NaOH
F4
6.0
15 min
Add 40 µl 0.4 M NaOH
G4
7.0
16 min
Add 40 µl 0.4 M NaOH
H4
9.0
3. After the last (16 min) aliquot of NaOH has been added, add 70 µL of distilled water and 40
µL of 0.4 M NaOH to well A4.
NOTE: For this experiment, a 10 minute incubation is used. This assumes that in Experiment 2,
the initial reaction rate was linear for this length of time.
64
BIOC2181
Enzymes
20172
Results
1.
Read the absorbance at 405 nm (after all experiments have been completed) and record
your results for this section in the space provided in Table 9. The amounts of p-NP liberated
can be obtained from your standard curve.
2.
Draw a graph of enzyme activity (i.e. column 4 in Table 9 below: nmol p-NP per well per 10
mins) plotted against pH and determine the pH optimum of the acid phosphatase.
Table 9: Absorbance readings for the effect of pH on reaction rate.
pH
Well
pH 3.0
B4
pH 4.5
C4
pH 5.0
D4
pH 5.5
E4
pH 6.0
F4
pH 7.0
G4
pH 9.0
H4
Absorbance at
405 nm
nmol p-NP
produced per well
per 10 mins
65
BIOC2181
Enzymes
20172
4. THE RELATIONSHIP BETWEEN THE INITIAL RATE OF REACTION AND ENZYME
CONCENTRATION
In this experiment, enzyme concentration will be varied and its effect upon the initial velocity of
the enzyme reaction will be studied.
Reagents




2.5 mM p-NPP
Acid phosphatase ___________ mg/ml (Record the concentration from the blackboard)
Citrate buffer pH 5.0
0.4 M NaOH
Method
1.
Ensure that you have already followed the instructions in the section “Setting up of the
microtiter plate” to add buffer and substrate in preparation for this experiment.
2.
Using Table 10, add the following amounts of water to the wells specified. This will
compensate for the varying volumes of enzyme solution added to each well.
NOTE: For this experiment, a 10 minute incubation is used. This assumes that in Experiment 2,
the initial reaction rate was linear for this length of time.
Table 10: Volumes of water to be added to the microtiter plate.
3.
Well
Distilled water
A5
70 µl
B5
55 µl
C5
40 µl
D5
25 µl
E5
10 µl
F5
0 µl
The varying volumes of enzyme solution are now to be added to the respective microtiter
plate wells at staggered times. Make the enzyme and NaOH additions as outlined in Table
11. Remember to change the volume of the pipettor between each addition and mix
thoroughly.
66
BIOC2181
Enzymes
20172
Table 11: The times and volumes of enzyme and NaOH to be added to microtiter plate.
Time
Procedure
Well
0 min
Add 15 µl enzyme
B5
1 min
Add 30 µl enzyme
C5
2 min
Add 45 µl enzyme
D5
3 min
Add 60 µl enzyme
E5
4 min
Add 70 µl enzyme
F5
10 min
Add 40 µl 0.4 M NaOH
B5
11 min
Add 40 µl 0.4 M NaOH
C5
12 min
Add 40 µl 0.4 M NaOH
D5
13 min
Add 40 µl 0.4 M NaOH
E5
14 min
Add 40 µl 0.4 M NaOH
F5
4.
After the last (14 min) reaction has been stopped, add 40 µl of 0.4 M NaOH to well A5.
5.
Read the plate at 405 nm.
Results
1.
Record your results in the space provided in Table 12. The amounts of p-NP liberated can
be obtained from your standard curve and the concentration of enzyme used in each
reaction can be determined using the concentration given on the blackboard.
Table 12: Absorbance readings for the effect of enzyme concentration on reaction rate.
Well
Final concentration
of enzyme in well
(mg/ml)
Absorbance
(405 nm *)
nmol p-NP
produced per well per
10 min
B5
C5
D5
E5
F5
2.
Plot V (the initial reaction velocity) in nmoles per well per 10 minutes against the
concentration of enzyme used.
67
BIOC2181
Enzymes
20172
Glue copies of your graphs onto this space
68
BIOC2181
Enzymes
20172
QUESTIONS
EXPERIMENT 2. VARIATION OF REACTION RATE WITH TIME
Q1. From your curve, what is the initial reaction velocity?
___________ nmol p-NP released/min
Q2. What is the specific activity?
___________ nmol p-NP released/min/mg protein
Q3. In the subsequent experiments, the amount of product formed after 10 minutes is
measured. Does this provide a valid estimate of the initial reaction velocity? Explain.
___________________________________________________________________________
___________________________________________________________________________
___________________________________________________________________________
EXPERIMENT 3. THE EFFECT OF PH ON THE ACTIVITY OF ACID PHOSPHATASE
Q1. From your results, what is the optimum pH of acid phosphatase? __________________
Q2. Give two reasons why enzyme activity may be affected by changing pH?
____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________
Q3. List three factors that should be considered when selecting a buffer?
____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________
69
BIOC2181
Enzymes
20172
EXPERIMENT 4. THE RELATIONSHIP BETWEEN THE INITIAL RATE OF REACTION AND
ENZYME CONCENTRATION
Q1. Is the relationship between the initial reaction velocity and concentration of the enzyme
linear? Should it be? Explain your answer.
____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________
_________________________________________________________________________
Q2. What would be the effect of increasing the enzyme concentration considerably?
____________________________________________________________________________
____________________________________________________________________________
_________________________________________________________________________
Assessment:
SATISFACTORY / UNSATISFACTORY
Demonstrator’s signature:……………………………. Date: .................................
70
BIOC2181
Oxygen Electrode Simulation
20172
PRACTICAL 4: OXYGEN ELECTRODE SIMULATION
A “Virtual” Laboratory
This computer-based “virtual” practical will take you through a series of experiments using an
oxygen electrode to closely study the process of oxidative phosphorylation.
Detailed notes on this activity and accompanying questions will be provided via Moodle and
lectures closer to the week of the practical.
In preparation for this practical, it is highly recommended that you revise your notes from your
lectures on oxidative phosphorylation and review any text book references that may assist you
with your understanding of this process. If you do not prepare for this activity in advance, you
will not receive the maximum learning benefit from the virtual experiments.
In order to gain a satisfactory mark for this virtual laboratory class, you must thoroughly
complete all questions provided with the activity. If you have completed the task to a satisfactory
standard, the course coordinator will award you with a satisfactory grade.
71
BIOC2181
Glycolysis
2017
GLYCOLYSIS PRACTICAL:
Introductory Lab Talk Instructions and Notes
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
………………………………………………………………………………………………………………………
72
BIOC2181
Glycolysis
2017
PRACTICAL 3: GLYCOLYSIS IN SKELETAL MUSCLE
AIMS

To demonstrate the function of the glycolytic pathway in a cell extract by measuring the
production of lactate (lactic acid) from glycolytic intermediates.

To investigate the cofactor requirements for glycolysis and the rate-limiting step(s) in the
pathway.

To provide an understanding of the significance of redox reactions in glucose catabolism
and NAD+/NADH cycling in anaerobic glycolysis and lactate formation.
SAFETY ISSUES
Safe practices for laboratory work must be observed at all times. This includes no eating,
drinking or smoking in the laboratory and wearing laboratory coats and suitable enclosed shoes
– NO THONGS or open sandals.
INTRODUCTION
There are two separate experiments in this practical session:

PART A - Investigating what components (cofactors) are required for glycolysis to take
place using a cell-free extract of skeletal muscle.

PART B - Investigating the conversion of different glycolytic intermediates to lactate by a
cell-free extract of skeletal muscle.
Students will work in pairs and will be assigned to one of these two experiments by their
demonstrator. The time allocation will be:
20 min
90 min
30 – 60 min
Initial discussion with demonstrators and allocation of practical tasks
Completion of practical tasks
Reporting and discussion of results within the group
73
BIOC2181
Glycolysis
2017
GENERAL INTRODUCTION
Glycolysis is a “universal” catabolic pathway for the conversion of glucose to pyruvate (pyruvic
acid). A cell-free extract can be prepared from skeletal muscle that contains all the glycolytic
enzymes and lactate dehydrogenase (LDH) for the subsequent reduction of pyruvate to lactate.
(See below for schematic of glycolytic pathway and conversion of pyruvate to lactate). A cellfree extract of this type, prepared from rat skeletal muscle, is used in this practical session to
investigate glycolytic function.
GLUCOSE
ATP
ADP
G-6-P
F-6-P
ATP
ADP
F-1,6-bisP
DHAP
GAP
NAD + + Pi
NADH + H+
2 x 1,3-bisPG
ADP
ATP
2 x 3-PG
2 x 2-PG
H2O
2 x PEP
ADP
ATP
2 x PYR
LDH
LACTATE
NADH + H+
NAD+
The experiments in this practical are conducted in two distinct stages:
1. Incubations with skeletal muscle extract
Initially rat skeletal muscle extract is incubated for 10 min in buffer and with different
components that may be required for glycolysis. In these incubations, glycolysis will result in
the production of lactate, and the amount of lactate formed is a measure of glycolytic “activity”.
These incubations are terminated by the addition of perchloric acid to denature and precipitate
the glycolytic enzymes and other proteins present in the skeletal muscle extract.
74
BIOC2181
Glycolysis
2017
2. Lactate assays
The incubation tubes from stage 1 are centrifuged to remove the precipitated proteins, and the
clear supernatants are assayed to measure the amount of lactate that has been formed. The
lactate assay uses the LDH enzyme to oxidise lactate to pyruvate, with NAD+ being reduced to
NADH in the process. NADH absorbs light strongly at 340 nm, while NAD+ and other assay
components do not absorb significantly at this wavelength. The increase in absorbance at 340
nm is therefore proportional to the amount of NADH formed, which is equal to the amount of
lactate present (assuming that the LDH-catalysed reaction goes to completion). Thus changes
in absorbance at 340 nm can be used to determine the amount of lactate produced in the initial
incubations.
RISK ASSESSMENT
Biological Risks - Rat skeletal muscle cell-free extract is an animal product and therefore
potentially hazardous.
Chemical Risks – Hydrazine (possibly carcinogenic) and perchloric acid (oxidising and corrosive
agent) are chemical hazards.
Procedural Risks – Safe operating procedures for microcentrifuges must be observed.
All spillages must be wiped up immediately. Solid and liquid waste must be disposed of as
directed by demonstrators and laboratory staff. Disposable gloves and safety glasses will be
provided for students who wish to use them and do not have their own.
75
BIOC2181
Glycolysis
2017
PART A. Investigating the Cofactor Requirements for Glycolysis.
This practical component should be attempted by 2-4 pairs of students in each group.
Reagents:








Reaction Mix “A” (containing triethanolamine HCl buffer (pH 7.4),
KCl, MgCl2 and fructose 1,6-bisphosphate (F1,6BP, 6 mM)
NAD+/ ADP/Pi mix (6/80/80 mM)
NAD+/ ADP mix (6/80 mM)
NAD+/ Pi mix (6/80 mM)
ADP/Pi mix (80/80 mM)
Rat skeletal muscle cell-free extract
Glycine/Hydrazine buffer, pH 9.4 (containing 6 mM NAD+)
Lactate dehydrogenase (LDH) solution
Experimental
In this experiment, rat skeletal muscle cell-free extract will be incubated with a glycolytic
intermediate (F1,6BP) and various combinations of NAD+, ADP and Pi. The muscle extract
contains all the enzymes required for the conversion of glucose to lactate via glycolysis. The
incubations are stopped by the addition of perchloric acid and the amount of lactate that has
been formed is measured using an enzymatic lactate assay. Analysis of the results will reveal
the cofactor requirements for glycolysis to occur using the muscle extract. Note that thorough
mixing of solutions are required to achieve good results.
1) Add 400 µl Reaction Mix “A” to each of 6 microcentrifuge tubes (labelled 1-6).
2) Add 100 µl RO water to tubes 1 and 2, and 100 µl NAD+/ ADP/Pi mix, NAD+/ ADP mix,
NAD+/Pi mix and ADP/Pi mix to tubes 3-6 respectively.
3) Add 200 µl skeletal muscle extract to tubes 2-6, noting the time of addition and mixing well
by inversion immediately after each addition, and then place each tube in a 37° C water
bath.
4) Exactly 10 min after the addition of the skeletal muscle extract, add 300 µl perchloric acid to
each of tubes 2-6 and mix well by inversion.
5) At a convenient time during these incubations, add 200 µl skeletal muscle extract to tube 1
and immediately follow this by the addition of 300 µl perchloric acid. This tube is a “zero”
time point.
6) Wait at least 2 min after the addition of perchloric acid to your last incubation tube before
centrifuging all six tubes for 3 min in a microcentrifuge.
7) Add 3 x 80 µl samples of each supernatant to three wells in a microtiter plate. Add 200 µl
Glycine/Hydrazine buffer to each well containing sample. Then add 20 µl LDH solution to
each well, keeping the time taken for these additions to the absolute minimum. (It should be
possible to make the 18 additions of LDH in less than 2 mins.) After adding the LDH to all
wells, carefully mix the contents of each using a micropipettor.
76
BIOC2181
Glycolysis
2017
8) Wait 20 min for the lactate dehydrogenase reaction to go to completion before reading the
absorbances at 340 nm. During this waiting time, check that you understand how to
calculate the experimental results.
Calculations
Calculate the average absorbance (A) values from each of your six incubations. Note that Tube
1 provides a “blank”, since the enzymes in the skeletal muscle extract were denatured before
any glycolysis and lactate formation could take place by the immediate addition of perchloric
acid. Therefore, the spectrophotometer has been programmed to automatically subtract the
absorbance measurement for Tube 1 from each of the other absorbance values for tubes 2 to 6.
A 1 mM solution of NADH in a microtiter plate well gives an absorbance of 5.5 in this
experiment. (The molar extinction coefficient of NADH is 6220, but the pathlength is only 0.9
cm.) Therefore the amount of lactate formed in each incubation is given by:
Amount of lactate formed = ∆A*0.3/5.5/0.08 µmol = ∆A*0.68 µmol
Make sure that you understand how this expression is derived, and use it to calculate the
amount of lactate formed in each of your incubations 2-6.
Incubation
Addition
1
water
2
water
3
NAD+/ ADP/Pi
4
NAD+/ ADP
5
NAD+/Pi
6
ADP/Pi
Average
Abs.
Lactate formed
(µmol)
0
0
77
BIOC2181
Glycolysis
2017
PART B. Investigating the Conversion of Different Glycolytic Intermediates to Lactate.
This practical component should be attempted by 2-4 pairs of students in each group.
Reagents:









Reaction Mix “B” (containing triethanolamine HCl buffer (pH 7.4),
KCl, MgCl2 NAD+, ADP and Pi)
Glucose (5 mM)
Fructose 6-phosphate (F6P) (5 mM)
Fructose 1,6-bisphosphate (F1,6BP) (5 mM)
Glyceraldehyde 3-phosphate (GAP) (10 mM)
1,3-bisphosphoglycerate (1,3BPG) (10 mM)
Rat skeletal muscle cell-free extract
Glycine/Hydrazine buffer, pH 9.4 (containing 6 mM NAD+)
Lactate dehydrogenase (LDH) solution
Experimental
In this experiment, rat skeletal muscle cell-free extract will be incubated with NAD+, ADP and Pi,
and a variety of glycolytic intermediates. The muscle extract contains all the enzymes required
for the conversion of glucose to lactate via glycolysis. The incubations are stopped by the
addition of perchloric acid and the amount of lactate that has been formed is measured using an
enzymatic lactate assay. Analysis of the results will reveal the major rate-limiting step in
glycolysis by the muscle extract.
1) Add 400 µl Reaction Mix “B” to each of 7 microcentrifuge tubes (labelled 1-7).
2) Add 100 µl RO water to tubes 1 and 2, and 100 µl glucose, F6P, F1,6BP, GAP and
1,3BPG to tubes 3-7 respectively.
3) Add 200 µl skeletal muscle extract to tubes 2-7, noting the time of addition and mixing
well by inversion immediately after each addition, and then place each tube in a 37° C
water bath.
4) Exactly 10 min after the addition of the skeletal muscle extract, add 300 µl perchloric
acid to each of tubes 2-7 and mix well by inversion.
5) At a convenient time during these incubations, add 200 µl skeletal muscle extract to tube
1 and immediately follow this by the addition of 300 µl perchloric acid. This tube is a
“zero” time point.
6) Wait at least 2 min after the addition of perchloric acid to your last incubation tube before
centrifuging all seven tubes for 3 min in a microcentrifuge. Note that you may need to
use an extra “balancing” microfuge tube.
7) Add 3 x 80 µl samples of each supernatant to three wells in a microtiter plate. Add 200
µl Glycine/Hydrazine buffer to each well containing sample. Then add 20 µl LDH
solution to each well, keeping the time taken for these additions to the absolute
minimum.
78
BIOC2181
Glycolysis
2017
8) (It should be possible to make the 21 additions of LDH in less than 2 mins..) After
adding the LDH to all wells, carefully mix the contents of each using a micropipettor.
9) Wait 20 min for the lactate dehydrogenase reaction to go to completion before reading
the absorbances at 340 nm. During this waiting time, check that you understand how to
calculate the experimental results.
Calculations
Calculate the average absorbance (A) values from each of your six incubations. Note that Tube
1 provides a “blank”, since the enzymes in the skeletal muscle extract were denatured before
any glycolysis and lactate formation could take place by the immediate addition of perchloric
acid. Therefore, the spectrophotometer has been programmed to automatically subtract the
absorbance measurement for Tube 1 from each of the other absorbance values for tubes 2 to 7.
A 1 mM solution of NADH in a microtiter plate well gives an absorbance of 5.5 in this
experiment. (The molar extinction coefficient of NADH is 6220, but the pathlength is only 0.9
cm.) Therefore the amount of lactate formed in each incubation is given by:
Amount of lactate formed = ∆A*0.3/5.5/0.08 µmol = ∆A*0.68 µmol
Make sure that you understand how this expression is derived, and use it to calculate the
amount of lactate formed in each of your incubations 2-7.
Incubation
Addition
1
water
2
water
3
glucose
4
F6P
5
F1,6BP
6
GAP
7
1,3BPG
Average
Abs.
Lactate formed
(µmol)
0
0
79
BIOC2181
Glycolysis
2017
QUESTIONS
Q1. What “cofactors” are required for conversion of F1,6BP to lactate by the enzymes in the
skeletal muscle extract?
Q2. Why is each of the “cofactors” necessary? (HINT: Look at the different steps in the
glycolytic pathway.)
Q3. From your results, which enzyme appears to be the rate-limiting step in glycolysis by the
skeletal muscle extract?
Q4. Does this conform with your expectations? (HINT: Consider what you have learned in
your lectures.)
80
BIOC2181
Glycolysis
2017
Q5. Why does some Glycolysis appear to occur starting from glucose and F6P, even though
no ATP has been added?
Q6. What is the explanation for the experimental results for lactate formation from GAP
compared to 1,3BPG?
Assessment:
SATISFACTORY / UNSATISFACTORY
Demonstrator’s signature:……………………………. Date:.............................
81
BIOC2181
Separation Techniques
2017
SEPARATION TECHNIQUES PRACTICAL:
Introductory Lab Talk Instructions and Notes
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
82
BIOC2181
Separation Techniques
2017
PRACTICAL 5: THIN LAYER CHROMATOGRAPHY (TLC) OF AMINO ACIDS
SAFETY ISSUES
Safe practices for laboratory work must be observed at all times. This includes no eating,
drinking or smoking in the laboratory and wearing laboratory coats and suitable enclosed shoes.
RISK ASSESSMENT
YOU MUST WEAR SAFETY GLASSES WHEN HANDLING THESE CHEMICALS
Chemical Risks
1. Isopropanol - flammable
2. 1M and 3M HCl - strong acid
3. n-butanol - respiratory irritant
4. Acetic acid - corrosive, respiratory irritant
Waste Disposal
1. Organic solvents (n-butanol and isopropanol) - into solvent bottle in the fume hood
2. Other solutions - combine into a beaker and then empty into the bucket provided in the
fume hood
3. Used tips - place in the labelled containers on top of the bench
AIMS

To identify the amino acids in an unknown mixture by separation using thin layer
chromatography.
INTRODUCTION
To identify different compounds that comprise a mixture you need techniques that will separate
and identify the various components. Thin layer chromatography (TLC) is one method that has
been used for this purpose. TLC can be used to separate different amino acids because of the
differences in the polarity of their side chains. This can be very useful as some metabolic
diseases involve anomalies in amino acid composition and these can be identified via
techniques such as TLC. Amino acids are also used in industry for the production of
biodegradable plastics, drugs, enzymes as well as flavor enhancers.
TLC is performed on a plate of glass, plastic or aluminium foil which has been coated with an
absorbent material such as silica gel or cellulose. This absorbent plate is known as the
stationary phase. It is possible to use material that has different polarities depending on the type
of mixture under analysis. The mixture of interest is applied to the absorbent plate and the
different components of the mixture will attach to the absorbent plate to varying degrees
depending on their individual polarity.
83
BIOC2181
Separation Techniques
2017
The mobile phase, which is a solvent of a different polarity flows up the absorbent plate via
capillary action. This movement carries the mixture up the plate and different components of the
mixture will travel at different rates depending on their polarity. This will provide deferential
separation of the components of the mixture.
A typical TLC set up
Once separated, the compounds have to be identified. Amino acids can be visualized through
their reaction with a chemical called ninhydrin to form coloured compounds. Identification can
be further helped by adding a chemical called dicyclohexylamine, which promotes some
differences in colour between amino acids. Amino acids are also identified by comparing their
position (Rf value) to that of the reference amino acids. If the distances moved by the two amino
acids are identical i.e. they share the same Rf value, they are assumed to be the same.
The Rf is the ratio of the distance travelled by the solute (amino acid) to the distance travelled
by the solvent, i.e. the mobile phase on the chromatogram. In general, the more polar the side
chain of the amino acid, the lower its Rf value and the larger the hydrophobic side chain, the
greater its Rf value (see page 91 for a diagram).
UNDERSTANDING A METABOLIC DISEASE USING TLC
Phenylketonuria (PKU) is one of the common defects of amino acid metabolism detected in
newborns. The majority of individuals who have Phenylketonuria have a mutation in the gene
coding for an enzyme, phenylalanine hydroxylase, which catalyses the conversion of
phenylalanine to tyrosine. Inactivation of the enzyme causes an accumulation of phenylalanine
in all body fluids; the blood levels of phenylalanine are at least 20-fold higher than in unaffected
individuals. Minor fates for phenylalanine, such as the formation of phenylpyruvate, become
major fates in phenylketonurics. Indeed the phenylpyruvate (a phenylketone) is excreted in the
urine (hence the name of the disease) and was the basis for initial tests for the disease. Filter
paper was placed in the nappies of newborns to collect enough urine to react with FeCl3 which
turns olive green in the presence of phenylpyruvate. In NSW, PKU is now detected by
electrospray tandem mass spectrometry. This technique is very accurate, rapid, requires very
little blood and has the capability to detect and quantify many metabolites simultaneously.
84
BIOC2181
Separation Techniques
2017
Almost all untreated PKU patients are severely mentally retarded and their life expectancy is
drastically shortened. The therapy for the disease is a low phenylalanine diet that only provides
enough phenylalanine to meet the needs for growth and replacement. This diet is commenced
immediately upon diagnosis; adherence to the diet is crucial in infancy and childhood and many
experts now recommend that it be followed for life. For a pregnant woman with PKU, it is
essential that the diet is initiated prior to conception and maintained throughout pregnancy to
ensure the normal development of the foetus. In this practical, we are going to investigate why
some soft drinks should be excluded from the diet of a patient with PKU.
REAGENTS AND MATERIALS


One plastic backed cellulose thin layer plate (6.6 cm wide x 10 cm high).
Samples (numbers correspond to the positions on the cellulose thin layer plate on which
they are to be applied, reading from left to right):
Plate I
1. Phenylalanine
2. Aspartic acid
3. Serine
4. Aspartame
5. Hydrolyzed aspartame
Plate II
1. Aspartame
2. Phenylalanine
3. Coca-Cola
4. Fresh Diet Coca-Cola
5. Aged Diet Coca-Cola

The solutions of the reference amino acids were made by dissolving 1 mmol in 2 to 2.5
ml of 1 M HCl and diluting to 10 ml with 10% isopropanol in H2O. The isopropanol
prevents the growth of microorganisms and the HCl assists in dissolving certain amino
acids that are amphoteric molecules. Hydrolysed aspartame is in 3M HCl.

Solvent System: n-Butanol: acetic acid: H2O (4:1:1 v/v/v)

Visualisation solution: 0.2% (w/v) Ninhydrin, 95% (v/v) ethanol, 5% (v/v) acetic acid and
2.5% (v/ v) dicyclohexylamine
85
BIOC2181
Separation Techniques
2017
METHOD
Work in a group of 4. One pair will set up plate I while the other pair will set up plate II.
NOTE: Take great care not to damage the cellulose layer of the plate. You must wear gloves at
all times. Do not handle the cellulose surface with your fingers as it leaves deposits of amino
acids which show up as spurious spots (fingerprints), instead hold it on a sheet of clean paper.
Use only a soft pencil to draw all lines and mark dots.
1.
Prepare the cellulose thin layer plate by drawing a line across the narrow end of each
plate, 1 cm from the lower edge. Beginning 1 cm in from the left hand edge, mark five
spots approximately 8 mm apart. Number the spots from 1 to 5 and mark your student
initials in the top left hand corner of the plate.
2.
You must wear safety glasses from this step onwards
Aspartame has been dissolved in 3M HCl in a small test tube. Heat the tube in the boiling
water bath for 5 min.
Make sure the liquid does not completely evaporate and use
appropriate HS equipment and precautions! Cool the test tube – this is the hydrolyzed
aspartame.
3.
Apply 2 µl of each of the reference amino acids and samples to the appropriate five
positions on the TLC plates (Table 1 will help you with the order). Keep the spot as small
as possible and dry the spots under the heat lamps between applications.
4.
The chromatogram is developed by ascending irrigation with n-butanol:acetic acid:water
(4:1:1, v/v/v) for about 1 hour. Use 10 ml of solvent in a 500 or 600 ml beaker, and cover it
with foil to contain the vapour so that it will saturate the gas phase of the tank. Run the
chromatogram on your bench.
5.
Remove the plate once the solvent reaches 0.5 cm from the top, taking care to handle it
only by the edges. Mark the solvent front with a pencil before it dries out. Air dry the plate
in the fume cupboard.
6.
Dip the dry cellulose thin layer plate into the visualisation solution and redry it under the
heat lamps for 10 - 15 minutes until the best differentiation of colour takes place. Place a
circle around each spot immediately, as the colour tends to fade with time, and place a
mark in the centre of the region of highest colour density for each spot.
86
BIOC2181
Separation Techniques
2017
RESULTS
By using the position (Rf value) and colour of the spots formed by the reference amino acids,
identify the amino acid(s) in your “unknown” and record your results in Table 1.
Rf = Distance travelled by amino acid mixture
Distance travelled by the solvent
Table 1: Reference amino acids and chromatographic data.
Plate I
Distance traveled (mm)
Solvent front (mm)
Rf
Distance traveled (mm)
Solvent front (mm)
Rf
Phenylalanine
Aspartic acid
Serine
Aspartame
Hydrolyzed aspartame
Plate II
Aspartame
Phenylalanine
Coca-Cola
Fresh Diet Coca-Cola
Aged Diet Coca-Cola
Solvent system: n-butanol:acetic acid:water (4:1:1) by volume
87
BIOC2181
Separation Techniques
2017
QUESTIONS
What is the function of the cellulose in cellulose thin layer chromatography?
__________________________________________________________________________
__________________________________________________________________________
_________________________________________________________________________
Why does the Rf change with different solvent systems?
__________________________________________________________________________
__________________________________________________________________________
__________________________________________________________________________
Aspartame is comprised of two amino acids, do your results support this?
__________________________________________________________________________
__________________________________________________________________________
_________________________________________________________________________
Describe what has occurred to the hydrolysed aspartame:
__________________________________________________________________________
__________________________________________________________________________
_________________________________________________________________________
Diet Coke has a shelf life of around three months, why might this be?
__________________________________________________________________________
__________________________________________________________________________
_________________________________________________________________________
88
BIOC2181
Separation Techniques
2017
What precautions does a woman with PKU have to take when she decides to have a baby?
Why?
__________________________________________________________________________
__________________________________________________________________________
__________________________________________________________________________
Why can someone with PKU drink Coke but not Diet Coke?
__________________________________________________________________________
__________________________________________________________________________
_________________________________________________________________________
Assessment:
SATISFACTORY / UNSATISFACTORY
Demonstrator’s signature:……………………………. Date: ...............................
89
BIOC2181
Glucose Tolerance Test
2017
GLUCOSE TOLERANCE TEST PRACTICAL:
Introductory Lab Talk Instructions and Notes
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
………………………………………………………………………………………………………………
90
BIOC2181
Glucose Tolerance Test
2017
PRACTICAL 5: GLUCOSE TOLERANCE TEST
AIMS
 To demonstrate the use of an enzymatic method for determining blood [glucose] in
conducting a Glucose Tolerance Test.
 To provide an understanding of how the Glucose Tolerance Test may be used in the
diagnosis of diabetes mellitus.
 To provide an understanding of factors that may affect the results obtained in a Glucose
Tolerance Test.
 To illustrate the role of insulin in blood [glucose] homeostasis.
SAFETY ISSUES
Safe practices for laboratory work must be observed at all times. This includes no eating,
drinking or smoking in the laboratory and wearing laboratory coats and suitable enclosed shoes.
ORGANISATION
Each laboratory group will be provided with sets of serum samples collected while conducting
Glucose Tolerance Tests on three different subjects. Students will work in pairs and will be
assigned one of these sets of serum samples by their demonstrator, for measurement of
glucose concentrations. The time allocation will be:
20 min
100 min
30 – 60 min
Initial discussion with demonstrators and allocation of practical tasks
Completion of practical tasks
Reporting and discussion of results within the group
91
BIOC2181
Glucose Tolerance Test
2017
INTRODUCTION
The Glucose Tolerance Test
The Glucose Tolerance Test was developed as a means of diagnosing diabetes mellitus. In the
test, the patient is given a large single dose of glucose and the degree and duration of the
hyperglycemia (increased blood [glucose]) that follows is compared to that for a normal human
subject.
After the ingestion of glucose, the blood [glucose] rises as the glucose is absorbed from the
small intestine. In a normal subject, this causes the pancreas to secrete additional insulin,
which in turn causes an increased rate of glucose uptake and storage and/or metabolism by
tissues, particularly liver, muscle and adipose tissue. In a normal subject, the postprandial rise
in blood [glucose] lasts for less than 2 hours. The main factor determining the duration of the
postprandial increase in blood [glucose] is daily carbohydrate intake, the duration being shorter
in subjects who consume a high carbohydrate diet.
Subjects who have diabetes mellitus have a decreased ability to remove excess glucose from
the blood after glucose ingestion, as a result of low insulin secretion (in Type 1 Diabetes) or
failure to respond to insulin (in Type 2 Diabetes).
Blood [glucose] (mM)
Glucose Tolerance Test
Untreated diabetic
Mild diabetic
Onset of diabetes
Normal
Time (hours)
The graph shows typical Glucose Tolerance Test results for normal and diabetic subjects. The
shaded area represents the normal range for fasted blood [glucose] and error bars indicate the
range of “normal” values at each time point in the Glucose Tolerance Test. The subjects fasted
overnight and were given a glucose load of 1 g glucose per kg body mass, taken in 150 ml of
water. Venous blood was collected at zero time and at 30 min intervals for a total of 3 hours.
The subjects remained supine and relaxed during the Test and were required not to drink
alcohol, tea or coffee, or smoke cigarettes during the Test. This was because these substances
(and excitement) affect the action of adrenaline, which increases blood [glucose].
For example, tea and coffee contain compounds such as theophyllin and caffeine which inhibit
the breakdown of cyclic-AMP.
92
BIOC2181
Glucose Tolerance Test
2017
The graph shows that the fasting blood [glucose] is much higher in subjects with severe
untreated diabetes, but that fasting blood [glucose] alone is not sufficient to diagnose diabetes
because there may be little or no increase in milder cases. However, even in the early stages
of developing diabetes, there are significant differences compared to normal subjects at around
two hours. In normal subjects, prolonged starvation or a prolonged intake of a low carbohydrate
diet can produce Glucose Tolerance Test results similar to those for “mild” diabetes.
The assay for blood [glucose]
The assay uses two enzyme-catalysed reactions:


glucose + ATP  glucose 6-phosphate + ADP
glucose 6-phosphate + NAD(P)+  6-phosphogluconolactone + NAD(P)H + H+
The first reaction is catalysed by hexokinase, the initial first enzyme in glycolysis, and the
second reaction is catalysed by glucose 6-phosphate dehydrogenase, the first enzyme in the
pentose phosphate pathway. The two enzyme-catalysed reactions result in the conversion of
glucose to 6-phosphogluconolactone, with one molecule NAD(P)+ being reduced to NAD(P)H
for every molecule of glucose. The formation of NAD(P)H can be measured because
spectrophotometrically because it absorbs light strongly at 340 nm. The human glucose 6phosphate dehydrogenase enzyme uses NADP+, but the enzyme used in this assay is not of
human origin and uses NAD+ as a cofactor. Both NADH and NADPH have similar absorbances
at 340 nm.
This assay procedure has the advantage of being highly specific for glucose.
RISK ASSESSMENT
Biological Risks – The enzymes hexokinase and glucose 6-phosphate dehydrogenase are of
biological origin. Note that the diluted serum samples have been replaced with simulated serum
samples that contain no material of animal/human origin, and are not a biological hazard.
All spillages must be wiped up immediately. Solid and liquid waste must be disposed of as
directed by demonstrators and laboratory staff. Disposable gloves and safety glasses will be
provided for students who wish to use them and do not have their own.
93
BIOC2181
Glucose Tolerance Test
2017
Determination of Blood [Glucose]
Students should work in pairs, using one of the 3 sets of “Patient” samples provided: A, B, or C.
Reagents:



Glucose Reagent (containing buffer, hexokinase, glucose 6phosphate dehydrogenase, NAD+, ATP and Mg2+).
Glucose Standard (2 mM).
“Patient” samples (serum samples collected at time intervals of 0,
30, 60, 90, 120, 150 and 180 min during a Glucose Tolerance Test
and diluted x10) for Patients “A”, “B” or “C”.
Experimental
In the experiment, a standard curve for glucose will be generated (indicating absorbance at 340
nm against amount of glucose). This will be used in determining the glucose concentration in a
set of serum samples from a Glucose Tolerance Test. NOTE: Each simulated patient serum
sample has already been diluted 10x (i.e. subjected to a 1-in-10 dilution). You must take this
dilution into account when determining the final concentrations of the samples.
1) For the standard curve, add 0, 10, 20, 30, 40 and 50 µl samples of the glucose standard to
duplicate* wells in a microtiter plate, and make the total volume in each well up to 50 µl
using R/O water. (*That is, pipette each volume into two wells so that you obtain replicate
absorbance values for each amount of glucose).
2) In separate duplicate** wells for the patient samples, add 50 µl of the 10x diluted serum for
each time point (i.e. 0, 30, 60, 90 120, 150 and 180 min). (**As above, you need to pipette
50 µl from each time point into two different wells to produce replicate data).
3) Add 200 µl Glucose Reagent to all the wells in the microtiter plate for both the standard
curve and the patient serum samples. These additions should be made as quickly as
possible, taking no more than 90 sec to complete the additions and working systematically
through the standard curve first and then the serum samples.
4) Incubate at room temperature for at least 15 min (and up to 30 min), before reading the
absorbance values at 340 nm. Note that there is an opportunity during this incubation to
complete the discussion of results from the previous practical.
Results
The 2 mM glucose standard contains 2 nmol glucose per µl. Therefore the 10 – 50 µl samples
of standard used for the standard curve contain 20 – 100 nmol glucose. Construct a Standard
Curve of A340 vs. nmol glucose.
94
BIOC2181
Glucose Tolerance Test
2017
For patient’s diluted serum samples, the amount of glucose (in nmol) read from the standard
curve is the amount present in 50 µl. Using this information, complete the Results Table below,
calculating the glucose concentrations (mM) in the original undiluted serum samples. Then plot
the serum [glucose] (mM) vs time (min) for the glucose tolerance test.
Addition
Mean A340
nmol
glucose
0 µl standard
0
0
10 µl standard
20
20 µl standard
40
30 µl standard
60
40 µl standard
80
50 µl standard
100
[glucose]
(diluted serum) (mM)
Serum [glucose]
(undiluted) (mM)
0 min serum
30 min serum
60 min serum
90 min serum
120 min serum
150 min serum
180 min serum
95
BIOC2181
Glucose Tolerance Test
2017
QUESTIONS
1. What can you conclude from the results for the Glucose Tolerance tests for the three
patients “A”, “B” and “C”?
2. What other biochemical tests might assist in the diagnosis?
3. Why were the patients fasted overnight prior to conducting the Glucose Tolerance Test?
4. Why is it important that the subjects remain supine and relaxed during the Test and do not
drink alcohol, tea or coffee, or smoke cigarettes during the Test?
Assessment:
SATISFACTORY / UNSATISFACTORY
Demonstrator’s signature…………………………………………..Date:…………….
96
BIOC2181
Appendix
2017
INSTRUMENTATION
INSTRUCTIONS FOR THE OPERATION OF INSTRUMENTS
The instructions below refer to instruments which you will probably use throughout the practical
course. Read through carefully and refer to them should you be in doubt as to the proper
procedures.
MICROCENTRIFUGE
Operation of a microcentrifuge is similar to a standard bench centrifuge. Most have steps
common to all centrifuges.

All centrifuges must be balanced. Tubes should be positioned opposite a tube of
similar weight and similar volume. A 1.5 ml tube cannot be balanced against a 0.5 ml
tube. For odd numbers of tubes a “balance tube” containing a similar volume of water
should be used.

Most microcentrifuges have a special locking device to prevent the lid being opened
during use. The power must be on to operate the lid mechanism. Usually once the
centrifuge has stopped there will be a pause being a distinctive click can be heard when
the locking mechanism is disengaged.
DO NOT ATTEMPT TO FORCE THE LID OPEN AT ANY STAGE
Check your time before altering the setting.
DO NOT ATTEMPT TO TURN BACK THE TIMER.
Check on the type of centrifuge to be used and how it is to be operated before using. Operating
instructions are given for the common centrifuges used in the Undergraduate teaching labs.
97
BIOC2181
Appendix
2017
Eppendorf 5415C
Turn on main switch to “l-on”. THE POWER MUST BE ON TO OPEN THE LID.
Open lid & unscrew rotor lid.
Load rotor symmetrically ie balanced tubes opposite.
Screw rotor lid on firmly & close the lid.
For timed runs, choose rotational speed and set the timer.
For quick spin, press the momentary switch.
ALWAYS WAIT UNTILTHE ROTOR HAS COME TO A COMPLETE STOP (THE PILOT LAMP
COMES ON) BEFORE ATTEMPTING TO OPEN THE LID.
DO NOT ATTEMPT TO FORCE THE LID OPEN.
DO NOT ATTEMPT TO TURN BACK THE TIMER.
Eppendorf 5410
Check power is on. THE POWER MUST BE ON TO OPEN THE LID.
Open lid by pressing down on the universal key completely & unscrew rotor lid.
Load rotor symmetrically ie balanced tubes opposite.
Screw rotor lid on firmly & close the lid by pressing down firmly on the lid.
For a timed spin, set the timer to desired spin time.
Start by pressing the universal key slightly for a short time.
For a quick spin, press down the universal key slightly for a longer time (at least
2 secs).
ALWAYS WAIT UNTILTHE ROTOR HAS COME TO A COMPLETE STOP
BEFORE ATTEMPTING TO OPEN THE LID.
DO NOT ATTEMPT TO FORCE THE LID OPEN.
DO NOT ATTEMPT TO TURN BACK THE TIMER.
TOMY Capsulefuge
Check that power is on.
Open the lid by pressing down on the stop lever.
Load the tubes symmetrically ie balanced opposite.
To start, close the lid by pressing down firmly until it locks into position.
It will begin spinning immediately the lid is closed.
To stop, press the stop lever.
Wait until the rotor has stopped spinning before attempting to remove the tubes.
98
BIOC2181
Appendix
2017
Beckman Microfuge E
Turn on power switch.
Open lid & load centrifuge tubes symmetrically i.e. balanced
opposite.
Close the lid by pressing down.
For a timed spin, set the timer to desired spin time.
For a quick spin, press down the momentary button.
It will run when being held down & stop when released.
ALWAYS WAIT UNTILTHE ROTOR HAS COME TO A COMPLETE STOP BEFORE
ATTEMPTING TO OPEN THE LID.
THE POWER MUST BE ON TO OPEN THE LID.
DO NOT ATTEMPT TO FORCE THE LID OPEN.
DO NOT ATTEMPT TO TURN BACK THE TIMER.
Sorvall MC-12V
Check power is on.
Open lid by depressing the latch on the right side. It should click open.
Load the centrifuge with tubes placed symmetrically ie balanced opposite.
Close the lid by pressing down firmly. The lock should engage with a click.
For a timed spin, set speed and then the timer to desired spin time.
For a quick spin, press down the pulse button. It will run when being held down
& stop when released.
ALWAYS WAIT UNTILTHE ROTOR HAS COME TO A COMPLETE STOP
BEFORE ATTEMPTING TO OPEN THE LID.
THE POWER MUST BE ON TO OPEN THE LID.
DO NOT ATTEMPT TO FORCE THE LID OPEN.
DO NOT ATTEMPT TO TURN BACK THE TIMER.
99
BIOC2181
Appendix
2017
MICROTITER PLATE READER
Fig. 1a Microtiter Plate Reader

Switch on - 1 minute is allowed for warming up and auto-calibration.

Select Measure Mode required.

The wavelength is selected and also the Blank requirement.

Press START key to measure absorbances
Numerous other options are available. These parameters are preset for the class.
Fig. 1b Microtiter Plate
*Plates must be clean and dry and free from any spillage. Wells should not be overfull.
100
BIOC2181
Appendix
2017
PIPPETORS / SAMPLERS
Glass pipettes have generally been replaced by hand held samplers or pipettors that can
dispense very small and highly reproducible volumes. The Gilson Pipetman P (see Fig. 2a) and
the Eppendorf (see Fig. 2b) pipettor will be used in practical classes. These should always be
placed in the racks provided on the benches. NEVER leave an automatic pipettor lying on the
bench. These are expensive items of equipment and should be treated with great care.
Fig. 2a Gilson Pipetman P
Fig. 2b Eppendorf
A range of models is available with maximum capacities varying from 2 - 10,000µL. The
capacity of the pipettor is indicated (in µL) on the push button on Gilson Pipetman P (see Fig.
4a) and on the side of the body of the Eppendorf P (see Fig. 4b). An example of the range for
these models is:
P20
- 2µL to 20µL – use yellow tips
P100
- 10µL to 100µL – use yellow tips
P200
- 20µL to 200µL – use yellow tips
P1000 - 100µL to 1000µL – use blue tips
NEVER ATTEMPT TO USE A PIPETTOR OUTSIDE ITS RANGE.
THIS WILL RESULT IN SEVERE DAMAGE TO THE INSTRUMENT.
101
BIOC2181
Appendix
2017
The method of changing the volumes in the variable volume samplers depends on the type of
pipettor. The Gilson Pipetman P has a direct reading digital volumeter that allows continuous
volume adjustment using the knurled adjustment ring. The Eppendorf pipettor is adjusted to
volume using the setting at the top of the pipette.
Appropriate disposable tips will be provided and these should be attached with a slight twisting
motion to ensure an airtight seal. Sufficient disposable tips will be provided for each experiment.
To prevent wastage you should arrange a piece of paper marked with the appropriate usage for
each tip and lay them out accordingly. This way if you need to repeat a sample the tip is still
available.
Care should be taken to hold the pipettor vertically during use and not to immerse the tip
more than 3 - 4 mm in the liquid. The push button should be operated slowly and smoothly
to avoid introducing air and inaccuracies into the dispensing procedure.
See over the page for illustrated instructions on how to use an automatic pipettor
102
BIOC2181
Appendix
2017
Step1
Ensure that the correct tip is fitted to the sampler. Depress
the plunger to the first stop. It is important to expel the air
from the tip before inserting it into the liquid otherwise
bubbles are forced into the liquid. This will make subsequent
samples difficult to take especially with protein solutions.
Step 2.
Immerse the tip in the solution
(only 3 - 4 mm – not the whole length of the
tip), then slowly and smoothly release the
plunger to fill the pipettor. Wait a second or
two before withdrawing the tip from the liquid.
Too fast will result in air being sucked in
= inaccurate measurement = splashing into
pipettor barrel = pipettor damage
Step 3.
To expel the solution, the plunger is depressed slowly and
completely to the first stop.
Wait for a second and then push slowly and completely to the
second stop.
Keeping the plunger depressed remove the pipettor and then
release.
103
BIOC2181
Appendix
2017
INSTRUCTIONS FOR USE OF DISPENSORS
A range of dispensors is available in the laboratory. They are precision instruments capable of
delivering aliquots with ±0.5% reproducibility and they are used for multiple dispensing of a
reagent. Maximum reproducibility is achieved when slow steady strokes are used. Excessive
speed will cause inertial movement of the fluid column to pre-dispense or post-dispense a
droplet.
1.
Place container in position below tip. Do not allow the tip to touch the container. This
could cause cross contamination.
2.
Smoothly raise the plunger until it meets the indicator stop. Pause slightly.
3.
Gently push the plunger down until it firmly bottoms. The correct will volume will be
dispensed. Slow delivery and the angling of the receiving container will reduce the
possibility of splash back.
N.B.
Any dispensors made available for class use will have been calibrated for you.
DO NOT ALTER SETTINGS!
Always check the settings on the dispenser before use.
They will be set up to :
a) Dispense sufficient solution for the whole experiment for you to then pipette on into the
required tube.
In this case you need to collect the solution in a container of appropriate volume for the
sample dispensed. Eg. for volumes up to 10 mL a plastic centrifuge is ideal. A 250 mL beaker
would not be ideal as the resulting depth of liquid would make it impossible to pipette accurately
from it.
OR
b) Set to an amount to dispense directly into the required tube.
104
BIOC2181
Appendix
2017
CALCULATION OF STANDARD DEVIATION AND % ERROR
Standard Deviation:
σ
=
Σ ( xi – x )2
N–1
Where:
Σ = “sum of”
xi = sample value
x = average (mean) of all sample values
N = number of sample values
Percentage Error:
% Error =
(
)
Theoretical value – Experimental value
Theoretical value
x 100
For Example, in your spectrophotometry practical in Week 2, % error would be calculated as
follows:
% Error =
(
)
Actual casein concentration – YOUR experimental result
Actual casein concentration
x 100
105