F LIMPOPO

UN YO
I VERSIT

Faculty of Science and Agriculture

SCHOOL OF MOLECULAR AND LIFE SCIENCES

Department of Biochemistry, Microbiology and Biotechnology

STUDY GUIDE

SBIA031
(Proteins, Enzymes and Biochemical Techniques)

2024
 SBIA031 STUDY GUIDE 2024
TABLE OF CONTENTS

1 MODULE INFORMATION ............................................................................................................... 3

1.1 Overview of module................................................................................................................ 3
1.2 Prerequisite module codes ..................................................................................................... 3
1.3 Ancillary module code............................................................................................................. 3
1.4 Important dates ...................................................................................................................... 3
1.5 Module activities schedule ..................................................................................................... 4
2 PURPOSE AND LEARNING OUTCOMES ..................................................................................... 4
2.1 Purpose ................................................................................................................................... 4
2.2 Learning outcomes.................................................................................................................. 4
3 CONTENT ASSUMPTIONS ............................................................................................................ 4
4 FACILITATION................................................................................................................................ 5
4.1 Facilitators ............................................................................................................................... 5
4.2 Learning hours ........................................................................................................................ 5
5 THEORY AND PRACTICAL TENTATIVE SCHEDULES ............................................................... 5
5.1 Theoretical work ..................................................................................................................... 5
5.2 Practical work.......................................................................................................................... 6
6 DETAILED CONTENT .................................................................................................................... 6
6.1 Theory ..................................................................................................................................... 6
6.1.1 Protein primary structure (PKC) ................................................................................... 6
6.1.2 Protein secondary and tertiary structures (PKC)............................................................. 6
6.1.3 Protein quaternary structure and structure-function relationships. (PKC) ....................... 6
6.1.4 Basic principles of biochemical techniques (PKC & MZM) ......................................... 7
6.1.5 Protein purification and characterisation techniques (PKC & MZM) .......................... 7
6.1.6 Enzymology, assays and kinetics (TMM & KWP) ........................................................ 7
6.2 Practical (DFM) ................................................................................................................. 7
6.2.1 Practical 1: Library Orientation .............................................................................................. 7
6.2.2 Practical 2: Determination of a sequence of an unknown peptide using TLC plates ............ 7
6.2.3 Practical 3: Lysozyme purification from chicken eggs........................................................... 7
6.2.4 Practical 4: Enzyme Kinetics: Characterisation of alkaline phosphatase ............................... 8
6.2.5 Practical 5: Detection of enzyme activity (metalloproteases) using gelatin zymography ..... 8
7 PRESCRIBED AND RECOMMENDED BOOKS............................................................................. 8
8 ASSESSMENT ................................................................................................................................ 9
8.1 Assessment criteria ................................................................................................................. 9
8.2 Assessment methods .............................................................................................................. 9
9 FEEDBACK ON MODULE ............................................................................................................ 10
10 MODULE POLICY ......................................................................................................................... 10
11 UNIVERSITY OF LIMPOPO’S GENERAL RULES ....................................................................... 10

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 SBIA031 STUDY GUIDE 2024

1 MODULE INFORMATION

1.1 Overview of module

The module will cover concepts on how amino acids interact with each other to form different levels of
protein structures (primary, secondary, tertiary and quaternary) and how protein structure influences or
affects protein function. Furthermore, enzymology: enzyme assays and kinetics concept as well as the
basic laboratory techniques commonly used in protein fractionation, purification and characterization will
be discussed. Protein primary, secondary and tertiary structures; Overview of use of Bioinformatics in
protein structure and function; Protein quaternary structure and Structure-Function relationships;
Enzymology: enzyme assays, inhibition and kinetics; Basic laboratory principles: statistics, safety, health,
environment, and quality; Basic laboratory techniques: fractionation; centrifugation; dialysis, ultrafiltration;
photometry; radioisotopes; protein purification and characterisation techniques; Overview of modern
protein structure determination techniques..

It is important that students understand materials presented in each lecture, as this information is usually
needed for the following lecture that will be presented. Therefore, students must ask questions during
lecture times or consult the lecturer before the next lecture begins in order to facilitate their understanding
of the taught concepts. It will be your responsibility to consult the appropriate lecturer if you have any
questions or do not understand any section of the work in relation to the module.

This study guide outlines topics to be presented in class. It will be the student’s responsibility to consult
the internet and textbook(s) for further information regarding a specific lecture. Note that lecturers expect
students to prepare for each lecture in advance. The approach of disseminating the information is
student-centred.

1.2 Prerequisite module codes

SBIA021 & SBIA022

1.3 Ancillary module code

None

1.4 Important dates

 Fourteen (14) weeks of theory classes: 29 January – 17 May 2023

 Quizzes – Every lecture
 Two (2) Ordinary Test Dates
Test 1: 27 March 2024
Test 2: 25 April 2024
 One (1) week of recess: 18-20 March 2024 (encompasses holidays on 21-22 March)
 One (1) week of examination preparations: 20-24 May 2024
 Examinations start: 27 May 2024

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 SBIA031 STUDY GUIDE 2024

1.5 Module activities schedule

Lecture venue and time

Blackboard (Students to log in at least 5 minutes ahead of class start time)
Monday: 07h30-09h10
Thursday: 09h20-11h00

Practical venue and time

1st Floor, New Life Science Laboratory
Tuesday: 11h00-17h00

2 PURPOSE AND LEARNING OUTCOMES

2.1 Purpose

 Acquaint students with terminologies, concepts, equipment and techniques commonly used in
Protein Biochemistry.
 Enable students to understand and apply knowledge on the basic structures and functions of proteins
using examples such as myoglobin, haemoglobin, antibodies and enzymes.
 Enable students to understand basic biochemical methods, be able to assess and know when to
apply them in different situations.

2.2 Learning outcomes

After successfully completing the module, the student should be able to:
 Understand protein structures and functions.
 Demonstrate an understanding on how the structure of a protein relates to its function.
 Apply information on protein structure and function to varying practical situations.
 Demonstrate an understanding of general homogenization and fractionation techniques, and other
general techniques used in Biochemistry. Demonstrate an understanding on the different methods
used to purify and characterize proteins.
 Use a multidisciplinary approach to evaluate information given on protein structure and function.
 Communicate an understanding of the subject matter in both oral and written forms.
 Formulate new information on protein structure and function using concepts learnt.
 Use biochemical techniques to solve problems on protein isolation and characterization.
 Demonstrate an understanding of basic enzyme kinetics.

3 CONTENT ASSUMPTIONS

When entering this module, students are expected to have good knowledge of general Chemistry and
Biochemistry as the basics of these will not be discussed in detail during lectures.
The module will be presented in theoretical classes. Information for this module is contained in Microsoft
PowerPoint notes that include tutorials, practical sessions, and a recommended textbook(s).
Supplementary notes will be posted on Blackboard.

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 SBIA031 STUDY GUIDE 2024
If necessary, any additional information will be communicated in class as well as through Blackboard
and/or University student email. All students are expected to know their Blackboard and e-mail login
details, which are obtained from the UL general Computer labs.

4 FACILITATION

4.1 Facilitators

Dr PK Chokoe (PKC) [Theory, Protein chemistry]

Office 3026, 3rd Floor, Life Sciences Building
Contact details: 015 268 2225; [email protected]
Consultation time: Monday-Friday (email or Blackboard)

Mr MZ Monama (MZM) [Theory, Protein chemistry]

Office 003, Biotechnology Unit
Contact details: 015 268 2172; [email protected]
Consultation time: Monday-Friday (email or Blackboard)

Prof. TM Matsebatlela (TMM) [Theory, Enzymology: Enzyme behaviours and their inhibition]
Office 1003, 1st Floor, Life Sciences Building
Contact details: 015 268 3013; [email protected]
Consultation time: Monday-Friday (email or Blackboard)

Dr KW Poopedi (KWP) [Theory, Enzymology: Enzyme behaviours and their inhibition]

Office 1008, 1st Floor, Life Sciences Building
Contact details: 015 268 2861; [email protected]
Consultation time: Monday-Friday (email or Blackboard)

Ms DF Mangokoana (DFM) [Practical]

Office 1005, 1st floor, Life Sciences Building
Contact details: 015 268 3018; [email protected]
Consultation time: Monday-Friday (email or Blackboard)

4.2 Learning hours

Ninety (90) minutes per lecture

Three (6) hours per practical session

5 THEORY AND PRACTICAL TENTATIVE SCHEDULES

5.1 Theoretical work

No. of lectures Topic/Activity

4 Protein primary structure (PKC).
3 Protein secondary and tertiary structures (PKC).
3 Protein quaternary structure and structure-function relationships (PKC).
Fractionation and centrifugation techniques, applications of dialysis, ultrafiltration,
4 chromatography, electrophoretic techniques, spectrophotometry and omics (PKC &
MZM).
5 Enzymology: enzyme behaviours, assays and kinetics (TMM & KWP).
6 Enzyme Inhibition (TMM & KWP).

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5.2 Practical work

Practical 1: Library Orientation and Highlighting Important Rules for Practical Sessions.
Practical 2: Determination of a Sequence of an Unknown Dipeptide.
Practical 3: Purification and Characterisation of Lysozyme from Chicken Egg White Using
Cation-exchange Chromatography.
Practical 4: Enzyme Kinetics: Characterisation of Alkaline Phosphatase.
Practical 5: Detection of Enzyme Activity (Trypsin & Metalloproteases) Using Gelatine
Zymography.

NB: Each practical is outlined in the practical manual. Further details regarding submission of reports will
be announced during the course of the module.

6 DETAILED CONTENT

6.1 Theory

6.1.1 Protein primary structure (PKC)

Amino acid structure, grouping, modifications and importance; oligonucleotide formation and titration
curves. Understand the importance of primary structures and different methods of determining the
primary structure (all 2nd year work is considered prior knowledge). Be able to sequence oligopeptides
using enzyme and chemical techniques and short polypeptides using the overlapping method.
6.1.2 Protein secondary and tertiary structures (PKC)
Review different types of secondary structures found. Determine the presence and feasibility of certain
secondary structures of proteins by understanding the structures, psi and phi angles and Ramachandran
plots. Describe different types of proteins with a high degree of secondary structure in them and
understand why certain amino acids are found in these structures - this relates to the importance of
primary structure in secondary structure formation.
Describe the tertiary folding of globular proteins, including the rules governing this procedure. Know
importance of primary structure in globular structure formation, thermodynamics of folding and the role of
disulphide bonds. Know and understand the kinetics of protein folding and disulphide bond formation, the
importance of proteins assisting in the folding process, motions and structure-function importance. Finally
students must be able to predict secondary structure, understand how the amino acids will lie in the
predictions and the consequences thereof. Know the different ways polypeptide chains can interact with
each other in quaternary structures.
6.1.3 Protein quaternary structure and structure-function relationships. (PKC)
Distinguish between structure/function relationships of tertiary proteins and quaternary proteins,
specifically using myoglobin and haemoglobin as examples. Know the mechanism of oxygen binding to
haem-proteins, and analysis of oxygen binding by myoglobin and haemoglobin using binding studies and
Hill plots. Allosteric models of haemoglobin, change in protein structure and a close look at haem
changes that cause the overall allosteric change of the protein. Understand the role played by allosteric
effectors on oxygen binding.
Protein evolution, protein mutations and their effects on structure/function relationships.
Functioning and structures of the immune system (limited to time constraints).

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 SBIA031 STUDY GUIDE 2024
6.1.4 Basic principles of biochemical techniques (PKC & MZM)
The section will briefly introduce students to the use of fractionation techniques, centrifugation technique,
applications of dialysis and ultrafiltration methods. Fractionation techniques: various choices and how to
determine the best one for your sample. Fractional Precipitation techniques: Heat, Acid, polyethylene
glycol (PEG). Centrifugation: Theoretical basis of centrifugation method, practical aspects of
centrifugation, apparatus used in centrifugal separations, the uses of centrifugation. Ultrafiltration and
Dialysis: Introduction to the use of dialysis apparatus, the diffusion theory, the practical applications of
ultrafiltration, and advances in membrane technology. Spectrophotometry: The origins of spectral
absorptions, Beer-Lambert Law, instrumentation and practical considerations of use of
spectrophotometers.
6.1.5 Protein purification and characterisation techniques (PKC & MZM)
This section will cover areas on the theory and practical aspects of Chromatography: Thin-Layer
Chromatography, Gas-Liquid Chromatography, High Performance Liquid Chromatography, Ion-
exchange Chromatography and Gel Filtration, and Affinity Chromatography. Use of Purification Tables
for monitoring enzyme protein purification. Electrophoretic Techniques to study the separations of
macromolecules (e.g. proteins) using gel electrophoresis. Polyacrylamide gel electrophoresis (PAGE);
its modifications and applications and uses, SDS-PAGE, Isoelectric focussing, 2- dimensional SDS-
PAGE.
6.1.6 Enzymology, assays and kinetics (TMM & KWP)
Enzyme structure, function, class, properties, definitions. Enzyme assays and quantitation, alternate
assay techniques and biosensors. Reaction order, rapid equilibrium and steady state hypotheses.
Function of V0, Vmax and Km, Haldane relationship and reversible reaction kinetics. Description and use
of various plotting methods. Multi-substrate Kinetics. Enzyme inhibition: competitive/non-competitive/un
- competitive. Mixed-type, feedback, irreversible, substrate, allosteric inhibitions.
6.2 Practical (DFM)

6.2.1 Practical 1: Library Orientation

 Writing of scientific reports.

 Searching for information on the internet using different databases.
 Proper referencing and the use of different referencing styles.

6.2.2 Practical 2: Determination of a sequence of an unknown peptide using TLC plates

 Understanding of protein primary structure.

 Amino acid and protein chemistry.
 Amino acid sequencing and prediction of the protein primary structure.
 Biochemistry techniques such as TLC and the use of different solvents for isolation of specific
compounds.

6.2.3 Practical 3: Lysozyme purification from chicken eggs

Part A: Buffer preparation

 Calculations required for buffer preparations.

Part B: Lysozyme isolation using cation exchange chromatography

 Protein isolation and characterisation.
 Protein chemistry.

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 Centrifugation technique, chromatography and spectrophotometer.

Part C: Protein Analysis

 Detection of protein concentration
 Principles of Lowry method
Part D: Lysozyme Activity assay
 Enzyme function
 Enzyme assay and enzyme kinetics

Part E: Analysis of lysozyme using SDS-PAGE

 Principles electrophoretic techniques
 Protein identification

6.2.4 Practical 4: Enzyme Kinetics: Characterisation of alkaline phosphatase

Part A: Preparation of a standard curve

 Setting up experimental controls

Part B: Enzyme reaction over time

 Understanding how an enzyme functions.
 Evaluation of the effect of time on the enzyme activity.

Part C: The effect of substrate concentration on the enzyme activity

 Understanding enzyme behaviour by looking at its relationship with the substrate.

Part D: The effect of pH on the enzyme activity

 pH as one of the factors that affect enzyme activity.
 Understanding how the active structure is affected by different parameters (pH).

Part E: The effect of temperature on the enzyme activity

 Optimal behaviour of an enzyme by looking at temperature as one of the factors that affect enzyme
activity
6.2.5 Practical 5: Detection of enzyme activity (metalloproteases) using gelatin zymography
 Enzyme characterisation.
 Principles of zymography.

7 PRESCRIBED AND RECOMMENDED BOOKS

Note: Students are expected to be in possession of the Biochemistry Course Outline, Biochemistry Study
Aids (not a substitute for the prescribed books) and Practical Manual.

Prescribed Text Books:

 Biochemistry, Berg, J.M., Tymoczko, J.L and Stryer, L. lnternational latest Edition (Reserve).
 Biochemical Calculations latest edition, I. H. Segal (Reserve).

Recommended Text Books:

 Biochemistry, Mathews, van Holde and Ahern, latest edition (Reserve).
 Biochemistry, Garrett and Grisham, latest edition (Reserve).

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 Analytical Biochemistry, Holme and Peck, latest edition.

 Principles and techniques of practical biochemistry, Wilson and Walker, latest edition. (Reserve).
 Biochemistry, Voet and Voet, latest edition (Reserve).
 Understanding Enzymes, T. Palmer, latest edition (Reserve)
 Principles of enzyme assays and kinetic studies, Tipton, K.F, latest edition.
 Many websites using “Protein structure and function”, “Enzymes”, “Enzyme kinetics”, or specific
words such as “Ramachandran” as key words
(Note: Reserve means that you can find these books in the reserve section of the UL library. Since the
library keeps a limited stock of the prescribed book, some books will be found on reserve. There are few
prescribed and other Biochemistry books on the OPEN shelves, 3 rd floor of the library that can be
borrowed).

8 ASSESSMENT

8.1 Assessment criteria

Students must be able to:

 Gain knowledge and application of protein structures and functions.

 Describe the Chemistry of protein primary structure and define the properties of amino acid side
chains/R-groups.
 Apply knowledge gained to synthesise the sequence and predict basic primary structures.
 Understand the formation of different levels of protein structures. Understand protein folding,
domains, thermodynamics and kinetics.
 Discuss allosteric models, changes occurring within the proteins when oxygen binds, allosteric
effectors and the applications and interpretations of them in real life situations.
 Describe evolutionary relationships and how base pair changes can affect amino acid sequence and
protein function.
 Create new information on protein structure using the knowledge gained on protein structure.
 Solve problems related to protein isolation and characterization. Understand how enzymes function,
know factors that can affect activity.
 Calculate kinetic parameters including catalytic constant, first order rate constant, catalytic efficiency
and evaluate the enzyme based on the parameters.
 Construct and interpret different enzyme kinetic plots.

8.2 Assessment methods

Quizzes 05%
Assignments 05%
Theory tests 30%
Practical reports 15%
Practical test 04%
Pre-lab quizzes 01%

Formative assessment (Theory & Practical, Semester) = 60%.

Summative (Examination) Assessment = 40%.

Minimum Formative Assessment mark for exam admission = 40%.

Final Exam mark = 60% Formative Assessment Mark + 40% Summative Assessment Mark.

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 SBIA031 STUDY GUIDE 2024
Minimum Final Assessment mark to Pass = 50%.

Summative assessment (Exam): designates an examination and it contributes 40% towards the final
mark.

Subminimum rule:
A minimum of 40% average for the theory and practical mark combined will allow entry into the
examination.
Irrespective of the final mark achieved, students must obtain at least 40% in the summative assessment
(examination paper) to pass the module. Students will be required to write a supplementary examination
(1) if they obtain less than 40% in the examination, but obtain 50% or more for final mark or (2) if they
obtain a final mark of 45-49%.

9 FEEDBACK ON MODULE

Students will be provided with regular feedback within ten (10) working days after date of assessment
and this will include one or combination of the following:
 Verbal feedback about problem areas.
 Individual feedback during consultation hours when such an appointment request was made.
 Display of memoranda and test marks on the Blackboard

NB: Students must query miscalculation or correction of the TEST marking within seven (7) working days
upon receiving feedback.

10 MODULE POLICY

Plagiarism is defined as using the exact words, and/or an opinion from another person without giving that
person credit. Therefore, students are expected to declare when submitting assignments and practical
reports that the work they are submitting was written in their own words and has not been pirated from a
fellow student or any other source. Students should thus indicate and acknowledge (by referencing) their
sources (published or unpublished). In addition, pictures, illustrations and material taken from published
or unpublished sources should be acknowledged; this practice would thus constitute academic honesty.
Failure to conform may lead to deduction of marks from student’s report or assignment. In severe
instances students may also be expelled from the institution for this academic piracy. Students are also
expected to attend all the lectures and practical sessions in order to meet the requirements of the module.

11 UNIVERSITY OF LIMPOPO’S GENERAL RULES

Each student is expected to know the University of Limpopo’s General rules (G1-27) as outlined in the
University of Limpopo General Calendar. This will also inform the student of rules G10 and G25, amongst
others.

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