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FACULTY OF SCIENCE SCHOOL OF BIOTECHNOLOGY AND BIOMOLECULAR SCIENCES BIOC2101 ADVANCED BIOCHEMISTRY SESSION 1, 2017 Table of Contents 1. Information about the Course.............................................................................................. 3 2. Staff Involved in the Course ................................................................................................ 3 3. Course Details .................................................................................................................... 3 4. Rationale and Strategies Underpinning the Course ............................................................ 7 5. Course Schedule ................................................................................................................ 9 6. Assessment Tasks and Feedback .................................................................................... 10 7. Additional Resources and Support.................................................................................... 11 8. Required Equipment, Training and Enabling Skills ........................................................... 11 9. Course Evaluation and Development................................................................................ 11 10. Administration Matters .................................................................................................... 12 11. UNSW Academic Honesty and Plagiarism...................................................................... 14 12. Practicals ........................................................................................................................ 15 Spectrophotometry pg 23 Enzymes pg 41 Enzyme Inhibition pg 58 Oxygen Electrode Simulation pg 68 Glycolysis pg 69 Separation Techniques 1 pg 79 Separation Techniques 2 pg 87 Glucose Tolerance Test pg 97 BIOC2101 - Advanced Biochemistry Course Outline 1. Course Information NB: Some of this information is available on the UNSW Handbook 1 Year of Delivery 2017 Course Code BIOC2101 Course Name Principles of Biochemistry (Advanced) Academic Unit School of Biotechnology and Biomolecular Sciences Level of Course 2 Units of Credit 6 Session(s) Offered S1 Assumed Knowledge, Prerequisites or Corequisites Prerequisite: BABS1201 and CHEM1011 or CHEM1031 or CHEM1051 and CHEM1021 or CHEM1041 or CHEM1061 Hours per Week 6 Number of Weeks 12 weeks Commencement Date Monday 27 February (Week 1) nd year undergraduate Summary of Course Structure Component Lectures, tutorials, and exams Weekday 1 Weekday 2 Weekday 3 (Exams on this day) Laboratory Lab – Option 1 Lab – Option 2 Lab – Option 3 Special Details Hours/week 2-3 3 2. Staff Involved in the Course Time Day Location 12 – 1 pm 1 – 2 pm 9 – 10 am Monday Thursday Friday Rex Vowels Rex Vowels Mathews Theatre A 10 am – 1 pm 2 – 5 pm 10 am – 1 pm Wednesday Wednesday Thursday Wallace Wurth 122/123 Wallace Wurth 122 Wallace Wurth 122/123 Staff Course Convenor Role Name A/Prof Kyle Hoehn Teaching Staff Lecturers Prof Marc Wilkins Dr Nirmani Wijenayake Prof Andrew Brown Dr Rebecca LeBard Dr Vladimir Sytnyk Dr Frances Byrne Dr Gus Maghzal Contact Details Room G17, Biosciences Building [email protected] Office hours on demand, x5-9399 [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] [email protected] 3. Course Details Course Description (Handbook Entry) 1 2 2 Introduces modern biochemistry, covers fundamental aspects of the structure-function relationships of proteins and an overall coverage of intermediary metabolism. Major topics covered include: the nature and function of proteins and enzymes; the metabolic working of cells, tissues and organs; the interrelationships between the pathways of carbohydrate, lipid and amino acid metabolism; the vital role of hormones in metabolic regulation; the energy-trapping mechanisms of animals; and interesting variations on the central metabolic pathways in various life forms. Practical work complements the lectures and introduces the principles of biochemical analysis. UNSW Online Handbook: http://www.handbook.unsw.edu.au UNSW Handbook: http://www.handbook.unsw.edu.au 3 Course Aims 3 Student Learning 4 Outcomes To introduce biochemistry with an emphasis on how humans convert food to useful energy. By the completion of this course students should be able to: - describe the fundamental aspects of the structure-function relationships of proteins; - outline intermediary metabolism in humans and its regulation; - perform standard biochemical laboratory techniques, and safe and efficient work practices, and; - communicate effectively and work constructively within a team. Graduate Attributes Developed in this Course Science Graduate 5 Attributes Research, inquiry and analytical thinking abilities Capability and motivation for intellectual development Ethical, social and professional understanding 5 Select the level of FOCUS 0 = NO FOCUS 1 = MINIMAL 2 = MINOR 3 = MAJOR 3 3 1 Activities / Assessment Lectures, tutorials, laboratory experiments, online discussions, computer exercises / instruction and formative self-assessment Multiple choice, short answer / essay, solving complex problems, assessed laboratory experiment Lectures, tutorials, laboratory experiments, online discussions, computer exercises / instruction and formative self-assessment Multiple choice, short answer / essay, solving complex problems, assessed laboratory experiment Laboratory experiments, lectures and tutorials Assessed laboratory experiment, HS online quiz Communication 2 Teamwork, collaborative and management skills Small group tutorials, laboratory experiments and online discussions Short answer / essay 2 Small group tutorials, laboratory experiments and online discussions 2 Large and small group tutorials, computer tutorial / instruction Information literacy Major Topics Introduction to Metabolism 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. 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. PROTEINS AND ENZYMES Protein Structure | Topics • Proteins and the central dogma of molecular biology 3 Learning and Teaching Unit: Course Outlines Learning and Teaching Unit: Learning Outcomes 5 Contextualised Science Graduate Attributes 4 4 • • • • There are 20 amino acids encoded in DNA and used in protein synthesis Amino acids are joined to form peptides and proteins Proteins can form secondary structures Protein amino acid sequence determines its 3-D structure Enzymes and Catalysis | Topics • What is an enzyme? • What is a substrate? • What is catalysis? • Free energy and activation energy • Transition states in catalysis • Induced fit model of catalysis Enzyme Kinetics | Topics • What is kinetics? Why study kinetics? • Enzyme reaction velocity • Test case: human foldase enzyme • The Michaelis-Menten value, KM • Implications of KM • The Michaelis-Menten equation • Lineweaver-Burk plots • The kinetically perfect enzyme • Enzyme engineering Enzyme Inhibition and Regulation | Topics • Competitive and non-competitive inhibition • Kinetics of (non)competitive inhibition • Allosteric inhibition • Zymogens • Enzyme phosphorylation CARBOHYDRATE CATABOLISM AND STORAGE Carbohydrate catabolism I | Learning Outcomes • Explain what carbohydrates are • Explain how monosaccharides are classed (number of carbons, aldose/ketose etc). • Explain the terms stereoisomer / epimer / diastereomer • Give an example of a disaccharide and explain the role of the glycosidic bond • Explain what polysaccharides are and give some examples Carbohydrate catabolism II | Overview • Carbohydrate digestion and transport • Glycolysis: an energy conversion pathway for glucose • Regulation of glycolysis • The fate of the products of glycolysis Glycogen | Learning Outcomes • Describe the general structure and function of glycogen. • List the three key enzymes involved in glycogen breakdown and provide a brief description of their functions. • Understand that glycogen is synthesised and degraded by different pathways. • List the three key enzymes involved in glycogen synthesis and provide a brief description of their functions. • Understand that the regulation of glycogen metabolism involves both allosteric control and hormonal control by covalent modification of regulatory enzymes. • Describe the way in which glycogen breakdown and synthesis are reciprocally regulated. Gluconeogenesis | Learning Outcomes • Provide a broad definition of the process of gluconeogenesis. • Appreciate that glucose can be synthesised from lactate, pyruvate, glycerol and amino acids. • Explain the Cori Cycle. • Describe the three enzymatic steps of glycolysis that are bypassed in gluconeogenesis. • Name the enzymes that are NOT common to glycolysis and gluconeogenesis. • Explain the energy requirements of gluconeogenesis. • Explain how glycolysis and gluconeogenesis are reciprocally regulated. PROTEIN CATABOLISM Amino Acid Catabolism | Learning Outcomes • Explain the process whereby an amino group is removed form an amino acid. 5 • • • • • Discuss the degradation of AA in muscle during prolonged exercise and fasting. Describe how amino acid degradation is linked to the TCA cycle. Discuss the Urea cycle e.g. ATP used, where performed, where the two N in urea are from. Explain what is meant by the terms keto- and glucogenic amino acids and provide examples. Give examples of conditions where there are errors in the degradation of amino acids. Protein digestion and turnover | Learning Outcomes • List the sources of amino acids for building proteins. • Describe what is meant by the term essential amino acids. • Describe the breakdown of dietary proteins • Discuss the process of protein turnover • Explain the function of ubiquitin and the proteasome BIOENERGETICS TCA Cycle | Learning Outcomes • State the primary function of the TCA cycle • Provide the net reaction of the TCA cycle Explain the role of NADH and FADH2 • • Explain why the cycle functions only in aerobic conditions • Describe how the TCA cycle is regulated at three levels • Discuss how cytoplasmic NADH are transported across the mitochondrial membrane and any impact this has on ATP yields Respiratory chain | Learning Outcomes • Outline to function of the respiratory chain • Describe the importance of the mitochondrial structure in reference to the respiratory chain • Explain the pathway of electrons through the respiratory chain, from their entry points to the reduction of oxygen to water • Describe the movements of protons across the membrane in the context of the respiratory chain and the creation of a proton gradient Redox biochemistry | Learning Outcomes • Understand where reactive species are produced • What are the major types of reactive species in the cell • Describe the major antioxidant defense systems in place Oxidative Phosphorylation | Learning Outcomes • Outline the chemiosmotic theory • Describe the structure and function of ATP synthase • Explain what the P/O ratio is • Explain what an uncoupler does and provide an example • Describe the ways in which an agent may inhibit oxidative phosphorylation • Discuss the role reactive oxygen species play in oxidative phosphorylation • Describe the process of oxidative phosphorylation giving reference to the mitochondrial membrane • Calculate the number of ATP equivalents generated from a glucose molecule SIGNAL TRANSDUCTION IN METABOLISM Signal Transduction | Learning Outcomes • Briefly describe the function of cell / organelle membranes and how proteins may be associated with them. • Briefly explain the structures of integral membrane proteins. • Describe the role of a receptor in signal transduction • Describe the structure of 7TM receptors and provide an example. • Outline the process of signal transduction. • Describe the features and action of 7TM and protein kinase receptors, giving examples. • Discuss the role of secondary messengers and give examples. • Explain what can occur if there is a defect in a signal transduction pathway. FAT METABOLISM AND STORAGE Fat catabolism I, II, III and IV | Learning Outcomes • List roles of fats in the diet. • List roles of fats in the body. • Briefly describe the structure and function of some of the major fats in the body. • Provide examples of fats that contain one or more fatty acids. • Describe the general structure of a fatty acid. • Explain the difference between saturated and unsaturated fatty acids. • List examples of lipid-soluble vitamins and their roles/functions. • Describe the steps involved in the digestion and absorption of fats. 6 • • • • • • • • • • • • • • • • • Outline the major steps that take place during the digestion of dietary lipids. Describe the process of lipid absorption and chylomicron formation in intestinal cells. Describe the general structure of a lipoprotein. Describe the main features of each of the different classes of lipoproteins. Explain the difference between exogenous and endogenous transport of lipoproteins. Briefly explain the difference between ‘good’ versus ‘bad’ types of cholesterol. Briefly explain the role of the liver in lipoprotein metabolism. Briefly describe the three main stages of fatty acid breakdown. Outline the overall stoichiometry & energy yield of fatty acid breakdown. Explain where, when and how ketone bodies are produced. Briefly describe the three main stages of fatty acid synthesis. Describe the first committed step in fatty acid synthesis. Describe the structure and function of the fatty acid synthase complex. Outline the overall stoichiometry of palmitate synthesis. Briefly explain why and how fatty acids can be modified. Compare and contrast the main features of β-oxidation and fatty acid synthesis. Briefly describe the main mechanisms of control of fatty acid metabolism. INTEGRATION OF METABOLISM Hormonal control of Fuel Metabolism | Learning Outcomes • Discuss the role of glucose transporters and comment on the differences between the various isoforms. • Describe the structure of receptor tyrosine kinases, using the insulin receptor as an example. • Outline the role of insulin on the liver, skeletal muscle and adipose tissues in the fed state. • Outline the role of glucagon on the liver in the fasted state. • Outline the role of adrenaline on the liver, skeletal muscle and adipose tissues. Metabolic Specialisation of Tissues | Learning Outcomes • Describe how different glucose transporters confer tissue-specificity • Explain how different hormones (e.g. insulin, glucagon) act on different tissues Fuel Supply in Fasting | Learning Outcomes • Explain the three steps of the starved-fed cycle. • Describe the effects of glucagon, cyclic-AMP and F-2,6-bisP during fasting. • Explain the strategies employed for maintaining blood glucose levels during fasting/starvation. • Explain what happens after liver glycogen is depleted. • Describe the various effects of prolonged starvation. • Describe the overall changes in fuel metabolism during starvation - particularly with respect to requirements of the brain. Relationship to Other Courses within the Program Fuel Supply in Exercise | Learning Outcomes • Outline the structure of muscle fibres and their requirement for energy. • Explain the difference between Type I and II muscle fibres in appearance, functional properties and metabolic profiles. • Explain the role of adenylate kinase and link this to the role of ATP/AMP as regulators of glycolysis and glycogen metabolism. • Explain the role of creatine phosphate, creatine kinase, and the fate of creatine. • Discuss how the energy sources for a sprinter, middle distance runner and marathon runner differ. BIOC2101 is a requirement for Medical Science programs. The course builds on many concepts introduced in first level courses, particularly BABS1201 Molecules, Cells and Genes. It provides the knowledge and skills required for the third level courses including BIOC3261 Human Biochemistry and BIOC3111 Molecular Biology of Proteins. 4. Rationale and Strategies Underpinning the Course Teaching Strategies Rationale for learning and Course content is initially presented in lectures. Key concepts from the lectures are incorporated into practical sessions, where students also learn laboratory techniques and safe workplace skills. Students are provided with venues for revision, practice and discussion of the course content through large and small group tutorials on each major content area. Lectures are used in the course to introduce and introduce new concepts and elaborate on 7 6 teaching in this course , intermediary metabolism and its regulation. Laboratory sessions are designed to complement the lecture material in addition to teaching professional technical skills and safe and efficient work practices. Tutorials are used to reinforce concepts presented in the lectures through problem solving, and to encourage further enquiry. Both laboratories and tutorials aim to promote effective communicate, discussion and teamwork. The integration of these main teaching areas of the course are in accordance with the UNSW 7 Guidelines on Learning that inform Teaching . Specifically: • • • • • • • • • • • • • Effective learning is supported when students are actively engaged in the learning process. Effective learning is supported by a climate of inquiry where students feel appropriately challenged and activities are linked to research and scholarship. Activities that are interesting and challenging, but which also create opportunities for students to have fun, can enhance the learning experience. Learning is more effective when students’ prior experience and knowledge are recognised and built on. Students become more engaged in the learning process if they can see the relevance of their studies to professional, disciplinary and/or personal contexts. If dialogue is encouraged between students and teachers and among students (in and out of class), thus creating a community of learners, student motivation and engagement can be increased. Students learn in different ways and their learning can be better supported by the use of multiple teaching methods and modes of instruction (visual, auditory, kinaesthetic, and read/write). Clearly articulated expectations, goals, learning outcomes, and course requirements increase student motivation and improve learning. When students are encouraged to take responsibility for their own learning, they are more likely to develop higher-order thinking skills such as analysis, synthesis, and evaluation. Graduate attributes – the qualities and skills the university hopes its students will develop as a result of their university studies – are most effectively acquired in a disciplinary context. Learning cooperatively with peers - rather than in an individualistic or competitive way - may help students to develop interpersonal, professional, and cognitive skills to a higher level. Effective learning is facilitated by assessment practices and other student learning activities that are designed to support the achievement of desired learning outcomes. Meaningful and timely feedback to students improves learning. 6 Reflecting on your teaching http://learningandteaching.unsw.edu.au/content/LT/course_prog_support/guidelines.cfm?ss=2 7 8 5. Course Schedule Some of this information is available on the Online Handbook 8 and the UNSW Timetable 9. Lecture Rex Vowels Theatre Monday 12 pm Practical Wallace Wurth 122/123 Wednesday 10-1 or 2-5 or Thursday 10-1 Lecture Rex Vowels Theatre Thursday 1pm Week Begins Week 1 27 Feb Lecture 1: Introduction (KH) Lecture 2: Proteins (MW) Week 2 6 Mar Lecture 4: Enzyme Kinetics (MW) Lecture 5: Enzyme Regulation (MW) Week 3 13 Mar Lecture 6: Carbohydrates (VS) Lecture 7: Glycolysis (VS) Week 4 20 Mar Lecture 9: Gluconeogenesis (NW) Lecture 10: Glycogen (NW) Enzymes Week 5 27 Mar Lecture 11: TCA cycle (VS) Lecture 12: OXPHOS (NW) Enzyme Inhibition Week 6 3 Apr Lecture 14: OXPHOS (NW) Week 7 10 Apr Tutorial - (interactive*) Writing a short answer 1 (KH) Health & Safety (Online activity) Biochemistry skills and calculations (dry lab) Spectrophotometry Oxygen electrode simulation (Online prac) Online activity Tutorial - (interactive*) Writing a short answer 2 (KH) Online activity Lecture 8: Regulation of glycolysis (VS) QUIZ 1 (Lectures 1-9) – does not include practical materials Lecture 13: OXPHOS (NW) Lecture 15: REDOX regulation (GM) Public Holiday – Good Friday . No Prac Week 8 24 Apr Lecture 16: Fats 1 (AB) Lecture 17: Fats 2 (AB) Week 9 1 May Lecture 19: Fats 4 (AB) Lecture 20: Protein Catabolism (RL) Separation techniques 1 QUIZ 2 (lectures 10-19) – does not include practical materials Week 10 8 May Lecture 21: Urea cycle (RL) Lecture 22: Hormones (FB) Separation techniques 2 Tutorial – Advice for writing the lab report (KH) Week 11 15 May Lecture 23: Exercise (RL) Lecture 24: Fasting (KH) Week 12 22 May Lecture 26: Metabolic Specialisation of Tissues (KH) Glucose tolerance test Online activity Biochemical calculations quiz *Interactive tutorials involve practice writing and marking and may not be recorded. 9 Lecture 3: Enzymes (MW) MID-SESSION BREAK . 8 Glycolysis Lecture Mathews Theatre A Friday 9 am UNSW Virtual Handbook: http://www.handbook.unsw.edu.au UNSW Timetable: http://www.timetable.unsw.edu.au/ 9 Lecture 18: Fats 3 (AB) Lecture 25: Integration of Metabolism (KH) Lecture 27: Outro (KH) (lab report due today) 6. Assessment Tasks and Feedback Task Knowledge & abilities assessed Assessment Criteria Includes materials learned from lectures 1-9. Multiple choice Quiz 2 Includes materials learned from Lectures 10-19. Multiple choice and short answer Biochemistry calculations quiz Laboratory report FINAL THEORY EXAM Practical theory, ability to perform biochemical calculations and lab safety Scientific calculations that are relevant to what you learned in the pracs. Prepare a written laboratory report about one of your practicals. Instructions will be provided on Moodle. All relevant materials related to lectures/tutorials (no calculations or content from practicals). Feedback Date Quiz 1 Pre-lab quizzes % On-line Short answer Aims (1 mark) Introduction (2 marks) Methods (2 marks) Results (4 marks) Discussion (2 marks) References (1 mark) Multiple choice questions, essay, and short response questions. WHO 20 24 March 20 5 May Mandatory Pre-lab quizzes must be completed by Weds 9 AM on week of your prac 8 During practical times 24-25 May Submit to Turnitin (Moodle) and drop a paper copy at the BSB Office by Friday 26 May at 12:00 pm 12 100 10 HOW Convenor Within 1 week Online Convenor Within 2 weeks Online Convenor Immediately Online Convenor Within 2 weeks Online Convenor Within 3 weeks Online See examination timetable 40 TOTAL WHEN 7. Additional Resources and Support th Text Books Biochemistry 8 edition, WH Freeman and Company, 2015. JM Berg, JL Tymoczko, GJ Gatto, and L Stryer Availability: UNSW bookshop, UNSW library: Open Reserve/High use collection Course Manual Course manual containing tutorials and practicals is available for purchase through the UNSW Bookshop or through download via Moodle. Recommended Internet Sites All students enrolled in BIOC2101 automatically have access to the course Moodle site http://telt.unsw.edu.au/. This site will be used to distribute course notes and information, and should be checked at regular intervals. In particular, the site will be used to provide: • Assessment marks • Practical notes • Lecture handouts • Information about examination arrangements • Wet practical exercise papers • Further assessment information resulting from special consideration • Self-management resources There are also a number of computer exercises and teaching aids available to students enrolled in BIOC2101 Principles of Biochemistry (Advanced). Links to the textbook companion websites and additional online animations and revision tutorial can be found on the course Moodle site. Computer Laboratories or Study Spaces Computers are available in Room G08, Biosciences Building. This room is open from 9-5 pm Monday to Friday and is available to all students enrolled in BIOC2101. Access to the room can be gained only by swiping your student card through the electronic lock. The room is not available to other students if it is being used for a class, even if additional seating is available. There is a student common area (BiBS) for study or relaxation with coffee and food preparation facilities in Biosciences 107. 8. Required Equipment, Training and Enabling Skills Equipment Required Personal protection equipment (PPE): safety glasses, lab coat and closed shoes. Calculator Timer Enabling Skills Training Required to Complete this Course Students need to complete the HS training (online or during the first lecture) and pass the HS quiz online. 9. Course Evaluation and Development MyExperience Students have the opportunity to provide feedback online, as instructed in the final week of term. 11 10. Administration Matters Expectations of Students Students are expected to be regular and punctual in attendance at all classes (lectures, practicals and tutorials). If students miss more than 1 practical, they may be refused final assessment or may receive a UF grade. Students are 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 Wednesday 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. Students must bring their own copy of the relevant laboratory experiment to each practical class. Failure to do so will result in exclusion from the class and an absence recorded. In some practical classes, raw data results will need to be confirmed by the Demonstrator. Failure to obtain confirmation may result in an absence being recorded. Illness and Misadventure In addition to the university's centralised procedure BIOC2101 Principles of Biochemistry (Advanced) has a local procedure that you will also need to follow. − If you are absent from a small group tutorial or practical due to a medical condition, it is sufficient to submit a copy of your medical certificate to your tutor/demonstrator at the next small group tutorial/practical. If you believe that the medical condition is confidential, then see the Course Coordinator. − Considerations for absences due to other reasons should be submitted to and discussed with a Course Coordinator within seven (7) days of the absence. Such matters will be treated in confidence. These procedures are designed to ensure that you are not penalised for absences for which there was an appropriate reason. A pass in BIOC2101 is conditional upon a satisfactory performance in the practical program. This consists of: (i) attendance at all of the practical classes (unless illness & misadventure documented); (ii) satisfactory performance in the marked practical assessment task, and; (iii) maintenance of an accurate and up-to-date laboratory manual, including the recording of all data and completion of calculations and questions. Assignment Submissions Occupational Health and 10 Safety Assessment Procedures UNSW Assessment 11 Policy Assignments in this course will require a submission cover sheet for submission to the BSB Office. Cover sheets available on Moodle or at the BSB office. Late submissions lose 25% per day late. Prior to commencing the practical component of the course you will need to complete and pass an HS Awareness quiz that covers information presented to you in week 1. Part of your laboratory practical manual comprises an Undergraduate Risk Assessment Guide for the School of Biotechnology and Biological Sciences. This addresses the hazards and risks you may encounter. To work with these correctly and safely, you will need to follow the safe work practices provided in the manual or by academic or technical staff involved in the course. Additional information on Health and Safety at UNSW can be found at: http://www.HS.unsw.edu.au/ SPECIAL CONSIDERATION AND FURTHER ASSESSMENT SEMESTER 1 2017 Students who believe that their performance, either during the session or in the end of session 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 “Special Consideration” section of Moodle for specific instructions related to each BABS course they are studying. Further general information on special consideration can also be found 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 10 11 UNSW HS Home page UNSW Assessment Policy 12 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 - download here). 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. 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. 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 Semester 1 2017, BABS Supplementary Exams will be scheduled on: ► Thursday 13th of July 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 93854734, http://www.studentequity.unsw.edu.au/ or http://www.equity.unsw.edu.au/disabil.html 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 12 Procedure School Contact Faculty Contact University Contact A/Prof Louise LutzeMann, Room 241C Biological Sciences Bldg. Dr Gavin Edwards Associate Dean (Undergraduate Programs) [email protected] Tel: 9385 4652 Student Conduct and Appeals Officer Telephone 02 9385 8515, email: [email protected]. University Counselling and 13 Psychological Services Tel: 9385 5418 [email protected] 12 13 UNSW Student Complaint Procedure University Counselling and Psychological Services 13 11. UNSW Academic Honesty and Plagiarism Academic misconduct may apply to any work or document related to assessment that is submitted to the School of Biotechnology and Biomolecular Sciences; this includes quizzes, the marked practical, laboratory workbooks, and examinations. All work 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. 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 14 12. PRACTICALS 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, you MUST complete an online Laboratory Occupational Health and Safety Quiz that is accessed through the BIOC2101 Moodle site. Your final quiz mark will be checked prior to your lab in Week 2. If you have not scored 100% in the quiz by 9am on the day of your Week 2 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 to the course coordinator within three days of the absence. 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. We have found that a good way to prepare for the next laboratory exercise is to make a short summary of the actual techniques and manipulations that you will be using in the laboratory. This summary can take the form of some brief, written comments, or it can take the form of a flow diagram that maps out the steps that you will have to take in order to complete the experiment. Within this manual, a page has been provided at the beginning of each practical for the purpose of writing up a summary and notes of the experiment(s) to be performed. 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, preferably before the next practical class when 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 15 why and you will be given an opportunity to rectify any problems. By the end of your last practical of this session, ALL question pages are to be completed and marked ‘Satisfactory’ by your Demonstrator. Failure to do so will result in the deduction of marks from your final assessment mark in BIOC2101. REMEMBER: A pass in BIOC2101 is conditional upon a satisfactory performance in the practical program. 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 BIOC2101 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 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 and safe work space may result in your removal from the laboratory and disqualification from the prac (in extreme cases, but that will never happen right..). NOTE: You are liable for any negligent or intentional 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. 16 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 Students are expected to be regular and punctual. Professional documentation (e.g. medical certificates) explaining absences from formal assessments and laboratory practicals should be included with an online application for Special Consideration according to the guidelines provided in this manual (and online via Moodle). All such documentation should be submitted within three days of the absence in question. This procedure will ensure that you are not penalised for absences for which there were appropriate medical or compassionate reasons. IMPORTANT NOTE: IF STUDENTS ATTEND LESS THAN EIGHTY PERCENT OF THEIR POSSIBLE PRACTICALS, THEY WILL BE REFUSED FINAL ASSESSMENT. 17 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 have been set down by the NSW Environmentally Hazardous Chemical Regulation 2008, NSW WHS Regulation 2011, NSW Work-cover Publications, Work-safe National Codes of Practice and Guidance Notes and Australian Standards AS:2243 series Safety in Laboratories. These policies and standards apply 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 18 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. 19 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 20 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. 21 STUDENT SAFETY DECLARATION I,……………………………………………………………(Print Full Name Please) declare that I have completed the safety quiz for BIOC2101, that I have read the Safety in Laboratories and Appendix Instrumentation sections in my BIOC2101 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:…………………………….. 22 SPECTROPHOTOMETRY PRACTICAL Instructions: Complete online prelab quiz. Read and attempt to complete questions through page 30 prior to attending the prac. Notes ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… …………………………………………………………………………………………………………….. 23 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 = = 24 µ log10e or µ l g -1 cm -1 2.303 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. is the absorption coefficient for a 1% (1 g/100ml or 10 mg/ml) solution and 1 cm light path α1% 1cm 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: ε = -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. 25 Absorbance @ λ nm Concentration (µg/µl) 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. 26 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. 27 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. 28 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. 29 PRE-LAB QUESTIONS 1. What is the molar absorption coefficient (ε)? What equation is used to obtain its value? What are the correct units? 2. What is haemoglobin? 3. Show how you would dilute a sample of the dye 1/100, and then a further 1/100 to obtain an overall dilution of 1/10000. The final volume should be 5 mL. Write down your workings and a diagram. 30 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. 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? 31 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 have pipettors that can only dispense more than 20µL. 5. What is the concentration of the diluted Amido Black 10B dye (in mM and mg/mL)? 6. How many nanomoles of Amido Black 10B dye is in the diluted sample? 32 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 i.e. 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: Signed: Mastery of basic biochemistry calculations: 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. ☐ ☐ 33 C. PIPETTING 34 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. 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 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. ☐ ☐ ☐ 35 PRACTICAL 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. 36 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. 37 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. Plot space provided on the next page 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. 38 Glue a copy of your labelled standard curve onto this space (about 10 cm high x 12 cm wide would be a good size) 39 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. ☐ ☐ ☐ 40 ENZYMES PRACTICAL Notes ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… 41 PRACTICAL: 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- acid phosphatase OH + H2O R O 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. 42 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. acid phosphatase 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. 43 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. 2. Microtiter plate – once the liquid is discarded, throw into a biological waste bin. 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. 44 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 Experiment 2 Time Course Experiment 3 Effect of pH 3 4 2 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 Experiment 2 Time Course Experiment 3 Effect of pH 3 4 2 A pH 3.0 (citrate) pH 4.5 (citrate) B C D pH 5.5 (citrate) pH 6.0 (citrate) pH 7.0 (Tris) pH 9.0 (Tris) E F G H 45 Experiment 4 Effect of Enzyme Conc. 5 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 Experiment 2 Time Course Experiment 3 Effect of pH 3 4 2 A B C D E F G H 46 Experiment 4 Effect of Enzyme Conc. 5 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. 47 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. 48 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. 49 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 50 Absorbance at 405 nm * p-NP (nmol per well) 0.00 0 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. 51 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 52 nmol p-NP produced per well per 10 mins 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. 53 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. 54 Glue copies of your graphs onto this space 55 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? ____________________________________________________________________________ ____________________________________________________________________________ ____________________________________________________________________________ ____________________________________________________________________________ 56 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: ................................. 57 PRACTICAL: ENZYME INHIBITION BEFORE CLASS PREPARATION ☐ Complete the online pre-work for the practical on Moodle. ☐ Read through the practical notes, including the risk assessment. ☐ Answer the pre-lab questions below. LEARNING OUTCOMES To demonstrate the digestion of casein, the major protein in milk, by protease enzymes. To describe how the zymogen precursors of protease enzymes are activated in the small intestine. To explain the specific inhibition of proteases and the role of pancreatic trypsin inhibitors. PREWORK Read through the practical notes to determine the answers for each of the following questions. What is the substrate used in Part A _________, Part B _________ and Part C _________? What is the enzyme added to act on the substrate in Part A ________? What is the enzyme added to act on the substrate in Part B ________? How is each reaction stopped? ________________________________________________ Write an aim for each Part of the experiment. INTRODUCTION There are four parts to this practical session: A. Investigating the digestion of casein by trypsin. B. Investigating the digestion of casein by chymotrypsin. C. Investigating the activation of the zymogen chymotrypsinogen by trypsin. D. Investigating the properties of pancreatic trypsin inhibitors. The workload will be distributed between the students in each demonstrator group, with each student completing at least one component. Comparison of the results from experiments A and B will provide some information about a pancreatic trypsin inhibitor protein, for use in conjunction with information obtained in experiment D. Experiments A and B also act as controls that are required for interpreting the results of experiment C. The time allocation is: 15 min 80 min 20 min Initial discussion and allocation of practical tasks Completion of practical tasks Reporting and discussion of results within the group 58 BABS Teaching Laboratory Student Risk Assessment Practical: Enzyme Inhibition Demonstrator: Hazards General Risks See below Biological agent (biological name) Exposure to skin may result in irritation / itching / soreness. Eye exposure, may cause damage. May be an environmental hazard. Vapours may constitute a respiratory hazard Biological material for research use only Trypsin, Chymotrypsin, chymotrypsinogen, Pancreatic trypsin inhibitor Casein Chemical (chemical name) Trichloroacetic acid (10%) Bradford’s Reagent (phosphoric acid, methanol, brilliant blue G) 100mM Phosphate buffer pH 7.6 Procedural (plant and equipment) Pipettes Water baths Controls At all times follow demonstrators instructions when handling all biological substances, chemicals and equipment items. Notify demonstrator immediately of any spills or other incidents. PPE (lab coats, closed in shoes & gloves). Adhere to aseptic techniques. Proper handwashing before leaving the laboratory. NON- HAZARDOUS (see MSDS) Hazardous (corrosive & harmful to aquatic organisms). Causes burns PPE (lab coats, closed in shoes & gloves as required). Awareness of safety showers and eye wash facilities. Harmful by inhalation, in contact with the skin and if swallowed, possibly irreversibly. Irritating to eyes and skin. PPE. In case of contact with eyes wash with plenty of water and seek medical advice. Spillage may occur where pipette tips are not securely fasted to pipette, Incorrect pipetting techniques such as sucking up liquids too quickly or dispensing liquids too quickly may result in spills. PHYSICAL INJURY – Loose clothing, body parts may get caught in water baths that have a “moving tray” mechanism. Place pipette barrel into pipette tip, apply enough force that a snug fit is achieved but not so much force that pipette tip holder flips or pipette tip cracks, use correct technique. PHYSICAL INJURYELECTRICAL SHOCK Water baths are an electrical appliance containing moderate to large volumes of liquid EXPOSURE- CHEMICAL AGENTS Shake samples 59 Tie hair back, secure loose clothing. Temporarily switch off water bath when retrieving or placing a sample within it. Switch off if incident occurs. Avoid dripping / splashing liquids on external surface of water bath or electrical components Ensure samples are sealed correctly and are properly secured so they don’t topple can be easily spilt in water bath, resulting in spillage of chemicals and biological agents and spill Personal Protective Equipment Emergency Procedures In the event of an alarm sounding, stop the experiment, turn off electrical equipment and wait for confirmation from demonstrators to evacuate. Then pack up your bags and wash your hands. Follow the instructions of the demonstrators regarding exits and assembly points. The BioScience assembly point is in the lawn in front of the Chancellery. In the event of an injury inform the demonstrator. First aiders and contact details are on display by the lifts. Clean up and waste disposal All used microfuge tubes should be placed in the biohazard bins. Used test tubes should be placed in the appropriate container on the front bench of the lab. All solutions should be placed in the appropriate liquid waste containers. Used pipette tips should be placed in approved biohazard sharps containers. Declaration – will be checked by demonstrator at the beginning of the practical class. I have read and understand the safety requirements for this practical class and I will observe these requirements. Signature: Date: 60 BACKGROUND Experiments A, B and C all use an assay for protease enzyme activity. In this assay the substrate is the milk protein casein and the enzyme is a pancreatic protease – either trypsin or chymotrypsin. When casein is incubated with trypsin or chymotrypsin, the enzyme hydrolyses internal peptide bonds within the casein protein, progressively releasing more low molecular weight peptides from the casein. The protease reaction can be stopped by adding trichloroacetic acid which denatures and precipitates the protease enzymes, intact casein molecules and large casein fragments. However, small casein fragments remain in solution. Centrifugation therefore removes the enzyme and substrate, leaving only the small molecular weight peptide products in the supernatant. These can be measured by adding a sample of the supernatant to Bradford’s Reagent which produces an intense blue colour with all peptides and proteins, A. INVESTIGATING THE DIGESTION OF CASEIN BY TRYPSIN. This experiment involves incubating casein with trypsin for different time intervals in microcentrifuge tubes, both with and without pancreatic trypsin inhibitor. The trypsin enzyme reaction is “stopped” by the addition of trichloroacetic acid and the tubes are then centrifuged to sediment the precipitated trypsin, pancreatic trypsin inhibitor, casein and large casein fragments. Samples of the supernatants are added to Bradford’s Reagent in a microtitre plate and the absorbance read at 620 nm to measure the amount of soluble small peptides that have been released from the casein. This experiment should be attempted by two pairs of students. Reagents: Casein solution (10 g/l in 100 mM phosphate buffer, pH 7.6) Trypsin solution (0.05 mg/ml) Pancreatic Trypsin Inhibitor (0.05 mg/ml) Phosphate buffer (100 mM, pH 7.6) Trichloroacetic Acid (10%) Bradford’s Reagent 61 Method 1. Add 200 µl casein and 120 µl phosphate buffer to each of 8 microcentrifuge tubes (labelled 1– 8). 2. Add 50 µl RO water to tubes 1–4, and 50 µl pancreatic trypsin inhibitor to tubes 5–8. 3. Add 30 µl trypsin to tubes 2-4 and 6-8, 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. At the following times (after the addition of trypsin) add 600 µl trichloroacetic acid to each of tubes 2-4 and 6-8, mix well by inversion and place on ice: Tubes 2 and 6 10 min Tubes 3 and 7 20 min Tubes 4 and 8 30 min 5. At a convenient time during these incubations, add 30 µl trypsin to tubes 1 and 5, and immediately follow this by the addition of 600 µl trichloroacetic acid and place on ice. These tubes are the zero time points. 6. Leave on ice at least 3 min after the addition of trichloroacetic acid before centrifuging all tubes for 3 min in a microcentrifuge. 7. In triplicate, add 100 µl samples of each supernatant to wells in a microtitre plate and 100 µl RO water to three additional wells (blanks). Then add 200 µl Bradford’s reagent to each well containing sample or RO water, wait 5 min for the colour to develop and read the absorbance at 620 nm. Results Calculate the average absorbance values for your triplicate “blanks” and experimental results. Subtract the “blank” from the other absorbance values. On the same graph, plot A620 against time for the hydrolysis of casein by trypsin in the absence and presence of pancreatic trypsin inhibitor. 62 B. INVESTIGATING THE DIGESTION OF CASEIN BY CHYMOTRYPSIN This experiment involves incubating casein with chymotrypsin for different time intervals in microcentrifuge tubes, both with and without pancreatic trypsin inhibitor. The chymotrypsin enzyme reaction is “stopped” by the addition of trichloroacetic acid and the tubes are then centrifuged to sediment the precipitated chymotrypsin, pancreatic trypsin inhibitor, casein and large casein fragments. Samples of the supernatants are added to Bradford’s Reagent in a microtitre plate and the absorbance read at 620 nm to measure the amount of soluble small peptides that have been released from the casein. This experiment should be attempted by two pairs of students. Reagents: Casein solution (10 g/l in 100 mM phosphate buffer, pH 7.6) Chymotrypsin solution (0.05 mg/ml) Pancreatic Trypsin Inhibitor (0.05 mg/ml) Phosphate buffer (100 mM, pH 7.6) Trichloroacetic Acid (10%) Bradford’s Reagent Method 1. Add 200 µl casein and 120 µl phosphate buffer to each of 8 microcentrifuge tubes (labelled 1 –8). 2. Add 50 µl RO water to tubes 1–4, and 50 µl pancreatic trypsin inhibitor to tubes 5-8. 3. Add 30 µl chymotrypsin to tubes 2-4 and 6-8, noting the time of addition and mixing well by inversion immediately after each addition, and place each tube in a 37° C water bath. 4. At the following times (after the addition of chymotrypsin) add 600 µl trichloroacetic acid to each of tubes 2-4 and 6-8, mix well by inversion and place on ice: Tubes 2 and 6 10 min Tubes 3 and 7 20 min Tubes 4 and 8 30 min 5. At a convenient time during these incubations, add 30 µl chymotrypsin to tubes 1 and 5, and immediately follow this by the addition of 600 µl trichloroacetic acid and place on ice. These tubes are the “zero” time points. 6. Leave on ice at least 3 min after the addition of trichloroacetic acid before centrifuging tubes for 3 min in a microcentrifuge. 7. In triplicate, add 100 µl samples of each supernatant to wells in a microtitre plate and 100 µl RO water to three additional wells (“blanks”). Then add 200 µl Bradford’s reagent to each well containing sample or RO water, wait 5 min for the colour to develop and read the absorbance at 620 nm. 63 Results Calculate the average absorbance values for your triplicate “blanks” and experimental results. Subtract the “blank” from the other absorbance values. On the same graph, plot A620 against time for the hydrolysis of casein by chymotrypsin in the absence and presence of pancreatic trypsin inhibitor - graph paper will be provided. 64 C. INVESTIGATING THE ACTIVATION OF THE ZYMOGEN CHYMOTRYPSINOGEN BY TRYPSIN This experiment involves incubating chymotrypsinogen with trypsin for different time intervals in microcentrifuge tubes. The activation of chymotrypsinogen by trypsin is “stopped” by the addition of pancreatic trypsin inhibitor. The formation of active chymotrypsin is then demonstrated by the addition of a substrate, casein, and a second incubation during which the active chymotrypsin can hydrolyse the casein. The chymotrypsin reaction is “stopped” by the addition of trichloroacetic acid and the tubes are then centrifuged to sediment the precipitated trypsin, chymotypsinogen, chymotrypsin, pancreatic trypsin inhibitor, casein and large casein fragments. Samples of the supernatants are added to Bradford’s Reagent in a microtitre plate and the absorbance read at 620 nm to measure the amount of soluble small peptides that have been released from the casein. This experiment should be attempted by at least two pairs of students. Completion of the experiment in the allocated 80 min will require making a prompt start and working quickly and efficiently. Reagents: Casein solution (10 g/l in 100 mM phosphate buffer, pH 7.6) Trypsin solution (0.05 mg/ml) Chymotrypsinogen solution (1.0 mg/ml) Pancreatic Trypsin Inhibitor (0.05 mg/ml) Trichloroacetic Acid (10%) Bradford’s Reagent Method 1. Add 125 µl chymotrypsinogen to each of 6 microcentrifuge tubes (labelled 1-6) 2. Add 25 µl trypsin to each of tubes 2-6 at one minute intervals, noting the exact time for each addition. Quickly mix each tube well by inversion immediately after trypsin addition and place in a 37° C water bath, 3. At the following times (after the addition of the trypsin) add 50 µl pancreatic trypsin inhibitor to each of tubes 2-6 and mix well by inversion: Tube 2 Tube 3 Tube 4 Tube 5 Tube 6 25 min 20 min 15 min 10 min 5 min 4. At a convenient time during these incubations, add 25 µl trypsin to tube 1, and immediately follow this by the addition of 50 µl pancreatic trypsin inhibitor. This tube is the “zero” time point. Mix well. 5. As soon the pancreatic trypsin inhibitor has been added to Tube 2, start adding 200 µl casein to each of the six microcentrifuge tubes. Make the addition to Tube 2 last. It is important that the contents of the tube are allowed to incubate with the pancreatic trypsin inhibitor for at least a minute, to ensure complete inhibition of the trypsin before casein addition. 6. Incubate the six microcentrifuge tubes at 37° C for a further 15 min, and then add 600 µl trichloroacetic acid to each tube and mix well by inversion and place on ice. 65 7. Leave on ice at least 3 min after the addition of trichloroacetic acid before centrifuging tubes for 3 min in a microcentrifuge. 8. In triplicate, add 100 µl samples of each supernatant to wells in a microtitre plate and 100 µl RO water to three additional wells (“blanks”). Then add 200 µl Bradford’s reagent to each well containing sample or RO water, wait 5 min for the colour to develop and read the absorbance at 620 nm. Results Calculate the average absorbance values for your triplicate “blanks” and experimental results. Subtract the “blank” from the other absorbance values. Plot a graph of A620 against time for the hydrolysis of casein by chymotrypsin resulting from the activation of chymotrypsinogen by trypsin - graph paper will be provided. 66 D. INVESTIGATING THE PROPERTIES OF PANCREATIC TRYPSIN INHIBITORS This practical component should be completed by whoever finishes Experiments A and B first (including calculating and plotting results). The task is to use the resource materials provided by the demonstrators to answer the following questions about pancreatic trypsin inhibitors, and to present the information to the rest of the group. Q1. At a protein structure level, what does a trypsin inhibitor do? Q2. What is their role(s) in the normal function of trypsin in digestion? Q3. What size of molecules are they? Q4. What relationship is there between pancreatic trypsin inhibitors and pancreatitis? Q5. What foods contain trypsin inhibitor proteins, and what is the role of the inhibitor in these foods? DISCUSSION Q1. What can you deduce from the results for Experiments A and B? Q2. Why do the enzymes hydrolyse casein but not themselves? Q3. Why are Experiments A and B important controls for Experiment C? Q4. What can you deduce from the results for Experiment C? 67 PRACTICAL: 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. 68 GLYCOLYSIS PRACTICAL: Notes ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… ……………………………………………………………………………………………………………………… 69 PRACTICAL: 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 70 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. 71 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. 72 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. 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. 73 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 74 Abs. Lactate formed (µmol) 0 0 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. 75 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 76 Abs. Lactate formed (µmol) 0 0 QUESTIONS What “cofactors” are required for conversion of F1,6BP to lactate by the enzymes in the skeletal muscle extract? Why is each of the “cofactors” necessary? (HINT: Look at the different steps in the glycolytic pathway.) From your results, which enzyme appears to be the rate-limiting step in glycolysis by the skeletal muscle extract? Does this conform with your expectations? (HINT: Consider what you have learned in your lectures.) 77 Why does some Glycolysis appear to occur starting from glucose and F6P, even though no ATP has been added? What is the explanation for the experimental results for lactate formation from GAP compared to 1,3BPG? Assessment: SATISFACTORY / UNSATISFACTORY Demonstrator’s signature:……………………………. Date:............................. 78 SEPARATION TECHNIQUES PRACTICAL: Notes ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… 79 PRACTICAL: 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. 80 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. 81 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 82 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. 83 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 84 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? __________________________________________________________________________ __________________________________________________________________________ _________________________________________________________________________ 85 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: ............................... 86 PRACTICAL: SEPARATION TECHNIQUES 2 BEFORE CLASS PREPARATION ☐ Complete the online pre-work for the practical on Moodle (if present). ☐ Read through the practical notes, including the risk assessment. ☐ Read the section on protein purification. A link to the online version: http://www.ncbi.nlm.nih.gov/books/NBK22410/ ☐ Read about sodium dodecyl (lauryl) sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at http://askabiologist.asu.edu/sds-page LEARNING OUTCOMES • • • To explain the basic principles of protein separation and visualization by polyacrylamide gel electrophoresis. To perform polyacrylamide gel electrophoresis. To perform a Bradford assay to quantitate the amount of protein present in a sample. INTRODUCTION The following techniques will be introduced to you: 1. Protein separation by polyacrylamide gel electrophoresis; 2. Bradford’s assay for protein quantitation. Students will work in pairs to perform all of the above techniques. Following completion of the techniques, demonstrator groups will meet to discuss the techniques performed and the results obtained. Items for discussion include: 1. 2. 3. 4. green fluorescent protein; overexpression vectors; SDS-PAGE; Bradford’s assay. BACKGROUND The Nobel Prize for Chemistry 2008 was awarded “for the discovery and development of the green fluorescent protein, GFP.” In this practical, you will investigate the overexpression of this protein. Green fluorescent protein (GFP) originally comes from the jellyfish Aequorea victoria. It is what we call a β-barrel protein because it consists of β-sheets that together form a barrel (Figure 1A). It has 238 amino acids and fluoresces green when exposed to UV light. In fluorescence, a photon of light is absorbed by the fluorophore and as the fluorophore relaxes to the ground state, light is emitted at a different wavelength (hence 87 the fluorescence you see). GFP absorbs light at 395 nm and emits green light at 475 nm. More details: http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2008/ A. B. Figure 1. A. X-ray crystal structure of A. victoria green fluorescent protein mutant. (PDB ID: 1 EMG). B. pGLO plasmid map (Bio-Rad). For this experiment, the GFP producing gene is carried on an engineered plasmid named pGLO (Figure 1B). This is a commercially available vector that can be used in recombinant DNA experiments performed in E. coli. The vector has a number of other features, namely, an origin of replication (ori), an antibiotic resistance gene (bla), inducible promoters and a gene for arabinose metabolism (araC). The presence of ori allows the vector to be replicated in E. coli cells, this is particularly important so that daughter cells receive copies of the plasmid. The antibiotic resistance gene (bla) enables the cells containing the plasmid to grow in the presence of ampicillin. This feature is termed a selectable marker, as it allows for only cells containing the plasmid carrying the marker selected i.e. when ampicillin is added to a growing culture of E. coli cells, those without the resistance gene not be viable. A multiple cloning site, allowing other DNA to be cloned into the vector, is located at the end of the GFP gene (not shown). The araC gene is present to allow control of GFP expression. When arabinose, a sugar, is present it binds with the AraC protein (produced by the araC gene). The AraCarabinose complex then binds with a sequence (termed an operator) upstream of the GFP gene to induce (switch on) its expression and lead to GFP production. 88 Figure 2: Arabinose mediated control of green fluorescent protein expression in pGLO. (http://www.bio-rad.com/LifeScience/pdf/Bulletin_9563.pdf). RISK ASSESSMENT Bradford’s reagent is TOXIC and CORROSIVE (contains phosphoric acid and methanol) Proteins are biological hazards Coomassie stain - Slightly hazardous in case of skin contact (irritant), of eye contact (irritant), of ingestion, of inhalation. Destain – methanol, Acetic acid- Highly flammable. - Toxic by inhalation, in contact with skin and if swallowed Electrophoresis apparatus Gel loading buffer (mercaptoethanol and SDS, glycerol, bromophenol blue) – mercaptoethanol is very hazardous in case of skin contact (permeator) and of ingestion. Hazardous in case of skin contact (irritant), of eye contact (irritant), of inhalation. Severe over-exposure can result in death. Heating blocks – burning 89 1. SDS-PAGE FOR SEPARATION OF SOLUBLE CELLULAR PROTEINS Aim To identify the overexpression of GFP using SDS-PAGE and staining with Coomassie blue. Introduction SDS-PAGE is a method for separating and visualising proteins. A protein same is loaded into the gel matrix and an electrical current applied. Proteins vary in size, but also in charge (dependent on pH). In this experiment we use the detergent SDS to “coat” the protein, giving them an overall negative charge, so they will migrate according to their size only. The gel loading buffer contains a dye to assist you in pipetting the protein samples into the wells. This is not bound to the proteins, so runs through the gel according to its own properties i.e. it does not represent the position where you overexpressed protein is running. The loading buffer also contains glycerol, which is heavy and ensures the samples fall to the bottom of the wells rather than floating. It also contains a reducing agent, beta-mercaptoethanol, which eliminated disulfide bonds (recall these features of protein structure from your Proteins lecture). A standard is also added that enables you to correlate the sizes of you sample proteins. The proteins in this standard have been pre-stained, so you can see them migrate through the gel. You can measure the relative migration distance (Rf) of the standards. The logarithm of the molecular weight of the standards (y axis) and their relative migration distance (Rf) (x axis) is plotted into a graph and should result in a linear plot. If your sample is fully denatured (it should be) you can read its size off the plot. To visualise the proteins in your samples, you will stain the gel with Coomassie blue stain. Reagents and Materials E. coli JM109 cells containing the pGLO plasmid (Bio-Rad); induced and uninduced SDS-PAGE loading buffer (Bio-Rad 4x Laemmli sample buffer) Gel electrophoresis apparatus containing a 4-20% mini-PROTEAN TGX gel, 1x Tris /glycine /SDS Running Buffer (Bio-Rad) Kaleidoscope protein molecular weight standard (Bio-Rad) Coomassie blue stain (Coomassie Brilliant Blue, 50% methanol, 10% acetic acid, 40% water (v/v) Destain solution: 40% methanol, 10% acetic acid, 50% water (v/v) 90 Method Technical staff inoculated 10 mL Luria broths with a single colony of E. coli JM109 cells containing the pGLO plasmid (Bio-Rad) with a final concentration of 100 µg/mL ampicillin. After approximately 16 hours growth at 37˚C, these were used to inoculate (1:100) flasks containing 50 mL Luria broth with a final concentration of 100 µg/mL ampicillin. The cultures were then grown to an optical density of 0.2 at 600 nm on the day of the experiment, before some flasks were treated with arabinose to induce expression of the GFP producing gene Take 500 µL of induce and uninduced cultures and transfer to separate microcentrifuge tubes. 1. Centrifuge for 2 minutes at 13,000 rpm. 2. Remove and discard the supernatant from each tube and resuspend the cell pellets in 100 µL SDS-PAGE loading buffer. 3. Place the samples on the heating block at 95˚C for 5 minutes. 4. Load 15 µL of each sample onto the SDS-PAGE gel using the gel loading tips. A Kaleidoscope protein molecular weight standard (Bio-Rad) must be added to each gel. Proteins are separated by electrophoresing at 150 V for 45 minutes. 5. 6. 7. 8. Once completed, your demonstrator will assist you to remove the get and place it in a tray. Before staining, measure the distance the standards have migrated. Each gel needs to be taken to the fume hood and 10-20 mL Coomassie blue stain will be added. The gels are to be left on the rocker platform overnight. Technical staff will remove the stain after approximately 16 hours and destain until your next class. 91 Questions 1. What is the current (mA) when the gel is running? ______________________________________________________________________ 2. What are the sizes of the proteins in the Kaleidoscope protein molecular weight standard (Bio-Rad)? ______________________________________________________________________ 3. What does PAGE stand for? ______________________________________________________________________ 4. What was PAGE and not agarose gel electrophoresis performed? ______________________________________________________________________ 5. Why was SDS used? ______________________________________________________________________ 6. When was beta-mercaptoethanol used? Why? ______________________________________________________________________ Results THIS WEEK: Plot a graph of the logarithm of the molecular weight of the standards (kDa) (y axis) and their relative migration distance (Rf) (x axis). You will use this next week to determine the size of the GFP. NEXT WEEK Label the gel photo with a title, lane numbers, the sizes of the standard proteins, the direction the samples travel and indicate GFP. Examine your protein gel and the printout of a sample gel and answer the following questions: 1. Can you identify the GFP on the gel? Using the protein size standard, determine the size of GFP. Is this as expected? 2. Was the GFP overexpressed in cells cultured with or without arabinose? ______________________________________________________________________ 92 Discussion questions 1. Which plasmid was present in the E. coli cells that carried the GFP gene, allowing GFP to be produced? ______________________________________________________________________ 2. Which gene regulates GFP synthesis? ______________________________________________________________________ 3. What role does araC have in switching GFP on/off in pGLO? ______________________________________________________________________ 4. What role does arabinose have in switching GFP on/off in pGLO? ______________________________________________________________________ Draw two diagrams showing the GFP gene carried by pGLO, one showing what occurs in the presence of arabinose and one in the absence. 93 BRADFORD’S ASSAY Aim To perform a Bradford’s assay and quantitate the amount of protein in a sample. Introduction In this experiment you will measure the protein concentration of the cell lysates used in experiment one using the “Bio-Rad Protein Assay” kit is used for this experiment. This is a commercially available variant of Bradford’s assay (Bradford, 1976). The principle behind the assay is the differential colour change of Coomassie Brilliant Blue G-250 dye in response to varying concentrations of protein. The absorbance maximum for an acidic solution of Coomassie Brilliant Blue G-250 dye shifts from 465 nm to 595 nm when bound to protein and the absorbance change will be measured at 620 nm. The dye binds primarily to basic and aromatic amino acid residues, particularly arginine. Reagents Bradford’s reagent Bovine serum albumin (5 mg/mL) as the protein standard E. coli JM109 cells containing the pGLO plasmid (Bio-Rad); induced and uninduced (cell lysates were diluted prior by______________________________________) Method The method is suitable for protein samples containing 0.0005-0.005 mg protein (0.5-5 µg) in 100 µl. Add 0-100 µl protein standards (“standards” and “unknowns”) to wells in a microtitre plate (you can choose the increments you use) and make up the total volume to 100 µl with RO water. Include RO water samples as controls, “blanks”. For your samples, add 20, 50 and 100 µL in triplicate. When all the “blanks”, “standards” and “unknowns” have been prepared, add 150 µl diluted, filtered, Bradford’s reagent to each well (this will normally be supplied at the appropriate 1:3 dilution and filtered). Note: it is important to add the reagent last to ensure that the incubation time is similar for all wells. Mix thoroughly using the mixer in the plate reader for 5 sec. Leave at room temperature for at least 5 minutes and read absorbance at 620 nm. Note: the absorbance will increase over time and should be read within 30 min of adding the Bradford’s reagent. 94 When finished, empty the microtitre plate into the waste bucket provided and dispose of in the bin. Results Construct your standard curve and use this to calculate the protein present in the samples you added. Remember to take into account that you samples were diluted prior to you receiving them. What was the concentration of the protein in each sample (mg/mL)? Did the protein concentration differ between the induced and uninduced samples? 95 Discussion What other methods can be used to measure the protein concentration of a sample? ____________________________________________________________________________ The standard you used in the assay was not the same protein that you were measuring the concentration of; what assumption is made about the standard and the samples? __________________________________________________________________________ Measuring the protein concentration of cell lysates could be used to normalise results in an experiment (recall your workshop on controls, normalization etc.). If you had purified the GFP protein that was overexpressed, you could also perform a Bradford’s assay to determine how much GFP was produced from a given volume of cell culture. REFERENCES M.M. Bradford. (1976) Analytical Biochemistry 72 pp. 248–254. G. Khalsa. (2010, April 12). Mama Ji's Molecular Kitchen. ASU - Ask A Biologist. Retrieved May 10, 2015 from http://askabiologist.asu.edu/sds-page. 96 GLUCOSE TOLERANCE TEST PRACTICAL: Notes ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… ……………………………………………………………………………………………………………… 97 PRACTICAL: 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 98 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). 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. 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 99 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. 100 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. 101 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) 0 min serum 30 min serum 60 min serum 90 min serum 120 min serum 150 min serum 180 min serum 102 Serum [glucose] (undiluted) (mM) 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:……………. 103 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. 104 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. 105 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. 106 CALCULATION OF STANDARD DEVIATION AND % ERROR 107
