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