glocH=wIsTRy LABORATORY MANUAL F ieCONT¢?NTS Breface.......... AMIGA Tiss 35 85 583 ariwietm tates Mil Acknowledgments.......... merece eer eroe sje nie wine x Part I: Laboratory Operations and Safety ....... 0.0. s cscs ee eee xi Part Il: Laboratory Course Policies, Procedures, and Grading System, DISCUSSION 1: Structure - Function Relationship of Cellular Parts. DISCUSSION 2: Review of Buffers, Acidosis, and Alkalosis 3 EXPERIMENT 1: Proteins ........ see eeeed” Pre-laboratory Review Sheet ............ sms so. as bar He Post-laboratory Data and Report Sheet ........ gtd oie no oS EXPERIMENT 2: Carbohydrates... . 16 Pre-laboratory Review Sheet Postrlaboratory Data and Report Sheet... . . aio rebiocs f Bok a eset a7 EXPERIMENT 3: Lipids . . Pre-laboratory Review Sheet... 5. . Post-laboratory Data and Report Sheet EXPERIMENT 4: Enzymes and Digestion . . Pre-laboratory Review Sheet . . . Post-laboratory Data and Report Sheet . . EXPERIMENT 5: Deoxyribose Nucleic Acid Extraction From Plant Tissue... 58 Post-laboratory Data and Report Sheet ..........0- bene cig 6 EXPERIMENT 6: Metabolism and Energy Production: Analysis of Urine. ... 63 Pre-laboratory Review Sheet ..... 0.0.0. eeeeeeeeeeeee a7 Post-laboratory Data and Report Sheet»... 4. ss 00eeeeeee ees 2 plerencesss . 6 ons, SIME oe tan 8 About the AuthorsPREFZSCE It is an irrefutable fact that physical and chemical changes take place unceasingly in all living organisms. Hence, living organisms can be considered ‘complex laboratories, although the changes are not experimental but rather a series of chemical reactions taking place with precision and accuracy under very specific conditions. Living organisms are able to maintain an internal environment necessary for their survival. The physical and chemical changes intheir systems follow the same chemical principles that govern all matter. It is for this reason that students in allied health courses must know and understand concepts fundamental to the field of biochemistry. A laboratory manual in a science subject such as Chemistry is an invaluable tool in the teaching-learning process. This manual is designed to conform with the ordering of the lecture material. The first two laboratory activities tackle topics vital to the understanding of biochemical reactions: the structure-function relationship of cellular parts; and a review of buffers, acidosis, and alkalosis. ‘The experiments that follow focus on important biomolecules, carbohydrates, lipids, proteins, and nucleic acids. They show the reactions of functional groups in these molecules, which are necessary in understanding enzyme-catalyzed transformations such as digestion. The experiment on nucleic acids is preceded by a discussion of DNA recombinant technology, particularly the biochemical structure of DNA and the health benefits brought about by this branch of genetic ‘engineering, Finally, analyses of samples of urine from healthy and diseased individuals are included because the results obtained from the comparative tests done on these samples are indicative of the urinary system's responses to ‘metabolic changes in the body. Prior to the performance of an experiment, students are required to answer the questions in the Pre-Laboratory Review Sheets. Doing so will familiarize them with the safety measures and enable them to organize a system by which the procedures can be done efficiently (i.e, no wastage of materials and time). Provided are data sheets on which the students can record their observations. ‘Since the general objectives of the experiment are already given, students are expected to know the specific objectives of each procedure. study questions enable students to grasp and better understand the concepts under investigation, To be able to answer these questions, students must rely not only on their lecture notes and researches but also and even more so on their critical thinking ability. ~The Authors vilACKN§@WLEDGMENTS Countless faculty members throughout the years have provided groundbreaking ideas for the creation of these laboratory procedures. They have also made numerous suggestions/revisions regarding their implementation. To them, we are eternally indebted, We are also especially grateful to the administrators of the UST College of Nursing for their unfailing support and encouragement to us in preparing a laboratory manual especially tailored for students (and would-be professionals) of Nursing. In addition, we wish to thank our farnilies for their unselfish love and support. Finally, we give thanks to the Almighty for His boundless grace of wisdom and knowledge, —The AuthorsTo the Student: Since a significant amount of your training in chemistry will take place in the laboratory, it is important that the following instructions be read carefully before you attend your first laboratory session, These instructions will not only guide you on proper laboratory procedures but also help you make use of your laboratory time efficiently, They will also promote laboratory safety. A. SAFETY RULES: PROTECTING YOURSELF IN THE LABORATORY 4, The laboratory is a place for serious work, DO NOT ATTEMPT ANY UNAUTHORIZED EXPERIMENTATION. Report all injuries—no matter how minor—to your instructor immediately. Beware of touching hot glass. Glass cools very slowly and may be very hot without appearing to be so. Immediately wash the injured area with cold water or have it cooled with an ice pack. Do not point your test tube at your seatmate or yourself when heating substances. Suddenly formed bubbles of vapor may expel the contents violently. When diluting sulfuric acid, pour the acid slowly and carefully into the water, stirring constantly. NEVER ADD WATER TO THE ACID. Remember the "AA Rule”: “Add Acid to water.” ‘Neutralize spilled acid and bases as follows: + Acid on clothing - use dilute ammonia solution, + Base on clothing - use dilute acetic acid followed by dilute ammonia solution. + Acid or base on the desktop or floor ~ wash off with plenty of water. Use solid crude sodium bicarbonate to neutralize large amounts of either acid or base. Then wash off the mixture with water. When inserting glass tubing (including thermometers, thistle tubes, and funnels) through a rubber stopper, first lubricate the tube and stopper with water/petroleum Jelly. Hold the tube with a cloth near the end where the insertion is to be made and insert the tube with a twisting motion. ‘Never taste a chemical or solution unless directed to do so. When directed to taste a solution, place a minute portion of the solution on a stirring rod, suspend it in the air, and touch it with your tongue. Then wash your mouth with water. When identifying the scent ofa liquid, do not place your face directly over the container. Waft/Fan a little of the vapor towards you by sweeping your hand over the top of the container. xiLABORATORY OPERATIONS AND SAFETY 10. 1. iz, 1B. 4. Im an experiment where poisonous ot otherwise objectionable gases or vapors are discharged, perform the operations under the hood. The hood provides the suction necessary to remove such gases or vapors from the laboratory. To protect your clothing from corrosive chemicals, always wear a laboratory gown when experimenting, Ladies must clip or tie their long hair and bangs to prevent possible accidents. Ifyour clothing catches fire, use a safety shower or a wet blanket. Ifa fire breaks out in the bench area, use the fire extinguisher. B. SAFETY RULES: FIRST-AID MEASURES IN CASE OF LABORATORY ACCIDENTS i 5. 6. Burns For burns caused by dry heat in which the skin did not break out, apply butesin picrate ointment. Acids on the Skin Wash immediately with plenty of water, then with saturated sodium bicarbonate solution, and again with water, For serious acid burns, follow the same procedure and call for medical aid at once. Alkali on the ©" Wash immediately with plenty of water, then with 1% acetic acid solution, and again with water. For serious alkali burns, follow the same procedure and call for medical aid at once. Bromine on the Skin Wash the affected part immediately with plenty of light petroleum (bp. 80 °C) and then rub glycerine well onto the skin. After a short while, remove the glycerin and apply butesin picrate ointment, Sodium on the Skin If a small fragment of sodium metal can still be seen, remove it carefully with a forceps. Wash thoroughly with water, followed with 1% acetic acid solution. Cover the area with gauze soaked in olive oil or acriflavine jelly or Vaseline. Organic Substances on the Skin Wash freely with rubbing alcohol, then with soap and warm water. xiiLABORATORY OPERATIONS AND SAFETY 7. Cuts If the cut is a minor one, allow it to bleed for a few seconds. Ifthe cut is caused by glass, see to it that no glass pieces remain lodged in the skin. Then apply a disinfectant and bandage. For serious cuts, check the bleeding by applying pressure above the injured area (eg., by using a tourniquet). Call for a doctor at once, 8. Eye Accidents Inall cases, the patient must see a doctor. For acid in the eye/s, wash the eye/s repeatedly with 1% sodium bicarbonate solution. Ifthe acid is concentrated, wash the eye/s with copious amount of water and then with bicarbonate solution. For caustic alkali in the eye/s, wash the eye/s with copious amount of water and then with 1% boric acid solution, For bromine water in the eye/s, wash the eye/s thoroughly with water, then with 1% sodium bicarbonate solution. 9. Fires For burning clothing, prevent the person from running. Let him/her roll on the floor or wrap another piece of clothing around him/her (a laboratory gown or blanket) to cut off the supply of oxygen. For burning chemicals in a beaker or any other container, cover the mouth of the vessel with a clean damp cloth so that the flame will be put out. For larger flames, sand may be employed. It is advisable to make use of a fire extinguisher and NOT water. 10. Poisons a. Acids If an acid is swallowed, dilute it by drinking plenty of water followed by limewater or Milk of Magnesia. Milk ‘may also be given. Do not give any emetic. b. Caustic Alkali Dilute the acid by drinking plenty of water, followed by vinegar, lemon, or orange juice. Milk may also be given. Do not give any emetic. c. Salts of Heavy Metals Ifa salt of heavy metal is swallowed, one may give milk or raw egg white. d. Arsenic or Mercury Compounds Give an emetic immediately such as one tablespoonful of salt or zine sulfate ina glass of warm water,| ARORATORY OPERATIONS AND SAFETY C. SAFETY RULES: LABORATORY WASTES AND ENVIRONMENTAL PROTECTION i. Never put solid waste materials in the sink. Place such materials in solid waste containers or special receptacles designated for such a purpose. Pour aqueous liquids directly into the drain followed by a liberal amount of water. Never pour non-aqueous solutions and organic solvents directly into a sink. The laboratory usually provides a container for such materials. Ifthe waste is a mixture of an undissolved solid in a liquid, decant the liquid into a trash container and place the residue in a designated receptacle. Corrosive reagents must be put in a designated waste bottle. DO NOT POUR SUCH REAGENTS INTO THE SINK. After every experiment performed in the laboratory, always make sure that the workplace you have occupied is left clean and spotless for the next laboratory class. D. LABORATORY GUIDELINES AND REGULATIONS To the Student: Below are the implementing guidelines and regulations observed in the laboratory especially during experiments. These are for strict implementation so be sure that you understand all of them. Ifsome items are not clear to you, ask your respective instructor. i, 8. Eating, smoking, as well as bringing in audic equipment (with or without earphones) are STRICTLY NOT allowed inside the laboratory, Students are allowed to work in the laboratory only during their officially scheduled laboratory period, No unauthorized overtime is allowed, Donot take the reagent bottles from the side shelves to your desk. Carry liquids in clean vials, test tubes, or beakers; carry solids in clean beakers or watch glasses or on small squares of papers. Read the label twice before taking anything from the bottles. Use alittle reagent as is convenient to perform your experiment. Two or three milliliters are usually sufficient in test tube experiments. NEVER return unused chemicals to the stock bottles. Throw these in their respective containers, DO NOT INSERT YOUR OWN PIPETTES, MEDICINE DROPPERS, OR SYRINGES INTO THE REAGENT BOTTLES. Pour out the solution instead in clean vials. This will avoid any possible contamination of the stock solution. Do not lay the stopper of the bottle down. Impurities may be picked up and thus contaminate the solution when the stopper is returned. xiva) 10, u. 12, 1B. Mu 15, vw. LABORATORY OPERATIONS AND SAFETY Do not heat graduated cylinders or bottles; they break easily, Test tubes may break if heated above the level of the liquid in them. Evaporating dishes and crucibles may be heated red hot, if necessary. Return all Bunsen burners, burettes, and other items borrowed from the laboratory counter at the end of every laboratory period. Make sure that you leave your pieces of glassware clean and dry inside your respective group locker at the end of each laboratory period. Wash and wipe the desktop so that it will be clean for the next class. Put all solids and waste paper in the waste can, NEVER THROW MATCHES, PIECES OF LITMUS PAPER, OR ANY SOLID INSOLUBLE CHEMICALS IN THE SINK. Place used chemicals into the waste bottles provided. See to it that your lockers are properly padlocked before leaving the room. Many incidents of loss have occurred due to padlocks left open. All members of the group MUST have a duplicate key to the group's padlock. When borrowing laboratory items from the counter, surrender to the laboratory technician your ID card together with a % sheet of paper containing the list of items needed. Do not pay for any losses, breakages, or repairs of apparatus to any laboratory or office personnel, Breakages/losses are charged against your laboratory deposits. If payments have to be made at the end ofthe semester, pay only to the Accounting Office. Demand an official receipt from the University. Keep all your student copies of the Laboratory Division forms issued during the distribution and returning of apparatus, You may need them for clearance of your accounts in the future. Notify the Laboratory Division Office immediately ifyou intend to drop any laboratory subject/course.A, LABORATORY POLICIES 1. Attendance Attendance will be checked at the start of each meeting for the entire duration of the course. a. ABSENCES A student should not exceed a total of 4 absences (12 hours) in the laboratory. A grade of FA (Failure due to Absences) will be given if the said limit is exceeded. b. TARDINESS ‘Three marks for coming late to class will be equivalent to one absence. 2. Special Examir tion A student can take only ONE special examination during the First and Second Grading Periods. No special quizzes will be given during the Third Grading Period. The scope of the Long Special Examination will cover topics discussed from the beginning ofthe quarter. a. Long examination ‘A request for a special examination with its corresponding reason/s and supporting documents (e.g., medical certificate) must be forwarded to the Subject Coordinator by the student concerned within five days upon returning to class. The Subject Coordinator will then schedule a special examination. Failure on the part of the student to take the scheduled Special Test without justified reason will result in a grade 20% less than the lowest score for that particular examination (this is not applicable during the Third Grading Examination). During the Third Grading Examination, a grade of INC (Incomplete) shall be given. b. Quizzes Missed quizzes should be requested as soon as possible. A make-up quiz automatically incurs a deduction of 5 points and a corresponding one point deduction for every succeeding day that follows from the time of the scheduled test. Failure of the student to take the scheduled make-up quiz will result in a grade of zero for that particular test. A student is only allowed to take ONE special quiz per semester. xviLABORATORY COURSE POLICIES. PROCEDURES, AND GRADING SYSTEM ¢. Grading system Lecture = 60% Laboratory 2 40% TOTAL 100% B. LABORATORY COURSE REQUIREMENTS 1. Individual Requirements Laboratory gown with affixed embroidered name (Last name, initials of first name) Laboratory manual Laboratory safety goggles Black/blue and red writing pen Pencil with eraser White board marker Hair pins (to clip bangs whose length is BELOW eyebrow level) or headbands, rubber bands or hair net for ladies 2, Group Requirements 1 pc. padlock with key (all members of the group must have a duplicate key) 1 pe.5" x8” index card 1 pc. long clip folder (without cover) 1 pe. ruler (12 inches) 1 pair of scissors 1 pc. stapler with staple wires 1 pe. permanent marker (black) 1 pe. masking tape (4 inch wide) 2rolls tissue paper 2 pes. rags 2pes. hand towel 1 bottle 500 mL. 70% alcohol Detergent powder in a jar or liquid detergent Scrub sponge Hand soap (with germicidal action) in a soap case or antibacterial hand-washing liquidLABORATORY COURSE POLICIES, PROCEDURES, AND GRADING SYSTEM + Lpack cotton balls + 3small boxes of matches or 1 big box of matches + 12pes. glass slides and cover slips + 12pcs, 50-mL wide mouth transparent vials with cover + 12pes. 20-mL transparent vials with cover + 12:pcs. long glass tip droppers (with at least 6 rubber bulbs) + 12 pes. droppers with graduation (medicine dropper) + 1 pe, small trash can + 1 pc. empty shoe box (preferably shoe box for males) + 5 pcs. 5-mL syringe with gauge 21 needle + 5 pcs. S-mL syringe with gauge 23 needle + 5 pcs. Vacutainer® tube (lavender top) * 5 pcs. Vacutainer® tube (red top) + 20 pcs. plastic stirrer with spoon (white) C. LABORATORY FLOW OF ACTIVITIES ‘To the Student: The following are the steps to be undertaken in the laboratory before, during, and after the conduct/performance of the experiments. Familiarize yourself with these steps as advanced instructions are seldom conducted. The activities in the laboratory work in a cycle, and students are expected to anticipate them. Finally, carefully read the experiment/discussion to be performed/conducted before you come to the laboratory. 1. Pre-laboratory Preparation You are required to complete the Pre-laboratory Review Sheet found before the Post-laboratory Data and Report Sheet of each experiment and submit it BEFORE ‘you begin your laboratory work. The questions have been chosen to draw attention to specific techniques and precautions that you should be aware of before you begin the experiment. The experiments are designed to allow you to collect data in 3 hours or less. Make sure you arrive on time because your laboratory instructor will provide additional instructions for the experiment/s as needed. Ifa discussion is scheduled instead of an experiment, make sure that you have read your lecture book to better prepare yourself for the discussion ahead. A discussion is ‘supposed to clarify ambiguities and NOT to learn about the concept for the first time. xviLABORATORY COURSE POLICIES. PROCEDURES. AND GRADING SYSTEM, 2, Experiment/Discussion Proper The procedures of the whole experiment are usually divided among the members of the group. The group leader of that certain experiment assigns the division. All members will take turns as group leader (e.g., member number 1 is assigned to experiment no. 1, member number 2 is assigned to experiment no. 2, and so on). After you have completed your assigned procedure, you must submit your actual experimental result (eg., solutions, precipitate, residue) with your accomplished Post-laboratory Data and Report Sheet which contains the following information that must be completed: Your complete name, block and group number, the specific objective/s of your assigned procedure/s and the obser vation/s data you have gathered. Attach these sheets to your clip folder when presenting your results. 3. Post-laboratory Conference Allaboratory conference is conducted the following meeting after every experiment has been performed, Topics that do not require an experiment are usually tackled after the previous experiment's laboratory conference. There are 10 topics to be discussed per semester. The topic is picked out by the group at the start of the semester. Two topics are usually assigned per group. ‘Twenty four hours before the scheduled laboratory conference, copies of the written report must be given to ALL the members of the class (ie., Groups 1-5 and Groups 6-10) to enable them to read the proceedings in advance and to prepare questions about Item/s which needs/need clarification/s during the actual oral report. Before the actual oral report begins, the reporter group submits the ORIGINAL copy of their written report to their respective instructors following the format of the report given below, NO WRITTEN REPORTS WILL BE ACCEPTED DURING OR AFTER THE ORAL REPORT. Group members who did not contribute in the formulation of the written report must be reported to the professor concerned. This must be clearly noted in the written report's title page. 4. Quizzes There will be a maximum of 3 quizzes per grading period. These quizzes are all scheduled at a specified date, Students who will have a conflict of schedule with their quizzes because of unforeseen family matters/school extracurricular activities must consult their respective instructor for a change in the date of the quiz prior to the original schedule given to the class.LARORATORY COURSF POI ICIES. PROCEDURES. AND GRADING SYSTEM D, GUIDELINES AND FORMAT OF THE WRITTEN REPORT FOR THE LABORATORY CONFERENCE 1. Use short bond paper (size: 8.5” x 11” inches). 2. Format: line spacing: double; font type: Arial; font size: 12 points 3. Parts of the written report a. Title Page i, Number and Title of Experiment or Topic ii, Names of Members iii, Section and Group Number b. Introduction c. Forevery procedure i. Objectives ii, Presentation of results fii, Dis ssion/Interpretation of results (with answers to study guide questions incv. porated in the discussion) d. References 5, Online journal article critique paper Choose an available online journal article related to your assigned experiment. Make a one-page critique or evaluation of your chosen article, sites of articles can be downloaded through the following: www.ebsco.com or www.sciencedirect.com. The articles are available FREE of charge from the UST CENTRAL LIBRARY. Submit the journal article and critique paper together with the written report. E, GUIDELINES IN PREPARING YOUR POWERPOINT PRESENTATION Strictly follow the following form: a. Title page Objectives of the experiment Procedures with actual pictures taken during the experiment Experimental results Discussion of results (i.e., equations, computations, graphs, etc.) “Answers to the study guide questions must be incorporated in the discussion. me eos xxDISCUSSION NO.1 _ STRUCTURE = FUNCTION RELATIONSHIP OF CELLULAR PARTS INTRODUCTION One of the most important courses in any health sciences program is biology—the study of life, Studying about life requires explorations from a global (macroscopic) to a microscopic perspective, In addition, all biology studies need chemistry since all living things are made up of the same building blocks—around 25 elements from the periodic table—and depend on the same biochemical processes in order to survive (Campbell, Williamson, & Heyden, 2009). As the structure of an atom determines the chemical properties of an element, so do the molecules of life —carbohydrates, proteins, lipids, and nucleic acids—determine the structure as well as function of a part of a cell. Understanding how a cell functions will make you better understand how an organ or an organ system works in your later study of human physiology. EEE Atthe end of the laboratory discussion, the students should be familiar with the organization and classification of living cells as well as with the structure and function of the different cellular parts that work in harmony to sustain cellular survival. fuRed er NET WN Rela econ A, Six Levels of Organization of Living Things 1. Chemical Level Cell Level Tissue Level Organ Level Organ System Level 6. Organism Level B. The Cell Theory of Matthias Schleiden and Theodore Schwann (1839) , Prominent Scientists Involved in the Systematic Classification of Living Things: 1. Carl von Linné - Binomial Nomenclature 2. Robert Whittaker - 5-Kingdom System 3. Carl Woese - Domains of Life (Bacteria, Eukarya, and Archaea) 4, Lynn Margulis - The Endosymbiotic Theory D, Similarities and Differences of Prokaryotes and EukaryotesSTRUCTURE - FUNCTION RELATIONSHIP OF CELLULAR PARTS E. Chemical Structure and Function of Eukaryotic Cells 1. The Cell Wall and Cell Membrane a. The fluid mosaic model b. Transport across the plasma membrane + The proteins of the plasma membrane + Types of animal cell junctions and the plasmodesmata of plants 2, The Cytoskeleton and the External Structures of Cells a, Appendages; Flagella, Cilia, Fimbriae, Pili, and Pseudopod (False feet) b. Glycocalyx: Capsules, Slime layer 3. The Nucleus a. Nuclear envelope b. Nucleolus ¢. Chromosomes and DNA replication ‘The Endomembrane System: a. Rough and smooth endoplasmic reticulum b. Golgi bodies c. Vesicles Other Membrane-bound Organelles a. Mitochondria b. Chloroplasts c. Lysosomes ‘osomes a. The synthesis of proteins: Transcription and translationTerese ‘Acid-base balance in the blood exists when the pH of blood is in the range of 7.35 to 7.45. A.decrease in the pH of blood is called acidosis, and an increase is called alkalosis. Either condition interferes with respiration, and, in extreme cases, a pH that falls below 6.8 or rises above 7.8 is lethal. The carbonate buffer, which is the chief buffer in blood, consists of HCOs"to neutralize H* and dissolved CO; to neutralize OH". In the lungs, bicarbonate ion is changed back to waste COz, which is expelled. The proper treatment of acidosis or alkalosis depends on knowing whether the underlying cause is a metabolic or a respiratory disorder. ‘nee At the end of the laboratory discussion, the students should be able to: 1. demonstrate how buffers control the pH of blood and its fluids quantitatively; 2, recognize how essential the ability to expel or retain COs to the control of acidosis and alkalosis is; and 3. differentiate clinical situations of respiratory and metabolic acidosis from respiratory and metabolic alkalosis. Care eee kelaietiscron | Blood Buffer Calculations using the Henderson-Hasselbalch Equation B, Acid-Base Balance of the Blood (causes, symptoms, and treatments) 1. Respiratory Acidosis 2. Respiratory Alkalosis 3. Metabolic Acidosis 4. Metabolic AlkalosisINTRODUCTION Proteins are complex biomolecules composed of alpha amino acids joined by peptide linkages. ‘A peptide bond is a carbon to nitrogen bond (an amide bond) formed by the loss of a molecule of Hz0 from the ~COOH group of one amino acid and the -NH: group of an adjacent amino acid. They perform many body functions which can be catalytic, contractile, structural, regulatory, protective, etc. in nature. AMINO ACIDS Amino acids are carboxylic acids with an amino group attached to the alpha carbon. Its general formula is: H:N—CH—C—OH. The twenty (20) amino acids found in nature are a - Il amino acids except proline. Each amino acid has a RO unique property due to the composition of its R group. Amino acids in the solid state exist as zwitterions. H3N*—-CH—COO- ‘The pH at which the number of positive and negative | charges in an amino acid or a protein are the same is R its isoelectric point. Azwitterion PROTEIN STRUCTURES AND DENATURATION Proteins have complex structures that are described at four levels: primary, secondary, tertiary, and quaternary. The primary structure is the linear sequence of amino acids held together by peptide bonds. The secondary structure is the result of regular folding patterns of specific regions of the polypeptide chain. It is maintained by hydrogen bonding between the amide hydrogen of one peptide bond and the carbonyl oxygen of another peptide bond. The tertiary structure is the three-dimensional structure that results from attractive forces among the side (R) chains. The quaternary structure exists in proteins containing more than one polypeptide chains such as hemoglobin. Disruption of the secondary, tertiary, and quaternary structures of proteins is called denaturation. Physical agents (e.g, heat, violent agitation) and chemical agents (eg., organic solvents, acids) cause the uncoiling of the protein molecule and the loss ofits biological activity. Denaturation may result in coagulation and precipitation of the protein out of solution.PROTEINS ENE eestsenass Im this experiment, the student should be able to: 1. observe several chemical properties of amino acids and proteins; 2. observe the effects of several denaturing agents on a protein sample; 3. extract proteins from blood plasma; and 4. identify some amino acids through their reactions with specific reagents. | MATERIALS AND EQUIPMENTS. Reagents Acetic acid, 3N Ninhydrin, 0.1% Alpha-naphthol, 0.2% Nitric acid, concentrated Copper sulfate, 0.5% 1-nitroso-2-naphthol in acetone Ethanol, 95% and 70% Silver nitrate, 1% Ether NaOH, 10%, 50%, and 6M Ferric chloride Sodium sulfate, 23% Hopkins-Cole reagent Sulfuric acid, concentrated Lead acetate, 10% Samples (to be provided by the students) Aspartame or Equal (5 tabs or 1 sachet/group) 1 fresh chicken egg/group 1 small tetrapack of evaporated milk/group 1 small bottle of bleach/class Freshly-drawn blood plasma (to be extracted by the laboratory instructor) Equipment Centrifuge Electric water bath Long, thin pipettes REMINDERS 1, Transfer an aliquot portion of reagent in a vial using a funnel and a graduated cylinder. 2. PIPETTES, DROPPERS, and SYRINGES cannot be used in. transferring reagents. Ss Use plenty of water when disposing of chemicals by rinsing them down the sink.PROTEINS PROCEDURES ‘A. PROTEIN DENATURATION Place 2.0 mL of egg white solution ina test tube. This will serve as the standard to be used by the members of the group working on denaturation, 1. Effect of Heat and Alcohol a. Place 2.0 ml. of egg white solution in a test tube and heat in a boiling water bath for 5 minutes. Compare the appearance of the heated sample with the standard. b. Label two tubes as 1 and 2, Add 2.0 mL. of egg white solution to each tube. To tube no, 1, add 95% ethanol and to tube no. 2, 70% ethanol. Compare the appearance of the resulting mixtures with the standard. Record your observation as (-) for no precipitation, (+) for slight precipitation (cloudiness), or (++) for heavy precipitation. ACAUTION Ethanol is highly flammable! Work away from open flames. 2. Effect of Heavy Metals a, Add 2.0 mL of egg white solution to each of the two test tubes labeled 1 and 2. b, Totest tube 1, add 1.0 mL of 1% AgNOs solution and to test tube 2, add 1.0 mL. ‘of 10% Pb(CHsCO0)2 solution. Mix well and note the color of the precipitates formed. Set aside for 5 minutes. ¢. Decant the supernatant liquid and test the solubility of a small portion of the precipitate in 5.0 mL of water. Potro Heavy metals are toxicl Wash with soap and water the skin that has come in contact with heavy metals, Dispose of waste solutions in a container labeled “Used Heavy Metal Solutions.” COLOR REACTIONS OF PROTEINS AND AMINO ACIDS. 1. Xanthoproteic Test a. Add 0.5 ml of concentrated HNOs to 1.0 ml of egg white solution in a test tube. b, Mix with a stirring rod and warm in a water bath for 5 minutes, Note the color of, the precipitate formed. ©. Cool the contents of the tube and make it alkaline by adding 50% NaOH. Note the changes in the mixture.PROTEINS Concentrated HNO; and 50% NaOH are both corrosive chemicals! Wash immediately with water the skin that has come in contact with these chemicals, DISPOSAL Pour used solutions into the sink and rinse them down with running water. 2, Biuret Test e Mix 1.0 mL of egg white solution and 10 gtts of 6M NaOH ina test tube. Add 1 drop of 0.5% CuSO, solution. Mix well and record your observation. Dissolve one tablet of aspartame or % sachet of Equal® in 2.0 mL of water. ‘Add to the resulting solution 10 gtts of 6M NaOH and 0.5% CuSO,solution. Mix well and record your observation. Compare the two results. Label one test tube TP (Total Protein), then add 3 gtts of blood plasma (or serum) and 3.0 mL of 23% Na2SO, solution, Mix well. Borrow a centrifuge tube from the counter and transfer half of the mixture from the tube marked TP to the centrifuge tube. Set aside the tube marked TP, ‘Add 1.0 ml of ether to the mixture in the centrifuge tube, mix thoroughly, and stopper it. Centrifuge for 5 minutes. Note the color of the precipitate formed at the junction of the two liquids (interface). Ether is highly flammable! Keep it away from open flames. Usinga very thin pipette, carefully draw out the lower aqueous layer, taking care not to disturb the precipitate at the interface. Wipe the pipette to remove any precipitate that adheres to it. ‘Transfer the contents of the pipette to a test tube and mark it as A (albumin). To each of the two tubes (TP and A), add 1.0 mL of 6M NaOH and 2 gtts of 0.5% CuSO, solution. Mix well and compare the color intensity in the test tubes. Record your observations. 3. Ferric Chloride Test Place 5.0 mi of milk on a watch glass and heat over a steam bath until a film is produced on the surface of the liquid. Using a clean and dry stirring rod, remove the film and place a small portion of it into a clean test tube. Add 5.0 gtts of FeCl; solution. Note the change in color of the film. 7~~ PROTEINS 4, Hopkins-Cole Test Add 2.0 ml of Hopkins-Cole reagent to 2.0 mL of egg white solution in a test tube. Mix thoroughly, b, Tilt the tube and carefully pour along one side of the tube 1.0 mL of concentrated 1,50 ACAUTION Concentrated sulfuric acid is highly corrosive! Wash thoroughly with soap and water the skin that has come in contact with this acid. Consult your instructor for more serious acid burns, ©. Hold the test tube in an upright position and observe the color of the ring formed at the interface of the two liquids. (If no ring is visible, gently agitate the tube to cause a very slight mixing at the interface.) 4. Record your observation 5. Ninhydrin Test a. Add 10gtts of ninhydrin solution to each of the two test tubes, one containing the 2.0 mi of egg white solution and the other with the 2.0 ml of the aspartame or Equal* aqueous solution, Mix thoroughly. b. Heat the tubes in a boiling water bath until a color change is observed. ©. Compare the results obtained. 6. Sakaguchi Test a. Strictly observe the order of addition of the reagents. To the 5.0 mL of egg white solution, add 1.0 mL of 10% NaOH solution. b, Mix, then add 1.0 mL of 0.2% a-naphthol solution. Mix thoroughly. ¢. After 3 minutes, add 5 drops of sodium hypochlorite (bleach). 4. Immediately note the color of the resulting solution as it fades quickly. Record your observation. 7. Lead Acetate Test a, Add 1.0 ml of 50% NaOH solution to a test tube containing 2.0 ml. of egg white solution, then mix. b. Add 5 drops of lead acetate solution. ©. Mix and heat the test tube in a boiling water bath until a change in color is observed. Record your observation.PROTEINS ‘Btyxetesuen A. PROTEIN DENATURATION 1. Define denaturation. 2. What physical and chemical agents are capable of denaturing proteins? Give the type of bonds or attractive interactions disrupted by these denaturing agents. 3. What concentration of alcohol is most effective as a disinfectant? Why? 4. Explain how protein denaturation using heat, alcohol, and heavy metal ions is used in the medical field. 5. What aminoacids ina protein are reactive with heavy metal ions? Explain and illustrate with an equation. B. COLOR REACTIONS 1. Xanthoproteic Reaction a. Give the principle involved in this test. Illustrate with an equation. b, What group in an amino acid is identified by this test? ¢. Name the amino acids that will be positive with this test. 4. Whatis the isoelectric point? Explain how pH changes ina protein solution affect its solubility, e. Given the tripeptide aspartylalanylglycine: e1. show the structure of the tripeptide in the presence of e.11. excess acid e412, excess base €2. What is its isoelectric pH (IpH)—acidic, basic, or neutral? 2. Biuret Test a, What is the principle involved in the biuret test? b. Whats the role of the reagents in the test? Illustrate with an equation and name the compound responsible for the visible result. ©. Willall proteins give a positive biuret test? Why? Will all peptides give a positive Biuret test? Explain. d. Account for the difference in test results between egg white and aspartame. Show the structure of aspartame to prove your point. €. Inthe extraction ofblood proteins, explain the function of the following reagents: @.1. 23% Na2SOq e.2. ether £ What is the chemical composition of the precipitate at the interface after centrifugation? Why should it not contaminate the tube marked A?PROTEINS 3. 6 1 & Account for the difference in color intensity between samples TP and A. h, What group in a peptide or a protein accounts for a positive Biuret test? Give the importance of the Biuret test in protein hydrolysis. Ferric Chloride Test a. Give the principle involved in the ferric chloride test. What is its purpose? b. Identify the role of FeCls in this test. . What group/amino acid is responsible for a positive FeCls test? d. Illustrate the reaction involved in this test with an equation, Hopkins-Cole Test a. Give the principle involved in the Hopkins-Cole test. What is its purpose? Give the composition of the Hopkins-Cole reagent and identify its role in the test. ¢. Show the equation involved in this test. d. What compound is responsible for a positive result? €. What group/amino acid present in a protein is identified by this test? Ninhydrin Test a. Give the principle involved in the ninhydrin test. What is its purpose? Identify the role of ninhydrin in this test. Show the equation involved in this test. What compound is responsible for a positive ninhydrin test? What group/amino acid in a protein is identified by this test? Did aspartame/Equal give a positive result? Why or why not? g Willall amino acids give a positive ninhydrin test? Explain. ‘Sakaguchi Test a. What is the principle involved in this test? What is its purpose? b. Why should NaOH be added first before the other reagents? Illustrate with an equation the effect of adding NaOH to arginine. . What is the purpose of the other reagents? d. What group in arginine responds to this test? me Bo Lead Acetate Test a. Give the principle involved in this test. What is its purpose? b. What is the role of NaOH in this test? ¢, Illustrate with an equation the reaction between Pb(CHsCO0); and the product obtained after heating with NaOH. d. What compound is responsible for the visible result? , What group/amino acid is identified by this test? 10PROTEINS eikelexel sen Given Polypeptide A - Arg-Leu-tyr-Glu-Lys-Ile-Ileu-Met-Gly-Pro-His-Trp Polypeptide B ~ Pro-His-CyS-CyS-1hr-Irp-Met-Glu-Asp-Tyr-Phe 1. Complete the table below by writing () for a positive result and (-) for a negative result ifthe given tests are done on A and B. Based on the results, what test/s can be used to differentiate the two polypeptides. Write >7, «7, OR <7. Test A [|B Test. A B. Biuret Sakaguchi Ninhydrin Lead Acetate Xanthoproteic Ferric Chloride Hopkins-Cole “Isoelectric PointPRE-LABORATORY REVIEW SHEE EXPERIMENT NO. 1 PROTEINS Name: Section Professor: Group No.: 1. Whatare the precautionary measures given in the experiment and for what specific chemicals are they given? 2. What is the isoelectric point? What is its importance in the preparation of cottage cheese (kesong puti)? 3. Give the names and symbols of the functional groups present in an amino acid that will be positive with the given tests. What are the positive visible results of these tests? Name of the Functional Group Symbol Visible Result a. Lead acetate test b. Sakaguchi test c. Xanthoproteic test a. FeCls 4, Give the formula of aspartame. Explain its structural similarity to a peptide. 5. Are all amino acids reactive with the Biuret reagent? Why or why not? 6, How is denaturation made use of in the hospital setting?Ue NAP Nake EXPERIMENT NO. 1 PROTEINS Group No: Date Performed: Section: Professor: ' A. PROTEIN DENATURATION i 1. Effect of Heat and Alcohol Specific objecti Observation Heat 95% ethanol 70% ethanol Performed by: 2, Effect of Heavy Metals Specific objective: Solubility in Water Silver Nitrate Lead Acetate Performed by:wnnennnaneny B. COLOR REACTIONS 1. Xanthoproteic Test Specific objective; “SM Observations: Color obtained (indicate if it is a precipitate or a solution) With HNOs With Nao Performed by: Biuret Test a. Specific objectives Observations: Visible result with egg white with aspartame Performed by: b. Specific objectives Color at the interface: Color and color intensity observed with TP Specific objecti with A, Performed by: nS 3. Ferric Chloride Test Specific objective: Color of the film: Result with FeCls Performed by: 4, Hopkins-Cole TestColor of the ring: Performed by:____ UNS - Ninhydrin Test Specific objective: Observations: Visible result with egg white with aspartame . Sakaguchi Test Specific objective: Observations; Color of solution :. Performed by: . Lead Acetate Test Specific objectivesEXPERIMENT NO. 2 CARBOHYDRATES INTRODUCTION Carbohydrates comprise half of all biological substances on earth. They serve as the main energy source for the body. Table sugar, starch, and cellulose in plants, and glycogen and glucose in human tissues are examples of carbohydrates. They are components of important biomolecules like DNA, RNA, ATP, cellular membranes, and receptors. Carbohydrates are polyhydroxy aldehydes and ketones, They may have as few as three or as many as thousands of carbon atoms. The simplest carbohydrates called monosaccharides are usually composed of three to six carbons with one carbon acting as aldehyde or ketone and the rest of the carbons with hydroxyl groups. Monosaccharides may combine via glycosidic linkages with another to form disaccharides, or with more to form polysaccharides, Furthermore, the saccharide units may assume cyclic and acyclic structures. The physical and chemical properties of carbohydrates depend on the presence of these groups and structures. At the end of the experiment, the student must be able to: 1. distinguish a carbohydrate from a non-carbohydrate; 2. be familiar with the different procedures and the underlying principles behind the comparative tests for the different simple sugars; and 3. be acquainted with the presence of simple sugars in clinical samples. MERE Samples/Materials to be brought by students « Urine samples ~ fasting sample (taken early morning from one who fasted for. ‘eight hours) - random sample (taken any time of the day from one who did not fast) ~ urine ofa known diabetic (taken any time of the day who from one did not fast) * Carageenan, eg., Alsa gelatin, Mr, Gulaman + Chicken liver, ( kilogram) Samples + Cooked potato starch solution + 10% (w/v) aqueous solution of glucose, fructose, xylose, lactose, galactose, sucrose, and maltoseCARBOHYDRATES Reagents Acetic acid, 0.1% Equipment + Alcohol lamp Barfoed's reagent * Glass slides and coverslips Benedict's reagent + Microscopes Bial’s Orcinol reagent*** + Warm water bath (at 60 °C) Fehling’s (A and B) reagents * Boiling water bath (at 100 °C) Iodine solution Molisch reagent Nitric acid, concentrated*** Phenylhydrazine hydrochloride crystals Seliwanoff's reagent*** Sodium acetate solution sulfuric acid, concentrated*** Distilled water ‘Use with extreme care REMINDERS 2, a. 3. 4. Carefully pour 5 mL-portion of the reagents and 10 mL-portion of the samples into labeled vials. Donotuse pipettes, droppers, and syringes in transferring reagents from stock bottles. ‘You may use droppers to add reagents and samples into test tubes. 1 mL = 15 drops Dispose of liquid chemicals by rinsing them down the sink with plenty of water. Return solid materials to the counter or throw them into the solid waste container. PROCEDURES ‘A. GENERAL TEST FOR CARBOHYDRATES Preparation of Sample (Chicken liver extract) a. Get a thumb-sized portion of chicken liver and finely cut or shred it with scissors. b d. e ‘Transfer the cut liver into a mortar and grind it with a pestle until it reaches a semi- liguid consistency. ‘Transfer the liver into a test tube and add 10 ml. water and 1 ml of 0.1% acetic acid. Heat the mixture for 30 minutes in a boiling water bath to precipitate the proteins, While warm, the mixture should be filtered using filter paper. Collect the filtrate to be used in the Molisch and Iodine tests,CARBOHYDRATES. 1. Molisch Test a, Prepare 3 test tubes and label them as follows: “glucose, “chicken liver.” b. Inthe 1* tube, put 2 ml of the 10% glucose solution. c. Inthe 2*4 tube, put a pinch (mongo size) of gelatin and add 2 mL. of hot distilled water to dissolve. Cool to room temperature. d. Inthe 3 tube, put 2 mL of chicken liver extract. e. Add two drops of the Molisch reagent into each tube and mix thoroughly. £ Usingadropper, slowly add 1 mL (15 drops) of concentrated sulfuric acid alongside each tube to create an interface with the mixture. g. Note what is formed in the interface. Record your observations. Concentrated sulfuric acid is very corrosive! Wash with plenty of water the skin that has come in contact with this acid. See First-aid Measures in case of accidents. ‘carrageenan,” and 2. Iodine Test a, Prepare 3 test tubes and label them as follows: “starch,” “carrageenan,” and “chicken liver.” b. Inthe 1* tube, put 1 mL of cooked potato starch mixture. ¢. Inthe 2" tube, put a pinch (mongo size) of gelatin and add 2 mL of hot distilled water to dissolve. Cool to room temperature. d, Inthe 3 tube, put 2 mL of chicken liver extract. e. Add a drop of iodine solution to each of the tubes and mix thoroughly. £. Note the color of the resulting mixture. g. Warm the three tubes in water bath (60 °C) until a change is observed in one of the tubes. h. Quickly remove all the tubes from the bath. Cool at room temperature. i, Note the color of each mixture and record the results. B. COMPARATIVE TEST REACTIONS OF CARBOHYDRATES 1. Fehling’s Test a. Ina 10 mL vial, put 2 mL of Feblling’s A solution. b, Add 2 mL of Fehling’s B solution and mix. c. Note and record the color of the resulting reagent. This serves as the Fehling’s reagent. d. Divide the Fehling’s reagent into four test tubes of 1 mL (15 drops) each. Label the tubes as follows: “glucose,” “sucrose,” “fructose,” and “lactos f. Add into each labeled tubes 1 mL (15 drops) of the 10% sugar solution (glucose, sucrose, fructose, lactose) respectively and mix thoroughly. 18CARBOHYDRATES g- Note any changes. h. Place all the tubes in a warm water bath (60 °C). i. Remove all the tubes once a change is observed in some of the samples. J. Note the changes and record your observations. Benedict's Test a. Prepare 7 test tubes. Label each tube as follows: “glucose,” “sucrose,” “fructose,” “lactose,” “fasting urine,” “random urine,” and “diabetic urine.” b, Into each tube, place 1 mL (15 drops) of Benedict's reagent. ©. Into the 4 tubes with sugar labels, add 1 mL (15 drops) each of glucose, sucrose, fructose, and lactose respectively and mix vigorously for a minute. d._ Note any changes. When no changes occur, place the tube in a warm water bath (60°C). e. Remove all the tubes once a change is observed in some of the samples. Allow to cool at room temperature. £ Note the changes and record your observations, & Into the other 3 tubes with corresponding urine labels (with Benedict's reagent), add 10 drops each of fasting urine, random urine, and diabetic urine respectively. h. Note any changes. When no changes occur, place the tube in a warm water bath for 5 minutes, Allow to cool at room temperature. Note the changes and record your observations. Report Color of Solution Glucose Concentration QO Blue <0.1% or < 100 mg/dL. @ Green 0.5% or 150 mg/dL. 4) Yellow 1.0% or 1,000 mg/dL (o44) Orange 2.0% or 2,000 mg/dL. Gores) Brick red > 2.0% or > 2,000 mg/aL C. SPECIFIC TESTS FOR CARBOHYDRATES 1. Barfoed’s Test a. Prepare 4 test tubes and label as follows: “glucose,” “sucrose,” “fructose,” and “lactose.” b. Place 1 mL (15 drops) of Barfoed’s reagent into each tube. ©. Add. m1 (15 drops) of each of the test sugars (glucose, sucrose, fructose, lactose) respectively and mix thoroughly for a minute. d._ Note any changes. When no change occurs, place the tube in a warm water bath (60°C) for not more than 3 minutes. e. Remove ALL the tubes once a change is observed in some of the samples. £. Note any changes and record your observations, 9CARBOHYDRATES: 2. Seliwanoff's Test a. Prepare 3 test tubes and label as follows: “glucose,” “sucrose,” and “fructose” b. Place 1 mL. (15 drops) of Seliwanoff’s reagent into each tube. ¢. Add 1 mL (15 drops) of each of the test sugars (glucose, sucrose, fructose) respectively and mix thoroughly for a minute. d. Note any changes. When no change is observed, place the tube in a warm water bath (60°C). €. Remove ALL tubes once a change is observed in some of the samples. £. Note any changes and record your observations. Seliwanoff’s reagent is corrosive! Immediately wash with plenty of water the skin which hhas come in contact with the chemical. 3. Bial's Orcinol Test a. Prepare 3 test tubes and label as follows: “glucose,” “fructose,” and “xylose.” b, Place 1 mL (15 drops) of Bial’s Orcinol reagent into each tube. ¢. Add1 mL. (15 drops) ofeach ofthe test sugars (glucose, fructose, xylose) respectively and mix thoroughly for a minute. 4. Note any changes. When no change is observed, place the tube in warm water bath (60°C), €. Remove ALL tubes once a change is observed in some of the samples. £. Note any changes and record your observations. Orcinol reagent is corrosive! Immediately wash with plenty of water the skin which has come in contact with the chemical. 4. Mucic Acid Test a, Prepare 3 test tubes and label as follows: “glucose,” “galactose,” and “lactose.” b, Add 1 mL (15 drops) of each of the test sugars (glucose, galactose, lactose) respectively. ©. Carefully place 1 mL (15 drops) of concentrated nitric acid into each tube and mix thoroughly for a minute, 4. Using a masking tape, label 3 glass slides with the name of the sugar samples. Holda slide with the wooden test tube holder along one end, Transfer some drops of the mixture onto the slide and heat over an alcohol lamp until they are almost dry, DO NOT SCORCH! Continue doing this until one mL or half of the sample has been transferred, £. Cool the slide to room temperature. 20CARBOHYDRATES g Examine the crystals formed under the microscope. h, Draw and describe the crystals (color and shape) formed under LPO. Write NONE on the field of vision (Post-laboratory Data and Report Sheet) if no crystals are formed with the given sugar. Concentrated nitric acid is very corrosive! Immediately wash with plenty of water the skin which has come in contact with the chemical. 5. Osazone Formation a. In a 50-mL vial, place 3 mL of sodium acetate solution and 2 grams of phenylhydrazine hydrochloride crystals, Dilute the resulting solution with 10 mL distilled water. Stir while warming in a water bath (60 °C) until the solution is CLEAR, This will serve as your reagent for the osazone test. b. Pour 2m of the prepared reagent into 4 test tubes labeled as follows: “glucose,” “fructose,” “sucrose,” and “galactose.” c. Add into tubes 1 ml. (15 drops) of the test sugars respectively and mix vigorously for a minute, Use a stopper with cotton plugs. d. Heat all the tubes in a boiling water bath (100 °C) for 30 minutes. Cool to room temperature. e. Observe for the formation of crystals. £, Examine the crystals that formed under the microscope, if there are any. & Draw and describe the crystals formed under LPO. Write NONE FORMED on the field of vision (Post-laboratory Data and Report Sheet) if no crystals are formed with the given sugar. | STUDY QUESTIONS A. GENERAL TEST FOR CARBOHYDRATES: 1. Molisch Test a. What is the principle involved in the Molisch test? b. What is the purpose of the test? c. Provide the type equation used in the test. d. What reagents were used in the Molisch’s test? State the purpose of each component. e. Provide possible explanation for: + the positive results obtained with your sample/s + the negative results obtained with your sample/s 21CAREOHYDRATES 2. Todine Test a. What is the principle involved in the iodine test? b. Explain the color noted when: * iodine was combined with the samples; + the mixtures of iodine and each sample were heated; + the heated mixtures of iodine and each sample were cooled. ©. What is the importance of the iodine test? What carbohydrates were identified in your samples? d. Structurally, how are amylose and amylopectin different from each another? From glycogen? From cellulose? How do their structural characteristics affect solubility in water? B. COMPARATIVE REACTIONS OF CARBOHYDRATI 1. Fehling’s and Benedict's Tests a, What is the principle involved in the Fehling’s and Benedict's tests? b. What is the purpose of the tests? ¢. What components make up the Fehling’s reagent? The Benedict's reagent? state the function of each component. d. Provide the type equations used in the tests. Show the sample equation involved in the reaction between the Fehling’s/ Benedict's reagent and your sample/s which gave a positive result. £. Provide a possible explanation for: + the positive results obtained with your sample/s + the negative results obtained with your sample/s Why is there a need to freshly prepare the Fehling’s reagent? Why is the Fehling’s test not used in analyzing carbohydrates in clinical samples? i. State the other advantages of using the Benedict's reagent instead of the Feblling’s reagent. j. Differentiate the 3 kinds of urine samples: fasting, post-prandial, and random. Which sample is the MOST suitable for use in a clinical laboratory test? Explain your choice. k. Whats diabetes mellitus? Differentiate Type 1 from Type 2 diabetes. Is it possible to have sugar diabetes even if no member of your family had it? Explain your answer. 1. Is one considered a diabetic if one obtains a positive result for sugar in a fasting urine sample? Explain your answer. ie 22CARBOHYDRATES. . SPECIFIC TESTS 1. Barfoed’s Test ‘a, What is the principle involved in the Barfoed’s test? b. What is the purpose of the test? c. What components make up the Barfoed’s reagent? State the function of each component. d. Provide the type equation used in the test. e. Show the sample equation involved in the reaction between Barfoed’s reagent and your sample/s which gave a positive result. £ Provide a possible explanation for: + the positive results obtained with your sample/s + the negative results obtained with your sample/s Why is the heating of the mixture limited to 3 minutes only? How does this test compare with Fehling’s and Benedict's tests? Which is more accurate? Fe 2. Seliwanoff’s and Bial’s Orcinol Tests a. What is the principle involved in the Seliwanoff’s and Bial’s Orcinol tests? b. What is the purpose of the tests? ©. What components make up the Seliwanoff’s reagent? The Bial’s Orcinol reagent? State the function of each component. d. Provide a possible explanation for: + the positive results obtained with your sample/s + the negative results obtained with your sample/s e. Compare these two tests with the Molisch’s test. 3. Mucic Acid Test a, What is the principle involved in the mucic acid test? b. What is the purpose of the test? . What components make up the mucic acid test? State the purpose of each component. d. Provide the type equation used in the test. . e. Show the sample equation involved in the reaction between the mucic acid reagent and your sample/s which gave a positive result. £. Provide a possible explanation for: + the positive results obtained with your sample/s + the negative results obtained with your sample/s 23CARBOHYDRATES & h, Explain why only galactose forms the white sandy crystals, Lactose has also been known to form crystals in the mucic acid test, Rationalize why this is possible. 4. Osazone Formation a b, o What is the principle involved in the osazone test? What is the purpose of the test? What components make up the osazone test? State the purpose of each component. Provide the type equation used in the test. Show the sample equations involved in the reaction between phenylhydrazine reagent and your sample/s which gave a positive result. Provide a possible explanation for: * the positive results obtained with your sample/s + the negative results obtained with your sample/s + the positive results obtained which yielded the same type of crystals 24Leditea Neel KOM LA eon ea EXPERIMENT NO. 2 Carbohydrates Name: Section: Professor: Group No: 1, Are there specific alerts (precautions) given in the experiment? List any that are given. 2, Are there any specific disposal directions given in the experiment? List any that are given. 3. What results are expected in the Molisch’s test? What is the test for? 4, What kinds of carbohydrates are identified by the iodine test? 5. What is the similarity between the Benedict's and Fehling’s tests? Their differences? 256. Draw the cyclic structure of sucrose; encircle the acetal link; and explain why it is non- reducing. 7. Why is heating in the Barfoed’s test limited to 3 minutes only? 8. Is glucose always present in the urine of normal individuals? Explain your answer. 9. Why is the mucic acid test specific for the presence of galactose? 10. What is the similarity of Molisch’s, Bial’s Orcinol, and Seliwanoff’s tests?POST-LABORATORY DATA AND REPORT SHEET EXPERIMENT NO. 2 CARBOHYDRATES Group No; ___________ ate Performed: Section: Professor: GSS UR accel ‘A, GENERAL TEST FOR CARBOHYDRATES. 1. Molisch Test Specific objectives Samples Observations Glucose Carrageenan Chicken liver extract Performed by: 2. Iodine Test Specific objective: Observations | Samples Before Heating | After Heating | _OnCooling | Potato starch Carrageenan Chicken Liver Extract Performed by: 27B. COMPARATIVE REACTIONS OF CARBOHYDRATES 1. Fehling’s Test Specific objective: Color of reagent: Samples e Observations Glucose Sucrose Fructose Lactose Performed by: 2, Benedict's Test for Test Sugars Specific objective: Color of reagent: ‘Samples Observations Glucose Sucrose Fructose Lactose Performed by: 282. Benedict's Test for Urine ‘Samples Specific objective: Color of reagent: Sampler, = Observations Fasting Urine Sample Random Urine Sample Diabetic Urine Sample Performed by: C. SPECIFIC TESTS 1. Barfoed’s Test Specific objective: Color of reagent: Samples = = Observations Glucose Sucrose Fructose Lactose Performed by: 292, Seliwanoff's Test Specific objecti Color of reagent: Samples Observations Glucose Sucrose Fructose Performed by: 3. Bial’s Orcinol Test Specific objective: Color of reagent: Samples : Observations Glucose Fructose Xylose Performed by: 4, Mucic Acid Test Specific objective; 30Color of reagent; OOO Glucose Galactose Lactose Performed by; —__3 5. Osazone Formation Specific objectives Color of reagents Glucose Sucrose Galactose Fructose Performed by: 3IINTRODUCTION ‘The term “lipid” refers to both true fats and fat-like substances. These are biomolecules that are insoluble in water but are soluble in organic solvents. The water insolubility is due to the fact that the polar portion of their molecule is much smaller than the non-polar portion. This property is important for the cell in the separation of components containing aqueous solutions from each other. Another important function of lipids isthe storage of energy in the form ofbody fat. Some lipids are important constituents of protoplasm. The brain and nervous tissues are rich in certain lipids, a fact that indicates the importance of these compounds in the maintenance of life. Lipids can be divided into five groups: (1) fats and oils; (2) waxes; (3) compound lipids; (@) sterols and steroids; and (5) derived lipids. Fatty acids, one of the building blocks of lipids, is just one of the examples of the hydrolysis products of fats and oils as well as of the compound lipids. Re=Nesyare ss seus At the end of the experiment, the student must be able to: 1. familiarize himself/herself with the different classes of lipids; and 2._ identify each kind of lipid based on the chemical properties of its hydrolyzed products. RSE easel ne ‘Samples to be brought by students (per group) + Vegetable oil + 4lipid-like products (¢g,, lotion, creams, hair wax, etc.) + 10 pieces of lecithin soft gel capsules + 1 pig's brain (To be brought one day before the scheduled laboratory experiment of the class) Reagents + Methylene chloride + Ethanol, 959% + Ether + Ammonium molybdate * Bromine water + Sulfuric acid, concentrated + Potassium bisulfate powder + Toluene * Nitric acid, 6N + Ninhydrin, 0.1% + Acetone + Acetic anhydride + Soda lime + Molisch reagent * Glycerol 32LIPIDS Equipment + Electric water bath + Litmus paper REMINDERS 1. Putan aliquot portion of reagent in a vial by pouring the reagent from a reagent bottle using a graduated cylinder. 2. PIPETTES, DROPPERS, SYRINGES are not allowed in transferring reagents. Dispose of used liquid materials (except aqueous solutions) in the receptacle provided by the laboratory. Aqueous solutions must be disposed of by rinsing them down the sink with plenty of water. Solid materials must be returned to the counter or thrown. into the solid waste container. Almost all solvents to be used in this experiment are highly volatile, flammable, and toxic! Avoid excessive inhalation and exposure to open flames! Observe proper laboratory safety precautions. PROCEDURES A. SPOTTING EFFECT 1. Obtain a piece of filter paper from the counter. With a pencil, draw five circles and write the names of your samples beside each circle. 2. Placeadrop of vegetable oil and the 4 other suspected lipid-like materials (solid samples must be dissolved in 1.0 mL methylene chloride before being tested) within each of the labeled circular areas. 3. Allow the spots to dry. When dry, hold the filter paper against the light and note the presence of translucent spots. Record your observations. B. SOLUBILITY 1. Prepare 4 clean and dry test tubes. Label as “water,” “methylene chloride," “ether,” and “toluene” accordingly. Place 1.0 ml of these solvents into their respective containers. In each tube, add 3 drops of vegetable oil and mix thoroughly. Compare the solubility of vegetable oil in the different solvents. Repeat the above procedure using a drop of lecithin from a soft gel capsule instead. Pierce the gel capsule with a pointed object, e.g,, a needle or copper wire. 33LIPIDS C, TEST FOR UNSATURATION (BROMINE WATER TEST) 1, Prepare 2 clean and dry test tubes. To 1 of the tubes, place 1.0 mL of vegetable oil and in the other, the contents of a lecithin soft gel capsule dissolved in 1.0 mL of methylene chloride. Pierce the gel capsule with a pointed object, e.g., a needle or copper wire, 2, Add 1.0 ml of bromine water to both tubes and mix thoroughly. Bromine water is corrosive! See First-aid Measures in case of an accident. 3. Compare the change in color of the bromine water. With vegetable oil, bromine water forms the upper layer while with methylene chloride, bromine water forms the lower layer. 4. Record your observations. D. ACROLEIN TEST 1. Prepare3 clean and dry test tubes. To the 1* tube, add a drop of glycerol; to the 2 tube, a drop of cooking oil; and to the 3% tube, a drop of lecithin from the soft gel capsule. Pierce the gel capsule with a pointed object, eg., a needle or copper wire. 2, Mixa pinch-sized amount of powdered potassium bisulfate to all the tubes. 3, Heat the mixture gently over a small flame. Note the odor of the vapor and record your observations, E, EXTRACTION OF BRAIN LIPIDS: (The pig’s brain must be incubated one day before the scheduled laboratory class) 1. Inan Erlenmeyer or Florence flask, place the homogenized brain and add enough ether to completely immerse the brain sample. Ether is very flammable! Stay away from open flames. See First-aid Measures in case of an accident. 2. Cover tightly and set aside until the next laboratory period. 3. Decant the incubated brain sample, ‘To the Decantate: a, Add acetone gradually until the precipitation is complete. The precipitation is complete when no turbidity can be observed after adding more acetone to the clear liquid above the precipitate. b, Filter the mixture. c. The residue, labeled as Residue B, is to be divided into 3 equal portions. 4. Each portion of Residue B will be used in the Ninhydrin Test, Soda Lime Test, and the Ammonium Molybdate Test. 34LupIDs e. Evaporate the filtrate until it dries using a steam bath (use an electric water bath). f. Label the residue obtained as Residue C and use this in the Leibermann- Burchard Test. To the Residue (After Decantation): 1, Get a small portion of the incubated brain sample and add 10 mL. of hot 95% ethanol (use an electric water bath in heating the alcohol). Mix thoroughly and decant. . Discard the residue and use the decantate, labeled as Decantate A in the Molisch Test. F. DETECTION OF BRAIN LIPIDS 1, Molisch Test a. To2.0 mL of decantate A, add 5 drops of Molisch’s reagent. Mix thoroughly. b, Tiltthe tube and carefully add 1.0 mL of concentrated sulfuric acid, drop by drop, by allowing the acid to flow by the side of the tube. DO NOT MIX! Concentrated sulfuric acid is very corrosive! See First-aid Measures in case of an accident. ¢. Return the tube to its original position and note the color of the ring formed at the junction of the liquids. d._ Record your observations. 2. Ninhydrin Test a. Dissolve a portion of residue B in 2.0 mL of water, b. Add1.0 ml of 0.1% ninhydrin reagent. Mix thoroughly and heat in aboiling water bath for two minutes. ¢. Note the color of the resulting solution. Record your observations. Ninhydrin can stain the skin! Avoid contact with your bare fingers. 3. Soda Lime Test a. Grind a pinch of soda lime and a portion of residue B. b, Transfer the mixture in a dry test tube and heat gently over a small flame. After a minute of heating, test the vapor by placing pieces of moistened red and blue litmus paper at the tip of a stirring rod. Note the change in color of the pieces of litmus paper. Record your observations. 35LIPIDS 4, Ammonium Molybdate Test a. Add 1.0 ml of 6N nitric acid to a portion of residue B. b. Heat the mixture in a boiling water bath for five minutes and add 1.0 mL of the ammonium molybdate solution. Continue heating for another 5 minutes. c. Note the change in color of the solution. Record your observations. 4. Repeat the test using the contents of a lecithin soft gel capsule instead. e. Record your observations. 5. Leibermann-Burchard Test REMINDER: All equipment and containers to be used for this test must be COMPLETELY DRY. a. Prepare two clean and DRY test tubes. b. Inthe 1 tube, dissolve residue C in 1.0 mL of methylene chloride. c. Inthe 2" tube, place 1.0 mL of vegetable oil. d. Add‘ drops of acetic anhydride, followed by three drops of concentrated sulfuric acid into both tubes. Mix thoroughly. ACAUTION Acetic anhydride and concentrated sulfuric acid are very corrosive! See First-aid Measures in case of an accident. e. Note the color of the solution and compare the resulting intensity. BY ReTesuite A, SPOTTING EFFECT 1. What is the purpose of this test? 2. What property of lipids is detected by this test? 3. Are the results of this test conclusive? Explain your answer. B. SOLUBILITY 1. With the aid of their structural formula, explain the demonstrated solubility/ insolubility of vegetable oil and lecithin to water, methylene chloride, ether, and toluene. C. TEST FOR UNSATURATION (BROMINE WATER TEST) 1. What is the principle involved in the bromine water test? What is its purpose? 2, Provide the type equation used in the bromine water test. 3. Show the sample equation involved in the reaction between bromine water and your sample/s which gave a positive result. 36LuPIDs 4. Provide a possible explanation for the difference in results between the vegetable oil and the lecithin, D. ACROLEIN TEST 1. What is the principle involved in the acrolein test? What is its purpose? 2. Show the sample equation involved in the rea a. glycerol and potassium bisulfate b. cooking oil and potassium bisulfate . lecithin and potassium bisulfate n between 3. What is the compound responsible for your observation? 4. What is the purpose of the potassium bisulfate used? 5. What group of lipids was identified by this test? E. EXTRACTION OF BRAIN LIPIDS 1, Draw a schematic diagram to summarize the steps done during the extraction process. 2. With the aid of your schematic diagram and the results of the different tests done to the residues, which type of lipid was extracted by (a) ether, (b) acetone, and (c) hot 95% alcohol? F. DETECTION OF BRAIN LIPIDS 1. Molisch Test a. What is the principle involved in the Molisch test? What is its purpose? b. What is the purpose of the reagent used? ¢. Show the equation involved in the reaction between Molisch’s reagent and your sample/s which gave a positive result. 4. What is the compound responsible for your observation? €. What group of lipids was identified by this test? 2, Ninhydrin Test a. What is the principle involved in the ninhydrin test? What is its purpose? b. What is the purpose of the reagent used? ¢. Show the equation involved in the reaction between the ninhydrin reagent and your sample/s which gave a positive result. d. What is the compound responsible for your observation? €. What group of lipids was identified by this test? £ Account for the precaution with the ninhydrin reagent. 373. Soda Lime Test x b. ch What is the principle involved in the soda lime tes‘? What is its purpose? What is the purpose of the reagent used? ‘Show the equation involved in the reaction between soda lime and your sample/s which gave a positive result. What is the compound responsible for your observation? €. What group of lipids was identified by this test? 4, Ammonium Molybdate Test a What is the principle involved in the ammonium molybdate test? What is its purpose? What is the purpose of the reagent used? Show the equation involved in the reaction between ammonium molybdate and your sample/s which gave a positive result. What is the compound responsible for your observation? €. What group of lipids was identified by this test? 5, Leibermann-Burchard Test a. What is the principle involved in the Leibermann-Burchard test? What is its purpose? What is the purpose of the reagent used? ©. Show the equation involved in the reaction between Molisch’s reagent and your sample/s which gave a positive result. What is the compound responsible for your observation? ©. What group of lipids was identified by this test? Why must one take the precaution of using only very dry containers and pieces of equipment? 38 LuPIDsAHO a aia esas EXPERIMENT NO. 3 LIPIDS Name: Section: Professor: Group No: 1. Are there specific alerts (precautions) given in the experiment? List any that are given. 2, Are there any specific disposal directions given in the experiment? List any that are given. 3. What property must a substance possess for it to be classified as a lipid? 4, What are the main types of molecules classified as lipids? 5. What compounds are commonly found among almost all lipids? 346, Inthe Leibermann-Burchard test, why is it important that all pieces of equipment to be used are totally dry? 7. What is being detected by the bromine water test? 8. What group of lipids is being detected by the Molisch test? 9. What is the ninhydrin test for? 10. What is lecithin? 40[xO ee Haare) Rea Ay WW WL hia) oa aL EXPERIMENT NO. 3 LIPIDS Group No: ___________ Date Performed: = Section: Professor: ‘A. SPOTTING EFFECT Specific objective: ‘With (#) or Without (-) Translucent Spot — Performed by: ¥ SOLUBILITY Specific objective: Simple oy] MethVlene chloride [= Ether Toluene Vegetable oil Lecithin Performed by: 41C. TEST FOR UNSATURATION: BROMINE WATER TEST Specific objective: Color of bromine water: Sample Change of Bromine Water Vegetable oil Lecithin Performed by: D, ACROLEIN TEST Specific objective: Glycerol Cooking oil Lecithin Performed by: E, DETECTION OF BRAIN LIPIDS 1. Molisch Test Specific objective: Color of ring: Performed by: 422. Ninhydrin Test Specific objective: Color of solution: Performed by: 3. Soda Lime Test Specific objective: Color of litmus paper: Performed by: 4, Ammonium Molybdate Test Specific objective: Sample Observations Residue B Lecithin Performed by: 5. Leibermann-Burchard Test Specific objective: Sample Observations Residue B Lecithin Performed by: 43INTRODUCTION ‘A. ENZYME Cells produce special proteins known as enzymes that catalyze biological reactions by lowering the activation energy ofa reaction. The reactant broken down by an enzyme is called the substrate. The active site of an enzyme is that portion of the molecule responsible for its catalytic action, One outstanding property of an enzyme is its high specificity—absolutely specific (.e,, it acts only on one substrate), group-specific (e.g, pepsin wi!!! hydrolyze only soluble native proteins), linkage-specific (i.e,, it breaks the bonds only between specific groups like thrombin breaking the bond between arginine and glycine in the fibrinogen molecule), or reaction-specific (eg,, hydrolases, oxido-reductases, polymerases). Chemically, enzymes are either simple proteins or a protein (apoenzyme) combined with a non-protein unit (a cofactor). If the cofactor is an organic unit, it is called as a coenzyme. If the cofactor is a metal-ion, itis called a metal-ion activator. The two parts (protein and non-protein) constitute a haloenzyme. ‘The rate of enzyme activity is influenced by many factors. These factors include temperature, hydrogen ion concentration, substrate concentration, and enzyme concentration. B. DIGESTION Most foods are complex substances that must be broken down into smaller molecules that can be absorbed and utilized by the cells of the body. Digestion involves hydrolysis of proteins to amino acids, of starches to monosaccharides, and of fats to fatty acids and glycerol, The enzymes needed are hydrolases. Digestion takes place in the mouth, the stomach, and the small intestine. The passage of food through the digestive tract occurs as follows: Mouth > esophagus > stomach —> small intestine (duodenum, jejunum, and ileum) — large intestine. The principal digestive juices are the following: 1. saliva gastric juice from the glands in the walls of the stomach pancreatic juices (secreted by the pancreas) bile (secreted by the liver) intestinal juice (secreted by the intestinal mucosal cells) 44ENZYMES AND DIGESTION Geers At the end ofthe experiment, the students should be abe to: 1. identify the factors that affect the rate of enzyme activity; 2. describe the role of enzymes in the digestive process; and 3. use chemical tests to identify the products of digestion of carbohydrates, fats, and proteins of various sites in the digestive tract. | MATERIALS AND EQUIPMENT Samples to be brought by the students (per group) + 1 fresh chicken egg + 1 small can of vegetable oil + saliva (per individual) + ice + distilled water, 1L Reagents + Barfoed’s reagent + Pancreatin, 5% + Benedict's reagent + Pepsin + Buffer solutions (pH 4, 7, 10) + pH paper + Copper sulfate, 196 + Silver nitrate, 19% + Hydrochloric acid, 3N + Sodium carbonate solution, 0.596 + Iodine solution + Sodium choleate + Lead acetate, 1% + Sodium hydroxide, 3N, 0.196 + Starch solution, 1% Equipment + Electric water bath, 37 °C + Group water bath, 100°C REMINDERS 1. Putan aliquot portion of reagent in a vial by pouring the reagent from a reagent bottle using a funnel and a graduated cylinder. 2. PIPETTES, DROPPERS, and SYRINGES cannot be used in transferring reagents. 3. Dispose of used liquid materials into the waste bottle provided. Solid materials must be returned to the counter or thrown into the solid waste container. 45ENZYMES AND DIGESTION “Pketel Soles ‘A. SALIVARY DIGESTION: FACTORS AFFECTING ENZYMATIC ACTIVITY 1. Fffect of Temperature a. Label 3 tubes as A, B, and C. Place 2.0 mL of 1% starch solution into each of the 3 test tubes. b. Label another 3 tubes with AX, B-1, and C-1. Add 1.0 mL saliva into each of the 3 tubes. ©. Place tube A-1 in an ice bath, B-1 in a 37 *C water bath, and C- ina boiling water bath for about 5 minutes. d. Transfer tube A into the ice bath and pour the contents of A-t to tube A. Mix and place tube A back into the ice bath. After 10 minutes, add 2 drops of the iodine solution, Note and record the color of the solution. e. Repeat the same procedure for B-1 to B and C-1 to ©, using instead the 37 *C water bath for tube B and the boiling water bath for tube C, Note and record the color of the solutions after the 10-minute incubation and addition of iodine solution. £. Rank the tubes from 1 to 3 (with 1 for being colorless signifying greatest digestion, and 3 which means least orno digestion). A dark blue solution indicates the presence of starch. Partial digestion of starch results in a violet or red colored solution. A colorless solution indicates the complete digestion of the polysaccharide. 2. Effect of pH a, Prepare 3 test tubes and label as 4, 7, and 10, Add the following solutions as. described below: Contents Tube 4 Tube 7 Tube 10 Starch solution, 1% 2.0 mL 2.0mL 2.0 mL Buffer solutions | pH4=20mL | pH7=20mL | pH 10-20mL Saliva 1,0 mL 1.0 mL 1.0 mL b, Add the solutions in this order: Starch solution, buffer solutions, and then the saliva, All tubes must receive the saliva at approximately the same time. . Warm all test tubes in a 37 °C water bath for 10 minutes. d. After heating, add 2 drops of iodine solution to each of the test tubes. . Record the color of the solutions and rank the tubes from 1 to 3 (1 ~ greatest digestion which is colorless, and 3 - which means least or no digestion). A dark blue solution indicates the presence of starch, Partial digestion of starch results in a violet or red colored solution. A colorless solution indicates the complete digestion of the polysaccharide, 46ENZYMES AND DIGESTION 3. Effect of Enzyme Concentration a. Prepare 4 clean and dry test tubes and label them from 1 to 4. b, Place 2.0 mL of 1% starch solution into each tube. Add a drop of iodine solution into all the tubes. Note and record the resulting color of the solution. c. Ina separate tube, warm 2.0 mL saliva in a 37 *C water bath for 5 minutes. 4. Tube 1 will serve as the control of the experiment. e. Add 3 drops of warm saliva to tube 2. Mix quickly and note the time needed for complete digestion to take place. £. Dothe same for tubes 3 and 4 but instead of 3 drops, add 6 drops of saliva to tube 3 and 10 drops of saliva to tube 4. g Rankthe tubes from 1 to 3 (1 fastest digestion and 3 - slowest digestion). Record all your observations. A dark blue solution indicates the presence of starch. Partial digestion of starch results in a violet or red colored solution. A colorless solution indicates the complete digestion of the polysaccharide. 4, Effect of the Substrate Concentration a, Prepare 4 clean and dry test tubes and label them from 1 to 4. Follow the table as to the contents of the tubes: Contents Tubei Tube? Tubes Tubea sian 1.0 mL 20mL 3.0 mL 5.0 mL solution Distilled water | 1.0L 10mL Lo mL 1.0 mL saliva LOmL 1.0mL 1.0mL Lo mL b, ALL TUBES MUST RECEIVE THE SALIVA AT APPROXIMATELY THE SAME TIME. Thoroughly mix all the tubes and set aside for 5 minutes in a water bath set at 37°C. c. Remove all tubes from the water bath at the same time. 4. Prepare another set of 4 tubes, labeled as 1a to 4a. Add 2.0 mt of Benedict's solution into each of the tubes. @. Add 5 drops from your incubated mixture, ie., tube 1 to tube 1a, tube 2 to tube 2a, and so on. Mix thoroughly. £, Warm all tubes in a boiling water bath for 5 minutes. Note the changes in the color of the solution and the resulting precipitation (if any). g Rank the tubes accordingly (1 ~ greatest digestion and 3 - least or no digestion). Record all your observations. A brickred precipitate indicates the complete 47ENZYMES AND DIGESTION digestion of starch, Partial digestion ranges from green to yellow to an orange colored solution. A blue solution is a negative result indicating that the starch was not digested. 5. Effect of Metal-ion Poisons on Enzyme Activity a. Prepare 3 clean and dry test tubes and label them from 1 to 3, Place 1.0 mL of saliva into each of the tubes. b. To the first tube, add 3.0 ml of distilled water; 3.0 mL of lead acetate solution to tube 2; and 3.0 mL of 1% silver nitrate solution to tube 3, Mix thoroughly. . Place all tubes in the water bath set at 37 °C for 10 minutes. Remove the tubes from the water bath at the same time. d._ Prepare 3 clean and dry vials and label as 4 to 6. Put 3.0 mL of 1% starch solution into each of the vials. Add 2 drops of iodine solution. Mix thoroughly. e. Transfer 10 drops of the solutions into the vials: tube 1 to vial 4, tube 2 to vial 5, and so on, Incubate at room temperature for 10 minutes while periodically mixing the contents of the vial. £. Record the color of the solutions and indicate if digestion took place (eg, with complete digestion - colorless; with partial digestion - violet or red; and no digestion - dark shade of blue). B. PANCREATIC AND BILIARY DIGESTION 1. Pancreatic Amylase a. Put 1.0 mL of 1% starch solution on each of 2 clean and dry test tubes. b. Totube 1,add 1.0 mL saliva and to tube 2, add 1.0 mL of 5% pancreatin and 1.0 mL 0.5% sodium carbonate solution. Mix thoroughly and place in a water bath set at 37°C for 10 minutes. Divide the solutions into two portions. ‘To the 1* portion, add 3.0ml Benedict's reagent and warm in aboiling water bath for 5 minutes. Note the color of the resulting solution and precipitate (if any). Record all your observations. A brick-red precipitate as well as a green to yellow cor to an orange colored solution is a positive result. A blue solution is a negative result. e. Tothe 2 portion, add 3.0 mL Barfoed’s reagent and warm in aboiling water bath for 2 minutes. Note the color of the resulting solution and precipitate (if any). Record all your observations. A brick-red precipitate indicates a positive result and a blue solution is a negative result. ° 48ENZYMES AND DIGESTION 2. Pancreatic Proteases a. Prepare 3 clean and dry test tubes and transfer the following solutions as follows: Solution TestTube1 | Test Tube2 Test Tube 3 Egg white solution 2.0 mL 2.0 mL 2.0 mL CuSO4, 1% 5 drops | 5 drops 5 drops oe Aika 20 mL ‘Add until basic to litmus Add until acidic Sue + to litmus ~ Pepsin Pr 2.0 mL om Pancreatin =” om 2.0mL b. Add the pepsin and pancreatin simultaneously. Quickly mix the contents of all tubes thoroughly. c, Immerse all tubes in a water bath set at 37 °C until a change in color of the solutions is observed. d. Remove all tubes from the water bath at the same time. Compare the intensity of the colored solutions produced. Record all your observations. 3. Pancreatic Lipase and Bile a, Prepare 3 clean and dry test tubes and transfer the contents as follows: =C }i Test Tube1 | Test Tube2 | Test Tube3 Vegetable oil 20 drops 20 drops 20 drops Pancreatin 3.0 mL - 3.0 mL Sodium choleate - 3.0mL 3.0 mL Distilled water 3.0mL 3.0 mL - b. Mix the contents of all tubes thoroughly. ¢. Note the initial pH using pH paper. Record the initial pH. Adjust the initial pH to 7 (or a little bit higher) by adding 0.1% NaOH dropwise. 4. Immerse all the tubes in a water bath set at 37 °C for 1 hour. Check the pH of your solutions after every 15 minutes. Remove all tubes from the water bath at the same time. Record the final pH. 49ENZYMES AND DIGESTION Buy acl sues INTRODUCTION, 1. What are enzymes? What comprises a functional unit of an enzyme? 2. How do enzymes function? Discuss the lock-and-key model in relation to their function. 3. What is a substrate? How do enzymes act on substrates? 4, How are enzymes classified? 5. What is digestion? Discuss the chemical basis behind digestion. 6. What are the enzymes found along the digestive system? Where are they synthesized? What triggers their synthesis and release? Tabulate your answer. ‘A. FACTORS AFFECTING ENZYMATIC ACTIVITY 1. Effect of Temperature a. What is optimum temperature? What is the optimum temperature of your enzyme? b. In this procedure, identify which is the enzyme, the substrate, and the manipulated variable. c. How would you compare the degree of digestion in the different temperatures? Provide an explanation for your observations in t's different temperatures. Drawa graph showing the relationship between enzyme activity andtemperature. How can iodine solution determine the degree of digestion in the given tubes? Explain the principle behind the use of this reagent. g What is the health implication of this procedure? a 2. Effect of pH a. What is optimum pH? What is the optimum pH of your enzyme? b. In this procedure, identify the enzyme, the substrate, and the manipulated variable. How would you compare the degree of digestion in the different pH? Provide an explanation for your observations in the different pH. Draw a graph showing the relationship between enzyme activity and pH. How can an iodine solution determine the degree of digestion in the given tubes? Explain the principle behind the use of this reagent. g What is the health implication of this procedure? 2 eoENZYMES AND DIGESTION 3. Effect of Enzyme Concentration a. In this procedure, identify the enzyme, the substrate, and the manipulated variable, b. Account for the result obtained after adding iodine in the tubes containing the starch solution, c. What is a control? What is its importance in the experiment? d, How would you compare the degree of digestion in the different amounts of saliva added? ¢. Provide an explanation for your observations in the different amounts of saliva. f. Draw the graph showing the relationship between enzyme activity and the amount of enzyme. g. How can iodine solution determine the degree of digestion in the given tubes? Explain the principle behind the use of this reagent. hh. What is the health implication of this procedure? 4. Effect of the Substrate Concentration a. In this procedure, identify the enzyme, the substrate, and the manipulated variable. . b. How would you compare the degree of digestion in the different amounts of starch solution added? c. Provide an explanation for your observations in the different amounts of starch solution. 4, Drawa graph showing the relationship between enzyme activity and the amount of substrate. e. Howcan Benedict's reagent determine the degree of digestion in the given tubes? Explain the principle and provide the equations behind the use of this reagent. f. In which tube did you observe the greatest digestion? Least digestion? Provide an explanation for your observations. g What is the health implication of this procedure? 5. Effect of Metal-ion Poisons on Enzyme Activity a. In this procedure, identify the enzyme, the substrate, and the manipulated variable. b, Howwould you compare the degree of digestion in the different solutions added? ¢. Provide an explanation for your observations in the different solutions. Use chemical equations, if necessary. . Inwhich tube did you observe the greatest digestion? Least digestion? Provide an explanation for your observations. e. What is the health implication of this procedure? 51ENZYMES AND DIGESTION B. PANCREATIC AND BILIARY DIGESTION 1. Pancreatic Amylase a. In this procedure, identify the enzyme, the substrate, and the manipulated variable. b. What is the principle behind the use of the Benedict's and Barfoed’s tests? ©. Draw the chemical equations involved in these tests and identify the substance responsible for the visible results. d. What is the purpose of the 0.5% sodium carbonate solution in the experiment? e. How does salivary amylase compare with pancreatic amylase in its ability to digest carbohydrates? 2. Pancreatic Proteases a. Inthis procedure, identify the function of every component used in the test. b. What is the name of the test used? What is the principle behind its use? c. Draw the chemical equations involved in the tests and identify the substance responsible for your visible results. 4. What is the purpose of adding 3N NaOH until it is basic to litmus? 3 HCl until it isacidic to litmus? . What is pepsin? Pancreatin? Pancreatic juice? Where are these synthesized and what triggers their release? £, What are the proteases found in pancreatic juice? Specify the peptide bonds broken by each type of protease. & How does pepsin compare with pancreatic protesases in its ability to digest proteins? 3. Pancreatic Lipase and Bile a. Inthis procedure, identify the function of every component used in the test. b, What is the principle behind the determination of the solution's pHi before and after incubation? . Draw the chemical equations involved in the tests and identify the substance responsible for your observations. d. What is sodium choleate? Where is it synthesized and what triggers its release? €. What is the purpose of adjusting the initial pi to pH7 or slightly above pH7? 52PRE-LABORATORY REVII EXPERIMENT NO. 4 ENZYMES AND DIGESTION Name: Section: Professor: Group No.: 1. Are there specific alerts (precautions) given in the experiment? List any that are given. 2. Are there any specific disposal directions given in the experiment? List any that are given. 3, Give the different classes of enzymes according to the reaction they catalyze and give examples of each, 4, What does “optimum temperature” mean? 5. Why are enzymes sensitive to temperature changes? 536. Define optimum pH. 7, Listall enzymes in the digestive tract that catalyze the hydrolysis of carbohydrates, fats, and proteins. Give their optimum pH. 8, What is the lock-and-key model for enzymes? 9. What is the positive (+) result of the following tests and what is its significance? a. Iodine test b. Benedict's test c. Barfoed’s test 10. What is bile? What is its role in the digestion of fats? 54POST-LABORATORY DATA AND REPORT SHEET EXPERIMENT NO. 4 ENZYMES AND DIGESTION Group No:____________ Date Performed: Section: Professor: Results and Observation: A. SALIVARY DIGESTION: FACTORS AFFECTING ENZYMATIC ACTIVITY 1. Effect of Temperature Specific objective: Temperature Color of Solution Rank Ice water bath 37°C Boiling water bath Performed by: 2. Effect of pH Specific objecti ‘Temperature Color of Solution Rank pHA pH7 pH 10 Performed by: 553. Effect of Enzyme Concentration Specific objective: Performed by: 4, Effect of Substrate Concentration Specific objective: Performed by: 5. Effect of Metal-ion Poisons on Enzyme Activity Specific objective: Performed by: 56ENZYMES AND DIGESTION B, PANCREATIC AND BILIARY DIGESTION 1. Pancreatic Amylase Specific objective: __-Barfoed’s Test Pancreatin and Sodium Carbonate Performed 2, Pancreatic Proteases Specific objective: ‘Color and Intensity of Solution ieserube 7] No enzyme present Pepsin Pancreatin Performed by: 3. Pancreatic Lipase/Bile Specific objective Initial pH Final pH Performed by: 57EXPERIMENT NO. 5 DEOXYRIBOSE NUCLEIC ACID EXTRACTION FROM PLANT TISSUE Ni XeleT eres felt Nucleic acids are biopolymers composed primarily of nucleosides. Two types of nucleic acids are known to occur in nature: the deoxyribose nucleic acid (DNA) and the ribonucleic acid (RNA). Both function specifically in the storage and expression of the genetic code. Various protocols for DNA preparation from various sources of tissues have been published over the last few decades. The extraction of nucleic acids, particularly the DNA, consists of three steps: (1) disruption of the cell membrane, including the cell wall for some organisms; (2) dissociation from and denaturation of proteins; and (3) separation of the DNA molecule from other cellular components, Plant DNA isolation differs from the most modern and generic techniques used in animal tissue isolation due to the cellular structure of plant material vis-a-vis that of animal tissues. Plants contain cell walls composed of cellulosic materials and some other complex polysaccharides, and the degree to which they must be separated from the nucleic acid material (particularly RNA) depends on the intended use of the nucleic acids. It is noteworthy to mention that primarily due to their large genome sizes, plants can yield large quantities of nucleic acids. Plants also contain two other genomes: mitochondrial and plastid. In both terrestrial plants and macroalgae, the plastid DNA genome is a double-stranded DNA circle, which contains the genes for plastid rRNA, tRNA, and some other proteins. Plant genomes can range from 120-217 kilobases. eens kelss eu ‘At the end of the experiment, the student must be able to: 1. extract DNA from plant sources; 2. determine the purity of the extracted DNA; and. 3. characterize the isolated DNA after acid hydrolysis. GSES Samples to be brought by students (per group) + 2pcs. onions + Crushed ice + Cheesecloth