Biochem Lab Finals Reviewer PDF

FINALS Polysaccharides ranges from less soluble to insoluble due to their CARBOHYDRATES...

FINALS Polysaccharides ranges from less soluble to insoluble due to their CARBOHYDRATES larger or more complex structure. Cn H2n On (1:2:1) Shape In every carbon atom, there is 2 hydrogen atoms and 1 oxygen monosaccharides and disaccharides appear atom as white crystalline Aka. Hydrate of Carbon these white crystalline reflects of Most important source of energy for the their simpler and more of their body structures Glucose while polysaccharide are amorphous. most important carbohydrate in Since they lack a defined shape the body which reflects their larger and less primary source of energy ordered structures It is a polyhydroxy aldehyde Taste (glucose) or polyhydroxy ketone (fructose) with increase complexity of the chemical ✓ means that glucose has structure, the level of sweetness decreases multiple hydroxyl group (- For example: OH group that is attached Monosaccharides like glucose or to its carbon atom fructose are known to be the sweetest which is followed by the disaccharides like sucrose Polysaccharides like starch lacked sweetness MEDICALLY SIGNIFICANT FUNCTIONS OF CARBOHYDRATES 1. Broken down to yield ATP – Adenosine Triphosphate (the biological energy fuel for all living system) Upon oxidation of carbohydrates There are exemptions to the general formula of carbohydrates, and these are: or its 1 gram, it will yield 4 kcal (kilocalorie) Rhamnose (C6H12O5) 2. Intermediates for the biosynthesis of biochemical entities It lacks 1 hydrogen atom compared to the Carbohydrates intermediates are typical carbohydrate formula used in biosynthesis of various Deoxyribose (C5H10O4) biochemical molecules like amino acids, lipids, nucleotides Found in DNA 3. Associated with other entities Deviates from general structure or formula 4. Precursor for the synthesis of DNA, RNA, which there is a 1 missing oxygen atom and ascorbic acid synthesis (vitamin C) They are essential for genetic These structural differences of carbohydrates, it material, cellular function and allows them to perform various roles in biological antioxidant activity systems. Includes glycosides and PHYSICAL PROPERTIES antibiotics. 5. Form structural tissue in plants and in Solubility microorganisms Structural tissue in cellulose, with increasing complexity of the chemical plants cell walls, chitin in fungal structure, the water solubility decreases. cell wall that contributes to their Ex: rigidity and support. Monosaccharides like glucose, 6. Biological transport, regulatory functions Disaccharides like sucrose -> they in cell-cell recognition are generally highly soluble in Including signaling and water communication between cells  For ex: glycoproteins transport consists of single sugar molecules like transferrin (found unit in iron) and ceruloplasmin (found Ex: glucose and fructose in copper) 7. Activation of growth factors and modulation of the immune system b. Disaccharides contributing to immune response Composed of 2 Ex: glycoprotein enzymes like monosaccharides which pepsin, TSH (thyroid stimulating are linked together hormone) and FSH (follicle Ex: sucrose and lactose stimulating hormone) 8. Function for protection and functionality c. Polysaccharides Such as forming mucus in the Complex carbohydrates respiratory and digestive system formed by long chains of which protects underlying tissues monosaccharide units Mucin is found in mucus and Ex: starch and cellulose epithelial cells 9. Contribute to the basis of blood typing B. FUNCTIONAL GROUPS Specific carbohydrates molecules a. Aldoses on the surface of RBC, helps to Monosaccharides that define blood types like type A,B, contain an aldehyde AB, O which are important in group (-CHO) such as blood transfusion and immune glucose reaction. Type A – N- b. Ketoses Acetylgalactosamine (GalNAc) Monosaccharides that ✓ This carbohydrate is part contain ketone group of the blood group (C=O) such as fructose antigen, when this GalNAc is present on the surface c. Sugar alcohols of RBC it contributes to Derived from the the identity of the blood reduction of the type A. monosaccharides which Type B – D-Galactosamine (Gal) are used as sweeteners ✓ This carbohydrate can be Examples: associated with other blood group antigen, but Xylitol in the context provided it seems to be considered Mannitol separately. osmotic diuretic which is ✓ More on type B. used to increase urine Type AB – both GalNAc and production galactosamine (gal) are present ✓ Indicates that the AB Sorbitol blood type which means both A and B antigens on commonly used as the RBC surface. sweetener in various Type 0 – both are absent pharmaceutical form when sorbitol undergoes CLASSIFICATIONS dehydration or removal of water, it is transformed - These classifications help in understanding isosorbide the structure, function, biological roles in when isosorbide undergo various carbohydrates in a living system or nitration, a chemical the body. reaction where nitrate groups like NO2 are A. NUMBER OF SUGAR UNITS a. Monosaccharides introduced into the molecule. This process Simplest form of modifies chemical carbohydrates which structures and enhances  the compounds property processes galactose and -> dito na nafoformed cause by a deficiency in mga gamot like Isosorbide one of the enzymes which Dinitrate (ISDN) or are needed to breakdown Isosorbide Mononitrate the galactose) (ISMN) -> this med is used to treat angina pectoris (a 3. Arabinose chest pain due to reduce Source: blood flow in the heart Gum Arabic wherein they dilate the Important information: blood vessels to improve Scientific name of Gum blood flow) arabic: Acacia senegal C. NUMBER OF CARBON ATOM 4. Xylose a. Trioses Source: Monosaccharides with 3 Corn Cob carbon atoms Important information: Ex: Glyceraldehyde Xylose is also known as “wood sugar” b. Pentoses Monosaccharides with 5 5. Fructose carbon atoms Source: Ex: Ribose Sucrose Inulin (type of prebiotic) c. Hexoses Important information: Monosaccharides with 6 Fructose is also known as carbon atoms “fruit sugar” Ex: glucose and fructose Known to be the sweetest sugar Glyceraldehyde – simplest form of aldose Dihydroxy acetone – simplest form of 6. Ribose ketose Important information: Glucose – commonest aldose Known as Nucleic acid Fructose – commonest ketose sugar MONOSACCHARIDE: HYDROLYSIS OF THE SUGAR: 1. Glucose Main Source: Starch Glycogen Amino acid Lactate Pyruvate Important information: Physiologic/blood sugar (this are evaluated when we had test in the sugar of blood) 2. Galactose Main Source: Milk Mammary gland/dairy products Important information: Milk Mammary Gland: “milk sugar” Galactosemia (rare genetic disorder which affects how our body IMPORTANT INFORMATION FOR SPECIFIC SUGARS: *When performing the test, few drops of Molisch Ribose test are added to the is a structural element of nucleic acids and sample followed by also of some coenzymes carefully layering the concentrated sulfuric Glucose acid. Must NOT be shaken and on oxidation yield energy which is required dapat pinadaan lang sa for various metabolic activities side ng test tube when Fructose dropping. (+) result: purple - violet ring at found in fruits, honey etc. which are the interface between the 2 liquid responsible for sweetness and can be (3 layers but the middle/second converted to glucose and utilized in the layer is the purple violet ring) body. Principle: Galactose The sulfuric acid acts as a strong dehydrating agent component of milk sugar-lactose, which breaks down the glycolipids and glycoproteins carbohydrates into furfural derivatives Mannose This furfural derivatives constituent of mucoproteins and will react with alpha- glycoproteins which are essential for the naphthol producing a body characteristic color Aldehyde undergoes condensation along with a-naphthol. TESTS 1. Molisch test 2. Iodine test 3. Tests for reducing sugars Benedict’s Fehling’s *These are the tests performed during lab. Other test for carbohydrates: 4. Barfoed’s 5. Tollen’s 6. Osazone 2. IODINE TEST 7. Seliwanoff’s Test for starch 8. Keller-killiani simple and effective test 9. Moore’s Reagents: Iodine solution 10. Mucic acid (+) result: 11. Bial’s complex blue solution blue to black colored 1. MOLISCH TEST complex General test for carbohydrates sometimes they are pink It is very effective for a broad or purple (but same lang range of carbohydrates including yung range ng color) monosaccharides, disaccharides, Principle: polysaccharides making it a starch (amylose reliable and widely used method. component) forms a Reagents: helical structure. When Molisch reagent (solution the iodine molecules of a-naphthol in 95% enter this helical ethanol) structure of starch, they Conc. Sulfuric acid bind and forms a starch iodine complex  This now results in the characteristic blue to black or purple color TESTS FOR REDUCING SUGARS: 2. Fehling’s 1. Benedict’s Test for reducing sugars Test for reducing sugars a type of sugar that can Reagents: donate electron to other Fehling’s A (CuSO4) molecules during a Fehling’s B (K Na tartrate chemical reaction C4H4O6. 4H2O) this property allows (+) result: Brick red ppt sugars to participate in Principle: reactions that can their aldehyde group of sugar change structures or is oxidised by complexed produce different copper ions to form acid. substances The red copper (I) oxide Common example: then precipitates, which is glucose, fructose, and an indicator for the redox lactose reaction (reduction Reagents: oxidation) Copper sulfate pentahydrate (CuSO4. 5H2O) Sodium citrate (Na3 C6H5O7 Sodium carbonate (Na2CO3) (+) result: Brick red ppt (due to Cuprous oxide Cu2O) Principle: Sodium carbonate creates alkaline Na2CO3 or basic environment which helps SUMMARY: converts the sugar into a more powerful/strong reducing agent called enediols This reaction causes the copper sulfate to change color indicating the presence of reducing sugars. OTHER TESTS: Their detection reagents (+) result Among the samples: Glucose is the only one have positive result. this means that glucose is the only reducing sugar in the samples. Osazone (aka Phenylhydrazine test) Is a chemical test used to detect reducing sugars. Allows the differentiation of different reducing sugars on the basis of: 1. the time of the appearance of the complex ✓ different reducing sugars will produce the color change or precipitate at different rates during test. ✓ The timing of this change can help identify which sugar is present 2. The shape and structure of the crystals ✓ When the red ppt forms, the resulting crystals can vary in shape ✓ The structure depending on the specific type of reducing sugars  EXPERIMENT 6: DETERMINATION OF THE OPTICAL ROTATION OF SUCROSE AND GLUCOSE [Did not discuss the image] Same MF, but different attachment ISOMERISM Isomers compounds with the same chemical formula but differ in spatial configuration. two or more molecules that share the Stereoisomers: same molecular formula. Describe the relationship between 2 or more entities Isomerism Can formed as right and left- handed Reason: The presence of asymmetric or chiral carbons allow the formation of isomer Cyclohexane and 1-hexene = isomers But 1-hexene and Propionic acid = not isomers Propionic acid and 1-hydroxy-2-propanone = isomers Isomers are determined through same molecular formula, but different in spatial configuration CONSTITUTIONAL ISOMERS VS. STEREOISOMERS Constitutional isomers different connectivity (e.g. 1- hexene vs. 2 E – Cis Ex: 2-Hexene (refer to the image) hexene) Z – Trans Same molecular formula, different 2 arrangement of Hydrogen in double bond (either connectivity/structure/arrangement same side or opposite side) Hint: same MF, different numbering and IUPAC name STEREOISOMERS: REVIEW CIS AND TRANS Stereoisomers Cis-Trans Isomers same connectivity, different arrangement in space are sometimes called geometric isomers a subset of diastereomers Stereoisomers CHIRALITY: can also arise from tetrahedral carbon Chiral Center atoms that are attached to four different Carbon atom with four different groups substituents attached in a tetrahedral orientation (i.e. a “chiral” or “asymmetric” carbon) Organic molecule which contains atom attached to different groups 2 (need tetrahedral orientation so that handedness will possess it) 1 3 Determined the chiral molecule and achiral molecule 4 Need different attachment Chiral Molecule Since magkamukha na yung dalawang Mirror image not superimposable –> have attachment handedness BASIC CONCEPTS TO CHIRALITY Achiral Molecule Monosaccharides Mirror image superimposable -> do not possess handedness simplest type of carbohydrates. They have specific structure and reactions. Another Property: Handedness Handedness Ex: Hands has right handed and left handed form If the 2 forms is same with the right and left hands -> mirror image (kapag pinagdikit dalawang kamay) Pero HINDI pwedeng pagpatungin, since CHIRAL MOLECULES: iba na yung itsura Mirror-Image Superimposable Coincide yung lahat ng point sa isang point Ex: plates na walang design (kapag Pinatong patong -> same lang sila) Nonsuperimposable Pinatong ang isang kamay sa isang (a) Bromochloroiodomethane kamay, yung thumb nasa Simple example chiral organic molecule magkabilang side Its mirror image is nonsuperimposable Since if pinagpatong sila like bromine and chlorine & chlorine and bromine (b) Glyceraldehyde Mirror image but Nonsuperimposable CH2OH is katapat ng OH and vice versa  1. ENANTIOMERS / D OR L ISOMERISM: They are Mirror images but their molecule are Non-superimposable The images are mirror image pero kapag pinagtapat they are nonsuperimposable. Why do we need to determine the handedness? The human body -> right handed and left handed form molecule are the ones elicit different responses in the human body. Sometimes both forms are biologically active Their each form gives different responses sometimes if both it is same response sometimes the one form has greater response compared to other leading to D and L Body responses of right handedness of hormones: Epinephrine is 20x greater than the response to the left handed form Naturally, monosaccharides are right handed Plants and dietary source of carbohydrates produces as right handed in the monosaccharides 2. DIASTEREOMERS Amino acids (building blocks for protein) are always left handed molecules Non-mirror images because of the Cis and Trans isomers and Non- superimposable STEREOISOMERISM Stereoisomers The twins have scar which means are Isomers that have the same molecular and enantiomers structural formulas but differ in orientation of atoms in space But if twin 1 is compared There is major structure features (chiral to another boy, they are diastereomers since they center and structural rigidity must be are non-mirror and non- present) superimposable Structural rigidity are caused by restricted rotation about the chemical bond that is why they are basis for Cis and Trans isomers Divided into 2 types: 1. Enantiomers 2. Diastereomers  A process known as mutarotation. Mutarotation is seen as change in the optical rotation of the solution due to the interconversion between alpha and beta anomers Common in sugars The glucose forms open chain and ring chain forms in the solution are at Other examples are erythrose and Threose equilibrium changing the observe optical rotation overtime, a process known as ring UNDER STEREOISOMERS (ISOMERIZATION) ARE: chain tautomerism 1. EPIMERS Kaya nagkakaroon ng alpha stereoisomers that differ only in (opp.side) and beta (same side) configuration about ONE chiral carbon For example, the D -glucose as reference. Two major aldohexose in nature, Glucose and Galactose, are epimers at C4. Glucose and Mannose are C2 epimers 3. KETO-ALDO ISOMERIZATION exhibited by glucose and fructose 4. OPTICAL ISOMERISM property of asymmetric or chiral compounds to rotate polarized light. Measurement of polarity uses an instrument called a polarimeter (Lippich type). Ex: D-Glucose (with 2 major aldohexose) Rotation is either: Difference between Galactose: epimer C4 Dextrorotatory (+) / (d) / Difference between Mannose: epimer C2 (r) which rotate the light Compound A and B -> first enantiomeric pair to the right or clockwise D-erythrose Levorotatory (-) / (l) / (s) L-erythrose which rotate polarized light to the left or Compound C and D -> second enantiomeric pair counterclockwise. D-threose L-threose This is independent of the D and L forms of a carbohydrate. 2. Α AND ß ISOMERISM α and ß anomers can interconvert using the open chain structure (straight chain) as intermediate. EXPERIMENT NO. 7 NUCLEIC ACIDS test tube, centrifuge for 5 minutes, and decant. HYDROLYSIS 6. Evaporate the supernatant liquid, filter if necessary. Cleaving of a molecule in the presence of 7. Cool the filtrate below 40ºC then pour 40 water mL of 95 % ethyl alcohol containing 2 drops Hydrolysis of nucleic acid (using sulfuric of conc. HCl. acid) 8. Allow RNA to settle, decant, and wash with Hydrolysis of nucleic acid will yield a 95% alcohol. Dry at room temperature. nucleoside (sugar, purine or pyrimidine base) and a phosphate group HYDROLYSIS OF RNA: The nucleoside will be further hydrolyzed to D-ribose and either a purine or 1. Place a small portion of the RNA in a test pyrimidine base tube. 2. Add about 10 mL of 10% sulfuric acid. Cover the test tube loosely and boil in a water bath for 30 minutes 3. Use the solution in no. 2 and the remaining unhydrolyzed RNA (dissolved in water) for the following tests and compare results. NUCLEOSIDE Presence of nitrogenous base and pentose sugar Nucleic acid breakdown (hydrolyzed) into Nucleoside (nitrogenous base -purine or pyrimidine and pentose sugar- ribose) and Phosphate Linkages: N-glycosidic bond EXPERIMENTAL PROCEDURES SEPARATION OF RNA FROM YEAST: 1. Dilute 10 mL of 1% NaOH with 50 mL water. 2. Add 6g of dried yeast and heat in a water bath for 30 minutes with occasional stirring. 3. Filter the mixture through a cheesecloth and centrifuge for 10 minutes. 4. Decant into a beaker and cool the supernatant liquid 5. Add glacial acetic acid to the supernatant liquid, drop wise until faintly acidic to litmus paper. Once turbid, transfer into a RNA Sugar: Ribose They both have phosphate group Nitrogenous bases: Adenine, Uracil, Guanine and Cytosine DNA Sugar: Deoxyribose (2-Deoxyribose) They both have phosphate group Nitrogenous bases: Adenine, Thymine, Guanine and Cytosine B. TEST FOR INORGANIC PHOSPHATE 1. Place 1 mL of dissolved RNA and 1 mL of hydrolyzed RNA into separate test A. TEST FOR RIBOSE tubes. 2. To each test tube, add 2 mL of NH3. 1. Place 1 mL of dissolved RNA and 1 Acidify with a few drops of 6N HNO3. mL of hydrolyzed RNA into 3. Add 1 mL of ammonium molybdate separate test tubes. reagent and heat with water. 2. To each test tube, add 5 drops of 4. Note and record the results. orcinol reagent and heat in a water bath for 3-5 minutes. Test for Inorganic Phosphate: 3. Cool the tubes immediately under the faucet. Compare the results. Reagent: Ammonium molybdate 4. Note and record the results. Principle: hydrolysis of the phosphate group is needed in order for it react TEST FOR D-RIBOSE: with ammonium molybdate in an acidic environment (addition of Nitric BIAL’S TEST acid), forming ammonium phosphomolybdate Reagent: Orcinol Result: (+) bright yellow/canary Principle: yellow ppt Sugar monomer will be dehydrated to form a furfural, which reacts with orcinol When pentoses (ribose) hydrolyze will yield to furfural which reacts with orcinol resulting in blue or blue-green color Result: (+) blue colored solution or blue-green colored precipitate (-) If hexoses are presence -> Hydroxy furfural -> brown colored ppt C. TEST FOR PURINE BASES (guanine and result with Benedict’s adenine) test. The color will vary 1. Place 1 mL of dissolved RNA and 1 mL depending on the of hydrolyzed RNA into separate test amount of the sugar tubes present. 2. To each test tube, add 1 mL of 2N HCl. *Fehling’s test is for non Mix and place in a boiling water bath reducing sugar for 20 minutes. Results: 3. Add 1 mL 2N NaOH and 2 mL of (+) Green- Traces of acetate buffer, mix and heat in a water reducing sugar bath. (+) Brick red - large 4. Add 0.5 mL of 10% CuSO4 and note the amount of reducing formation of a bluish brown sugars precipitate. 5. Add 10 drops of NaHSO4 solution and mix. 6. After a few minutes, note the color of the precipitate formed. 7. Note and record the results. Test for Purine Bases Blue: Negative result, Reagent: Copper sulfate and indicating no presence of Sodium bisulfate reducing sugar Principle: Green: Trace result, Purine bases with ions of signifying a minimal Copper and/or Silver. amount of reducing Sodium bisulfate sugar. reduces Cu2+ to Cu+ -- Orange: Positive (+) this will cause result, indicating a precipitation as it moderate presence of renders an insoluble reducing sugar. purine-copper complex. Brick Red: Strongly A pyrimidine base will positive (++) result, remain stable if exposed denoting a substantial in the same amount of reducing environment and sugar. conditions. Result: (+) yellow precipitate D. TEST FOR REDUCING SUGARS 1. Place 2 mL of dissolved RNA and 1 mL of hydrolyzed RNA into separate test tubes. 2. Neutralize each of the test solutions with Na2CO3. 3. Decant 1 mL of each sample and add 1 mL of Benedict’s reagent. Heat in a boiling water bath. 4. Note and record the results. [Study the range numbers] Test for Reducing Sugars MEDICATIONS AFFECTING DNA AND RNA Reagent: Benedict’s reagent PROCESSES [Did not discussed thoroughly] Principle: if a sugar has a reducing Methotrexate end, it will undergo redox Prevents DNA synthesis by inhibiting and produce a positive dihydrofolate reductase (DHFR) to convert into tetrahydrofolate Doxorubicin (Etoposide) Target: topoisomerase II poison which is used in DNA replication. Used as chemotherapeutic Induces cytotoxicity of DNA break Rivfloza A small-interfering RNA (siRNA) that is used to treat primary hyperoxaluria in adults Oxaliplatin A DNA crosslinker Daunorubicin A topoisomerase II poison Cyclophosphamide An alkylating agent 5-fluorouracil An antimetabolite Etoposide A topoisomerase inhibitor Bleomycin A cytotoxic antibiotic Cisplatin, Topotecan, Mitomycin C & Gemcitabine A DNA-targeting drug EXPERIMENT NO. 8 ENZYME ACTION 3. Peel off potatoes and cut three cubes (1cm3) 4. Drop the following to test tubes 1-4. DEFINITION OF TERMS: - test tube 1 – pebble or small rock - test tube 2 – 1 cube of potato 1. Catalyst - test tube 3 – mashed 1 cube potato a compound that speeds up a - test tube 4 (but with water) - mashed 1 reaction by lowering activation cube potato energy. 5. Observe the reactions in the 4 test tubes for 5 minutes and note the vigorous formation 2. Activation energy of bubbles in each test tube (release of amount of energy required for oxygen gas). Using a metric ruler, measure reactants to form products the height of the bubbles produced after 5 minutes. 3. Enzyme a biological catalyst that speeds up reactions and interactions between molecules in biological systems. 4. Catalase an enzyme normally found in many plant and animal tissues Enzyme that are present in potato Alpha amylase – mouth Lipase – small intestine How will we know if the catalase in the experiment 5. Hydrogen peroxide (H2O2) is reacting with hydrogen peroxide? a poisonous byproduct of metabolism that can damage cells If there is bubble formation or froth if it is not removed From the equation: ROLE OF CATALASE IN THE HUMAN BODY 2H2O2----------catalase-------> 2H2O + O2 Catalase is an enzyme that speeds up the breakdown of hydrogen peroxide into test tube 1 – pebble or small rock + water (H2O) and oxygen gas (O2) that is H2O2 = why during the experiment there is bubble test tube 2 – 1 cube of potato + H2O2 formation. test tube 3 – mashed 1 cube potato + Importance: Protects our cells H2O2test = HIGH BUBBLES from oxidative damages since it is mashed, it means it Living organisms are exposed to oxygen has greater surface area -> specifically these are liver, kidney and RBCs speed reaction -> catalase will breakdown hydrogen peroxide tube 4 - mashed 1 cube potato + H2O no reaction Even the potato is catalase but they don’t breakdown hydrogen peroxide into water only ENZYME ACTION: CATALASE ACTIVITY 1. Prepare 4 test tubes and label each as 1, 2, 3, 4 using a ruler mark at 3cm with a marker pen or pencil. 2. Fill test tubes 1-3 to the 3cm mark with hydrogen peroxide and test tube 4 with water.  Principle: At high temp. enzymes denature leading to decreased activity. At low temp. enzyme molecular motion decreases leading to decreased activity. May nagfoform na bubbles kaso matagal Optimum Temperatures temperature at which the enzyme activity is greatest. occurs in the human body around 37°C. THE EFFECT OF TEMPERATURE ON AN ENZYME 1. Prepare 3 test tubes and label test tubes 1, 2, and 3. Mark a 3 cm in each test tube using a marking pen or pencil. 2. Place a 1cm3 cube of potato into each test tube. 3. Add 5 drops of water to each test tube. 4. Place the test tube no. 1 in a hot water bath for 5 minutes. 5. Place the test tube no. 2 in the cold – water bath for 5 minutes 6. Place test tube no. 3 in the test tube rack at room temperature. 7. After the 5-minutes add H2O2 up to the 3cm mark on each test tube. 8. Observe for five minutes and record the height of the bubbles in each tube in cm. THE EFFECT OF PH ON ENZYME ACTIVITY 1. Prepare 3 test tubes and label each test tube 1, 2, and 3. Mark a 3 cm in each test tube using a marking pen or pencil. Place a 1cm3 cube of potato into each test tube. 2. Add 10 drops of HCl to the test tube no. 1, 10 drops of NaOH to test tube no. 2 while drops of distilled water to the tube no. 3. 3. Swirl gently to mix the contents. Test the pH of each using a pH meter or pH paper. pH of HCl = 2.9-3 (acidic) NaOH = 12.3 – 14 (strong bases) Water = 7 (neutral) 4. After 2 minutes, add H2O2 up to the 3cm mark in each test tube. 5. Observe for 5 minutes for reaction to occur and measure the height of the bubbles in cm. Record your results. Test tube 3 must produce high bubbles due to its optimal pH activity unlike test tube 1 Test tube 1 (Hot water) – no bubbles and 2 they are acidic/basic (standard) formed If there is a long time for observation, test Test tube 2 (Cold water) – no bubbles tube 2 have high formation too since the formed alkaline environment can change together Test tube 3 (Room temp.) – high with catalase formation of bubbles pH affects structure of enzyme and substrate affects how enzyme interacts with the substrate  Differences in optimal pH ✓ Ex: Atenolol (beta e.g. gastric enzyme in acidic blockers) – inhibit beta stomach pH adrenergic reducing heart rate and blood pressure PHARMACEUTICAL PRODUCTS THAT CONTAIN ENZYMES [no need to memorize] Products Enzymes Pancreatic Enzyme Replacement Lipase, protease, and Therapy (PERT) amylase Lactase Supplements Lactase Proteolytic Enzymes for Bromelain (pineapple Inflammation and Wound juice), trypsin, and papain Healing (in papaya) Urate Oxidase (Rasburicase) Urate oxidase (rasburicase) Asparaginase for Cancer Asparaginase Treatment Collagenase for Wound Collagenase Debridement IMPORTANCE OF ENZYMES IN THE Dornase Alfa (Pulmozyme) for Dornase alfa (a PHARMACEUTICAL INDUSTRY Cystic Fibrosis recombinant DNase enzyme) Drug any substance that brings about a change Pancreatic Enzyme Replacement Therapy (PERT) in biologic function through its chemical Aids in digestion of patient with pancreatic actions. insufficiency (cystic fibrosis) Molecules in the body in the proteins -> Helps to breakdown fats, proteins and therapeutic effect carbohydrates to improve the absorption Generally can be an : of nutrients Agonist ✓ activator, mimetic Lactase supplements ✓ bind in the receptors For lactose intolerance -> digestion ✓ mimics the action discomfort like bloating and diarrhea (ginagaya) – hormones and neurotransmitter Urate Oxidase ✓ Ex: morphine (opioid) – For High uric acid levels bind opioid receptors relieving the pain Asparaginase Antagonist for Cancer Treatment (acute lymphoblastic ✓ inhibitor, blocker leukemia) ✓ bind to the receptors or breakdown asparagine enzymes but do not activate Collagenase ✓ instead they inhibit Breakdown collagen in deade tissues action ✓ no biological response Dornase Alfa For cystic fibrosis Breakdown DNA to mucus to reduce viscosity and improvement for breaking and reduce infections in lungs MAJOR DIGESTIVE ENZYMES FACTORS AFFECTING ENZYME ACTIVITY Temperature pH Enzyme concentration Substrate concentration Effects of pH Effects of temperature Reaction product Enzyme inhibition