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LESSON : ISOLATION OF RNA FROM YEAST1OBJECTIVESAt the end of the experiment, you should be able: - To isolate RNA from yeast - To be able to identify products of hydrolysis of RNA - To be able to test qualitatively for the presence of inorganic phosphate, purine bases, and reducing sugars in the pro...

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Biochemistry (Laboratory) LESSON : ISOLATION OF RNA FROM YEAST OBJECTIVES At the end of the experiment, you should be able: • To isolate RNA from yeast • To be able to identify products of hydrolysis of RNA • To be able to test qualitatively for the presence of inorganic phosphate, purine bases, and reducing sugars in the products of RNA hydrolysis

DNA (DEOXYRIBONUCLEIC ACID) Structure: • Made up of a DOUBLE HELIX STRANDS OF NUCLEOTIDES which are antiparallel and complementary • There are hydrogen bond forces and base stacking interactions per turn (10 base pairs)

INTRODUCTION WHAT IS NUCLEIC ACID? NUCLEIC ACIDS - are biopolymers, or large biomolecules, essential for all known forms of life - are made from monomers known as NUCLEOTIDES • RNA (ribonucleic acid) • DNA (deoxyribonucleic acid)

Main function: Responsible for STORAGE and TRANSMISSION of genetic material

All nucleotides contain 3 COMPONENTS: • Nitrogen base • Pentose sugar • Phosphate residue

RNA (RIBONUCLEIC ACID) Structure: • Made up of a SINGLE STRAND OF NUCLEOTIDES • Polymer of Purine and Pyrimidine ribonucleotides linked through 3’-5- phosphodiester bridges Main function: Involved in the PRODUCTION and MANUFACTURE of proteins Pyrimidine base: URACIL Sugar: D-RIBOSE

Pyrimidine base: THYMINE Sugar: 2-DEOXY-D-RIBOSE EXPERIMENT: ISOLATION OF RNA FROM YEAST WHY YEAST AS SOURCE OF RNA? Ribonucleic acid preparations are usually obtained from YEASTS because the ratio of RNA-DNA is the highest in these microorganisms. OVERVIEW OF EXPERIMENT • Reagents used and importance • Nucleic Acid Isolation/Extraction (Yeast) • Hydrolysis of extracted nucleic acid (RNA) • Qualitative tests for RNA and its components REAGENTS USED AND IMPORTANCE 1. 1% NaOH (SODIUM HYDROXIDE) - Precipitates nucleic acids and other substances associated with nucleic acids like proteins. - Inactivates nucleases that can degrade nucleic acid. 2. 95% C2H5OH (ETHYL ALCOHOL) - Ensures complete precipitation of nucleic acids. 3. 10% H2SO4 (SULFURIC ACID) - Aids in the hydrolysis of nucleic acids. 4. Glacial CH3COOH (ACETIC ACID) - Precipitates protein from other compounds associated with it. NUCLEIC ACID ISOLATION AND EXTRACTION NUCLEIC ACID EXTRACTION or ISOLATION - is one of the most pivotal steps and routinely used method in many areas of the biological and medical sciences - Roughly divided into four steps: 1. Cell disruption 2. Removal of membrane lipids, proteins, and other nucleic acids 3. Nucleic acid purification/binding from bulk 4. Nucleic acid concentration

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Biochemistry (Laboratory) LESSON : ISOLATION OF RNA FROM YEAST - Can be either: • Mechanical (grinding, bead beating, shocking, etc.) • Chemical (detergents, lytic enzymes, chaotropic agent, etc.) PROCEDURE I. EXTRACTION OF RNA FROM YEAST 1. Dilute 10 mL of 1% NaOH with 50 mL water. 2. Add 6 g dried yeast and heat in water bath for 30 minutes, w/ occasionally stirring. 3. Filter through cheesecloth and centrifuges in 10 minutes. 4. Decant into beaker and cool the supernatant liquid. 5. Add glacial acetic, drop wise until faintly acidic to litmus paper, if turbid transfer in a test tube and centrifuge in 5 mins and decant. 6. Evaporate supernatant liquid, filter if necessary. 7. Cool the filtrate below 40 degrees then pour in 40 mL of 95% ethyl alcohol containing 2 drops of conc. HCl. 8. Allow RNA to settle, decant and wash with 95% alcohol. Dry at room temperature.

HYDROLYSIS OF NUCLEIC ACID (RNA) HYDROLYSIS REACTION • HYDROLYSIS - Chemical process involving the addition of water causing a molecule to cleave into two parts. • HYDROLYSIS OF RNA - A reaction in which a phosphodiester bond in the sugar-phosphate backbone of RNA is broken, cleaving the RNA molecule.

• Complete hydrolysis of RNA yields the following: 1. Pentose sugar (D-ribose) 2. Phosphoric acid 3. Nitrogen containing heterocyclic compounds (called BASES) such • Guanine (G) • Cytosine (C) • Uracil (U) • Adenine (A)

II. ACID HYDROLYSIS OF RNA 1. Place small portion of RNA from yeast in a test tube. 2. Add about 10 mL of 10% sulfuric acid. Cover the test tube loosely and boil in water bath for 30 minutes. 3. Use the solution in no. 2 and the unhydrolyzed RNA (dissolved in water) for the following tests and compare results.

QUALITATIVE TESTS FOR RNA COMPONENTS TEST FOR RNA COMPONENTS To qualitatively detect the presence of RNA and it’s components, the following tests are used in this experiment: • Orcinol test • Ammonium molybdate test • Test for Purine bases • Benedict’s test

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Biochemistry (Laboratory) LESSON : ISOLATION OF RNA FROM YEAST ORCINOL TEST FOR RIBOSE - Bial’s Orcinol test = for PENTOSES PRINCIPLE: The orcinol reagent reacts with pentose groups in the backbone of the RNA and depends on the formation of furfural when the pentose is heated with concentrated hydrochloric acid. REAGENTS: • Orcinol in HCl • Ribose + HCl -> furfural + orcinol = (+) result POSITIVE RESULT: BLUE–GREEN PRECIPITATE PROCEDURE: 1. Place 1 mL dissolved RNA in a test tube and into another test tube with 1 mL of hydrolysate from number 2. Add 5 drops of orcinol reagent and heat in water bath. 3. Cool the tube immediately under the faucet and compare results.

TEST FOR PURINE BASES - Test for GUANINE and ADENINE PRINCIPLE: • Hydrolysis of N-β-glucosidic bonds between purine bases and ribose or deoxyribose results in a release of purine bases(A and G) caused by NH4OH. • Ag+ precipitate causes the formation of foamy gelatinous substance. REAGENTS: • AgNO3 • NH4OH POSITIVE RESULTS: WHITE TO TAN FLOCCULENT PRECIPITATE

AMMONIUM MOLYBDATE TEST - Test for INORGANIC PHOSPHATES

PROCEDURE: 1. Dissolve a pinch of the prepared RNA in 1 mL water and to another test tube, 1 mL hydrolysate. 2. Then add 5 drops of ammoniacal AgNO3. 3. Add 1 mL NH4OH and then mix. 4. Note the formation of a white or tan flocculent precipitate forms.

PRINCIPLE: Phosphate reacts with nitric acid and ammonium molybdate to form yellow precipitate of ammonium phosphomolybdate REAGENTS: • 6N HNO3, Ammonium molybdate reagent • 6N HNO3 + Nucleic acid (phosphate) + ammonium molybdate reagent -> ammonium phosphomolybdate precipitate POSITIVE RESULT: Canary yellow precipitate PROCEDURE: 1. Add excess NH3 to 1 mL of the test solution. 2. Acidify with 6N HNO3 3. Add 1 mL ammonium molybdate reagent and heat with water bath. Compare results.

BENEDICT’S TEST - Test to detect the PRESENCE OF REDUCING SUGARS PRINCIPLE: • Cu2+ react with reducing sugars forming precipitates of cuprous oxide (Cu2O) • This produces a change in the Benedict’s reagent from blue to green or reddish orange, depending on amount of reducing sugar. • The cyclic sugar is reduced to linear aldehyde.

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Biochemistry (Laboratory) LESSON : ISOLATION OF RNA FROM YEAST REAGENTS: • CuSO4, Na2CO4 • Ribose + CuSO4 => Ribonic acid + Cu2O

AMMONIUM MOLYBDATE TEST

POSITIVE RESULTS: BLUE to GREEN: small amount of reducing sugars BRICK-RED to ORANGE: abundance of reducing sugars PROCEDURE: 1. Neutralize 2mL of the test solution with Na2CO3 and decant. 2. To 1mL of the neutralized sample, add 1mL of Benedict’s reagent. 3. Heat in boiling water bath and note results.

HYDROLIZED RESULT:

RESULT:

TEST FOR PURINE BASES

HYDROLIZED WITH HEATING RESULT: Cloudy White RESULTS TO BE USED IN ACTIVITY SHEETS

UNHYDROLIZED

UNHYDROLIZED WITHOUT HEATING RESULT: Cloudy White

BENEDICT’S TEST

ORCINOL TEST

HYDROLIZED WITH HEATING RESULT: Green-Orange-Purple Precipitate

UNHYDROLIZED WITHOUT HEATING RESULT: Light Brown

HYDROLIZED WITH HEATING RESULT: Cloudy Light Blue

UNHYDROLIZED WITHOUT HEATING RESULT: Royal Blue

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Biochemistry (Laboratory) LESSON : DIGESTION AND METABOLISM ENZYMES BIOLOGICAL CATALYSTS - cells rely on since it would be impossible for living cells to control and coordinate their many biochemical reactions by adjusting the temperature ENZYMES - biological catalysts in cells are proteins - just about every biochemical reaction in a cell has its own specifically shaped enzyme catalyst - By controlling the production and activity of enzymes (which are encoded by genes): cells control and coordinate their biochemical activity i.e. their metabolism.

Factors that can denature an enzyme and cause it to become non-functional – changes in: • TEMPERATURE • PH • SALT CONCENTRATION FOR EXAMPLE: Most human enzymes have evolved to function best at normal cellular conditions: 37o C, pH 7.4 and 0.9% NaCl.

As with any catalyst, an enzyme works by binding and positioning the reactant(s) for a specific reaction in a way that lowers the activation energy.

If the temperature, pH or salt concentration deviates significantly from the “normal” state, enzymes and other proteins will begin to denature and lose their function. Ø This is largely why high fevers and deviations in pH (acidosis, alkalosis), for example, can be so dangerous.

SUBSTRATE - biochemical reactant(s) that a given enzyme binds to ACTIVE SITE - part of the enzyme that binds the substrate

DIGESTIVE ENZYME FUNCTION In this experiment, you will observe how three different digestive enzymes catalyze biochemical reactions that break down their substrates into smaller molecules.

The diagram below illustrates this for the enzyme sucrase and its substrate sucrose (the suffix –ase denotes an enzyme, whereas –ose denotes a carbohydrate):

LIST OF EACH ENZYME WITH ITS SUBSTRATE AND RESULTING PRODUCT(S)

Each of these digestive enzymes is produced in the PANCREAS as part of a cocktail of digestive enzymes in what we call “PANCREATIC JUICE”.

Enzyme sucrase, a protein, binds directly to its substrate sucrose and positions it so the covalent bond between the monosaccharides glucose and fructose is strained in a way that lowers the activation energy enough to break the bond. This yields the products glucose and fructose, which are then released. The enzyme is free to repeat this process, catalyzing the reaction over and over again until it is no longer active. Like any protein, the action of an enzyme is dependent upon its unique three-dimensional shape. Anything that causes an enzyme to adopt a nonfunctional shape is said to DENATURE THE ENZYME.

Pancreatic juice is released into the first section of the small intestine, the duodenum, when it receives partially digested food called CHYME from the stomach. Ø The enzymes contained in pancreatic juice will complete the chemical digestion of a meal so that the monomeric nutrients it contains (e.g., amino acids, monosaccharide sugars, fatty acids) can be absorbed. EXPERIMENT • Digestion of triglycerides by the enzyme lipase • Digestion of proteins by the enzyme trypsin or pepsin • Digestion of starch by the enzyme amylase

Biochemistry (Laboratory) LESSON : DIGESTION AND METABOLISM Enzyme: LIPASE Digestion of Triglycerides by the Enzyme Lipase PANCREATIC LIPASE - is produced by the pancreas to catalyze the breakdown of lipids such as triglycerides into free fatty acids and glycerol:

TRIGLYCERIDES - Main form of lipid found in animal fats (such as milk cream) and vegetable oils - Must be digested to fatty acids and glycerol via pancreatic lipase to be absorbed Since lipids are not soluble in water, they will form large droplets to minimize contact with the surrounding aqueous environment. This limits their interaction with lipase thus making their digestion very slow and inefficient. - To avoid this problem, the liver produces bile, a greenish fluid with properties similar to soap that helps to emulsify lipids, i.e., break them up into smaller droplets. BILE - is stored in the gall bladder until your partially digested meal reaches the duodenum. - This triggers the release of bile into the duodenum to help emulsify the lipids. In the experiment, digestion of triglycerides to fatty acids and glycerol can be detected by a decrease in pH As their name implies, fatty acids are acidic (they release H+ into solution) due to their carboxyl groups. The release of fatty acids from neutral triglycerides will thus result in an increase in the H+ concentration (i.e., lowering of the pH value). In the experiment, you will detect such changes in pH by using a pH indicator that changes color in response to pH.

Digestion of cream (TG) by lipase OBJECTIVE: To test the ability of pancreatic lipase to digest triglycerides in milk cream, both with and without bile powder. PRINCIPLE: • The pH indicator litmus has been added to the cream. Litmus is red under acidic conditions, blue under basic conditions, and is purple at neutral pH. • The “litmus cream” that will be used is light purple color (neutral pH) and will gradually turn reddish-pink if free fatty acids are released due to the digestion of triglycerides in the cream. PROCEDURE: 1. Label four test tubes 1, 2, 3 & 4 and add 2 ml of litmus cream to each tube. 2. Add a pinch (~0.02 g or 20 mg) of bile powder to tubes 2 & 4 only. 3. Add 0.5 ml of water to tubes 1 & 2 and 0.5 ml of 1% lipase solution to tubes 3 & 4. 4. Mix each tube well and incubate them in a 37C water bath for 1 hour. 5. Record the results on your worksheet and answer the associated questions. Enzyme: TRYPSIN Digestion of Proteins by the Enzyme Trypsin TRYPSIN - is one of many enzymes your body produces to digest or break down proteins. - will catalyze the breakage of peptide bonds in proteins at lysine and arginine amino acid residues. This results in larger polypeptides being broken down into smaller polypeptides (commonly referred to as “peptides”). Gelatin - protein source that will be used to observe trypsin digestion - consists primarily of the protein collagen extracted from animal bones and - other connective tissues. - At room temperature and below: is a semisolid gel due to interactions between the collagen fibers that form a fishnet-like structure. Trypsin will partially digest the collagen fibers, disrupting their interaction and causing the gelatin to liquefy and remain liquid, even at cool temperatures.

Biochemistry (Laboratory) LESSON : DIGESTION AND METABOLISM OBJECTIVE: To demonstrate the ability of trypsin to catalyze the partial digestion of gelatin. PRINCIPLE: If digestion of the gelatin has occurred, you will see that the gelatin remains liquid even on ice. PROCEDURE: 1. Obtain two tubes of molten gelatin from the 37o C water bath and label them 1 & 2. 2. Add 0.5 ml of water to tube 1 and 0.5 ml of 1% trypsin solution to tube 2. 3. Mix well and place both tubes in the 37o C water bath for 30 minutes. 4. Place both tubes on ice for 15 minutes (or long enough for tube 1 to solidify). 5. Invert each tube to see if the gelatin is liquid or solid and record on your worksheet. Enzyme: AMYLASE Digestion of Starch by the Enzyme Amylase STARCH - is a large polymer of the monosaccharide glucose. - In order for your body to obtain glucose from the starch you eat, it must be digested by the enzyme amylase.

AMYLASE - is present in human saliva as well as pancreatic juice. In the experiment, there will be three reaction tubes. Two reactions will serve as controls, one lacking the enzyme and the other lacking starch. The third reaction will contain both the enzyme amylase and its substrate, starch. OBJECTIVE: To demonstrate the ability of amylase to digest starch PRINCIPLE: • Starch can be detected by the addition of a small amount of iodine solution. If starch is present the sample will turn dark blue or black when iodine solution is added, if there is no starch then the sample should be a clear light brown color.

• The complete digestion of starch to free glucose should result in no dark blue or black color when iodine solution is added. The experiment will use iodine solution to determine if starch is digested by amylase. PROCEDURE: 1. Label three test tubes 1, 2 & 3. 2. Add 0.5 ml of water to tube 1 and 2.5 ml of water to tube 2. 3. Add 2.5 ml of starch solution to tubes 1 & 3. 4. Add 0.5 ml of 1% amylase solution to tubes 2 & 3. 5. Mix well and incubate each tube at 37o C for 10 minutes. 6. Add 3 drops of iodine to each tube, mix and record the results on your worksheet

Biochemistry (Laboratory) LESSON : CHEMICAL AND PHYSICAL TESTING OF URINE INTRODUCTION URINE TESTING: Physical and Chemical - May indicate presence of abnormalities or health condition - Helps in the diagnosis of diseases such urinary tract infections, kidney disorders, metabolic problem like diabetes, albuminuria, proteinuria or Hematuria or even presence of pregnancy PROPER COLLECTION OF URINE

• About 15 ml of midstream sample (especially in female patients) is cleanly collected. • Collect clean catch sample. 24-HOUR SPECIMEN - Quantitative estimation of proteins, hormones and electrolytes URINE SPECIMEN FIRST VOIDED SPECIMEN - Most concentrated and has acidic pH, so formed elements (cells and casts) are well preserved. - It is preferred for urinalysis. RANDOM SPECIMEN - Single specimen collected at any time of day and is sufficient for routine urine examination (though not preferred, it is the most frequently received specimen) POST-PRANDIAL SPECIMEN - (collected 2 hours after a meal in the afternoon) Sometimes requested for estimation of glucose or of urobilinogen. PHYSICAL EXAMINATION 1. Volume 2. Appearance 3. Odor 4. Specific gravity

VOLUME - Total volume can be evaluated only from 24-hour urine sample - Main determinant of urine volume is water intake NORMAL INDIVIDUAL: 24-HOUR URINARY OUTPUT: 600 to 2000 ml - out of which about 400 ml is produced during night (Reference: Todd and Stanford) EXCEPTIONS: PREGNANCY – diurnal variation may be reversed YOUNG CHILDREN – 3-4 times more urine than adults (ml/per body weight) Ø Decreased in volume might indicate health conditions URINE pH: from 5.5 to 7 - Increase pH may indicate presence of ammonia from urease producing bacteria Color: Clear - Hazy or turbid appearance indicates high concentration of urine, presence of crystals, or blood or pus cells Odor Specific Gravity

Biochemistry (Laboratory) LESSON : CHEMICAL AND PHYSICAL TESTING OF URINE FEHLING’S SOLUTION – B - Aqueous solution of sodium potassium tartrate - Clear and Colourless solution SUGAR 1. Place 10 drops each of Fehling’s A and B in a test tube then add 10 drops of urine sample. 2. Heat in boiling water for two minutes. 3. Observe a color change.

CHEMICAL TEST FOR NORMAL CONSTITUENTS OF URINE ANIONS: chlorides, sulfates, and phosphates CATIONS: sodium, potassium, ammonium, calcium, and magnesium CHEMICAL TEST FOR PATHOLOGIC OR ABNORMAL CONSTITUENT OF URINE • PROTEIN • KETONE BODIES • SUGAR • BILE PIGMENTS - found in normal urine in small quantities, too small to be detected by ordinary laboratory procedures. - They become pathologic when excreted in abnormal amounts. - Blood, however, when present in urine (except menstrual blood) is always pathologically significant. FEHLING’S TEST and BENEDICT’S TEST - Detects the presence of sugar in the urine FEHLING’S SOLUTION TWO DIFFERENT TYPES OF FEHLING’S SOLUTION FEHLING’S SOLUTION – A - Aqueous solution of copper II sulphate - Blue colour solution

BENEDICT’S SOLUTION - serves as a reagent in this test. - The reagent is a blend of copper, sodium citrate and sodium carbonate and copper II sulphate pentahydrate (CuSO4.5H2O) - Detects all reducing substances like glucose & fructose RESULTS: 5ml of benedict’s reagent in a test tube, add 8 drops of urine sample • BLUE GREEN – NEGATIVE • YELLOW GREEN – + (2%)

Biochemistry (Laboratory) LESSON : CHEMICAL AND PHYSICAL TESTING OF URINE PRINCIPLES: • When the urine sample is boiled with the two different reagents, the CuSO4 found in Benedict’s and Fehling’s solution is reduced by the reducing agent, glucose for the formation of a coloured cuprous oxide precipitate. • Depending on the glucose concentration, a yellow, green and brick-red formatio...