Biochemistry Laboratory Experiment 10: Nucleic Acids PDF

## BIOCHEMISTRY LABORATORY ### POST-LABORATORY DISCUSSION #### Experiment 18-D ### EXPERIMENT 10-NUCLEIC ACIDS **NUCLEIC ACIDS** - are polymers of high molecular weight mononucleotides as the repeating unit, that are widely distributed in plant and animals cell, attached to proteins = nucleoprot...

## BIOCHEMISTRY LABORATORY ### POST-LABORATORY DISCUSSION #### Experiment 18-D ### EXPERIMENT 10-NUCLEIC ACIDS **NUCLEIC ACIDS** - are polymers of high molecular weight mononucleotides as the repeating unit, that are widely distributed in plant and animals cell, attached to proteins = nucleoproteins **NUCLEOPROTEINS** - any member of a class of conjugated proteins in which molecules of nucleic acid are closely associated with molecules of protein, can be DNA or RNA. Nucleic acids are the ultimate blue prints of life. Combined with proteins and nucleic acids (made up of nucleic acids & proteins). The building blocks of nucleic acid are nucleotides. Nucleotide is the first products of hydrolysis of the nucleic acids. Nucleic acids can be hydrolyzed in steps or completely by enzymes or inorganic catalyst. The Important role of nucleic acid plays in the transmission of hereditary traits, in the control of the functioning of living cells, and in the synthesis of proteins. Nucleic acids are defined as those complex biochemical substances which yield, upon hydrolysis, purine and pyrimidine bases, ribose or dioxyribose, and phosphate. They are found in all living cells where they are usually present at a level of less than 1% of the total net weight of the cell. Yeast cells and some bacterial cells may contain up to 5% nucleic acid. The bacteriophages may contain as much as 60% DNA. It has been estimated that if all the DNA in an average adult human were uncoiled and laid out in a straight line it would reach five billion miles. ### 2 MAJOR TYPES; * **deoxyribonucleic acid (DNA)** -located in the cell nucleus and present in the same amount in all cells, which is primarily responsible for the transfer of genetic information. * **ribonucleic acid (RNA)** -located in the cytoplasm and present in widely varying amounts, responsible for proteins synthesis. ### PROPERTIES: * Nucleic acids are large, acidic, chainlike molecule containing phosphoric acid, sugar and purine and pyrimidine bases. * Nucleic acids are polymer of mononucleotide * Mononucleotide is composed of base, sugar and phosphate. -Nucleoside is base + sugar * insoluble in alcohol, slightly in cold water * readily soluble in hot water and dilute alkalies * precipitate by HCI (hydrochloric acid) and excess acetic acid * Nucleic acid is catalyzed by acids, bases, or enzymes. Enzymes which catalyze the hydrolysis of only RNA. * Nucleic have high molecular weight, and it is accurate to say that nucleic acid are probably the largest molecules known. ### Data & Results: (yeast used in experiment as a source of nucleoproteins) 1. **TEST FOR NUCLEOPROTEINS** * Reagent: 10% NaOH & 1% CuSO4 * filtrate = creamy yetirmish brown in calor * violet color = contains proteins, therefore. form (+) reaction to Biuret reagent * NaOH (Sodium hydroxide) will make the nucleoprotein soluble forming salts 2. **MILD ACID HYDROLYSIS** (filtrate & 10% H2 SO4) * **TEST FOR PHOSPHATES** * Reagent: 10% HNO3 & 5% (NH4)2 M004 * (+) formation of yellow precipitate * **TEST FOR RIBOSE** * Reagent: Bial' Orcinol * production of green solution and precipitate indicating the presence of ribose * Test solution from acid mild hydrolysis * 0.1% ribose solution * 0.1% glucose solution * Green solution * Green solution * Yellowish or brownish solution * **TEST FOR PURINE** * Reagent: 10% NH4 OH & 5% AgNO3 * (+) formation of white precipitate ## Answers to Questions: 1. What is the basic structure of nucleic acids? Give its basic components The basic structure of nucleic acid is the nucleotide. The basic components are: * Phosphates * Nucleosides - such as D-ribose; D-deoxyribose; -pyramidine; and purines. 2. Give 4 basic differences between DNA & RNA Based on their functions, DNA is responsible for the storage and transmission of the genetic material while RNA is involved in the manufacture of proteins Based on their sugar composition. DNA contains D-deoxyribose while RNA has D-ribose Based on their pyramidine base composition. DNA contains thymine while RNA has uracil. Based on their structure. DNA has a double helix while RNA is made up of single strand. 3. How did Watson & Creek describe the basic structure of DNA? The molecule as proposed by J.D. Watson and F.H. Creek in 1953, consists of two parallel polynucleotide chains wrapped around common axis into helix and held together by hydrogen bonding between nitrogen bases spaced along the chain. The polynucleotide chain or nucleic acids, is formed by the repetitious union of the nucleotide unit, a unit formed by joining together a molecule of the pentose sugar deoxyribose, a molecule of phosphoric acid and a molecule of nitrogen base, thymine. ## EXPERIMENT NO. 11 ENZYMES **ENZYMES** are biologic catalysts that increase the speed of a chemical reaction but do not themselves change. -any of a group of catalytic proteins that are produced by living cells and that mediate and promote the chemical processes of life without themselves being altered or destroyed. Enzymes, we have said, are proteins that act as enormously effective catalysts. are proteins. The enzymes in the body function best at about 40°C temperature above or below body temperature will decrease the activity of enzymes. Each enzyme has a certain pH at which it can function best. An increase in the amount of enzyme will increase the rate of reaction. An increase in the amount of substrate will increase the rate of the reactions. (substrate the substance with which an enzyme reacts) a compound with which a reagent reacts. Compounds that increase the activity of an enzyme are called activators. Compounds that interfere with the activity of an enzyme are called inhibitors. Enzymes contain an "active site" that binds to the substrate to form an enzyme- substrate complex. This complex yields the products and regenerates the enzymes. Many enzymes contain two parts - a protein and a non protein part. The protein part of an enzyme is called the apoenzyme. Some enzymes require the presence of a substance called a coenzyme before they can act effectively. Coenzymes frequently contain the B vitamins or compounds derived from the B vitamins. Under the older system of naming enzymes the substrate was not mentioned; the newer system indicates the substrate being acted on. The names of enzymes under this system end in -ase. Some hydrolytic enzymes are found in the lysosomes of the cytoplasm. The cytoplasm also contains the mitochondria. These structural and functional units contain the most of the oxidative enzymes and are deeply involved in the electron transport system of oxidation - reduction. The mitochondria also produce ATP, the cells' chief source of energy. Abnormal plasma enzymes concentrations are of clinical significance in the diagnosis of certain disease. Isozymes are enzymes with the same functions but slightly different structural features. Allosteric enzymes are key metabolic enzymes whose activity can be changed by molecules other than the substrate. Zymogens are the precursors of enzymes. ### Data & Results: (from potato extract) * **Preparation of Catalase Enzyme** * **PROTEIN NATURE OF ENZYME (BIURET TEST)** * Reagent: Sodium hydroxide (NaOH) and Copper sulfate (CuSO4) * (+) blue violet solution * **CATALASE ACTIVITY TEST** * Reagent: 3% Hydrogen peroxide (H2O2) * -extract + hydrogen peroxide (H2O2) -----→ H2O + O2 * (+) oxygen support combustion of glowing splint * CATALASE an enzyme that catalyzes the decomposition of hydrogen peroxide into molecular oxygen and water. Hydrogen peroxide (H2O2) is used as a substrate (found in blood mucous membrane, liver and kidney) * **BENZIDINE TEST** - test for dissolved O2 * Benzidine formation of blue green color same with peroxidase (found in milk, plants, WBC or leukocytes) * (+) blue green solution * **TEST FOR SPECIFICITY OF ENZYME ACTION** * 2 test tube containing the mixture of 0.02M Phosphate buffer and 0.9% NaCl (NSS) * - Cooked starch + salivary amylase + iodine solution - turn to yellow solution to colorless. This means that starch has been completely hydrolyzed to glucose. * The high specificity of enzymes is due to its protein nature * AMYLASE an enzyme that hydrolyzes reserve carbohydrates, starch in plants and glycogen in animals. * Human enzymes has a pH 7 ## EXPERIMENT 12-URINE ### FORMATION OF URINE Blood flows to the kidneys through the renal arteries. From the renal arteries the blood passes into the arterioles and then into capillaries of the kidneys. These capillaries coil up to form a glomerulus, a rounded ball capillaries. Around the glomerulus is a structure called Bowman's capsule. Each Bowman's capsule is connected by a tubule to a large tube, which in turn carries the urine to the bladder, where it is stored until it is excreted. As blood flows into the kidney, the various soluble components diffuse into the glomeruli (there are over a million glomeruli in each kidney). The protein material in the blood cannot pass through the membrane (Recall that proteins are colloids, and colloids do not pass through membranes). The driving force for this diffusion of fluid through the walls of the glomerulus is the blood pressure. The liquid in the glomerulus thus has approximately the same composition as blood plasma except for the protein material. As the fluid in the glomerulus passes down the tubule, a large proportion of the water is reabsorbed into the bloodstream. Also reabsorbed are the glucose, amino acids, and the most inorganic ions. The remaining liquid containing urea and other waste products flows to the collecting tubules and to the bladder. Thus, the kidneys act as a very efficierit filter, removing the waste materials but not needed nutrients from the blood. Approximately 1 liter of blood is filtered through the kidneys every minute. Of this amount, most of the water is reabsorbed, so the amount of urine excreted per day is less than 1 percent of the total amount of liquid filtered. If the kidneys are not functioning normally, an artificial kidney machine may be used. | Constituent | Blood Plasma | Urine | |---|---|---| | Water | 90-93 | 95 | | Protein | 7 | 0 | | Sodium | 0.3 | 0.35 | | Glucose | 0.09 | 0 | | Ammonia | 0.004 | 0.05 | | Phosphate | 0.009 | 0.5 | | Urea | 0.03 | 2.0 | | Sulfate | 0.002 | 0.18 | ### GENERAL PROPERTIES OF URINE #### VOLUME Approximately 600 to 2500ml of urine is excreted daily, the amount depending on the fluid intake, weather condition, humidity, and certain diuretic substances. Drugs such as caffeine (in coffee or tea) and alcoholic beverages have a diuretic effect; that is, they increase the flow of urine * Oliguria - a decreased flow of urine; such a condition may occur during a high fever when most of the water lost by the body in the form of perspiration. Certain kidney diseases may also cause oliguria. * Anuria - means a total lack of urine excretion. Anuria indicates extensive kidney damage such as may be caused by blood transfusion of the wrong type. In this condition the blood cells disintegrate, releasing hemoglobin, which clogs the glumeruli and does not allow any excretion of urine. * Bichloride of mercury also affects the kidney and may cause oliguria or anuria. * Polyuria -is a condition in which the amount of urine excreted is much greater than normal. It may be due to excessive intake of water or to certain pathologic conditions. Polyurla may be caused by such diuretics such as alcohol or caffeine. Urea, a normal constituent of urine, is also diuretic. * A person on a high-protein diet will excrete more urea, which in turn causes the formation of more urine. #### DENSITY The density of urine varies between 1.003 and 1.030 g/ml. The density of urine depends on the concentration of the solutes. The greater the concentration of solutes, the greater the density.in cases of diabetes mellitus, the density will be higher because of a high concentration of sugar in the urine. In cases of diabetes insipidus, the density will be very low (close to 1.000 g/ml) because of the large amount of water being excreted. #### PH Urine is normally slightiy acidic, with a pH range of 4.0 to 8.0 and average value of about 6.3.however, the pH of urine varies with the diet. Protein foods, such as meats, increase the acidity of the urine (lower the pH) because of the formation of phosphates and sulfates. The acidity of the urine is also increased during acidosis and with fever. Conversely, the urine may tend to become alkaline on a diet high in vegetables and fruits or because of alkalosis, a condition that may be produced by excessive vomiting. #### COLOR Normal urine is pale yellow or amber. The color, however, varies with the amount of urine produced and also with the concentration of solutes in the urine. The larger volume of urine of urine excreted, the ligther the color. The greater the concentration of solutes, the darker the color. The color of urine is caused by urobilin and urobilinogen. Freshly voided urine is clear and usually contains no sediment. However, when it stands for a while it may become cloudy and develop sediment because of the precipitation of calcium and phosphate. #### ODOR Fresh urine has a distinctive odor, but this odor may be modified by the presence of other substances. Diet can also modify the odor of urine. #### Data and Result: URINE - ultra filtrate of blood "same composition of plasma without CHON" * Mainly H2O 99% H2O * 3 % solids (UREA), * salts. ions (Na, K, Cr) #### KIDNEY - main organ of excretion. Made up of nephron (functional unit of the Kidney) **REACTIONS:** 1. Excretion of waste products of metabolism /include toxic waste 2. Maintain H2O balance (acid-base) 3. Detoxification of the body | General Characteristics: | NORMAL | ABNORMAL | |---|---|---| | COLOR | urochrome | red-Hb, black - malaria | | a. Straw to amber | Mobella | heat | | b. Urea | Urinetyron | pungent urea | | (aromatic odor) | upon long stand | NH3 | | c. Transparency (clear) | Sweet smell (ICE, VAL, IEU) = Offensive odor = bacterial infection | urinod | | d. Reaction | 4.5 to 8.0 does not reach > 9.0 (specimen is improperly preserved) ABN. Acidosis of renal tubal = 6.5 or up | | | e. Specific gravity | determine Urine concentrating ability 1.015to 1.025 not greater than 1.032 | refract /TS meter Affected by CHON/glucose 1.010 failure of kidney to excrete | #### CHEMICAL EXAMINATION **GLUCOSE** * Reducing test * I.FEHLING'S * 2.BENEDICT'S * 3.NYLANDER'S * Sp. = (+) glucose = DM (diabetes mellitus) * (-) = oxidase * RESULTS * (+) reducing sugar---→brick red precipitate (glucose, pentose, fructose) * used to test for sugar in urine (galactose, lactose) Sign in pregnancy **ALBUMIN** * CHON = proteins * N= nitrogen * 1. HEAT * HAC SSA→ color ppt. * 2. Heller's Test (concentrated nitric acid) * 3. Exton's Test (Na₂SO₄ & sulfosalicylic acid) * **Test Albumin** * RESULTS * = proteinuria * = can proteinuria (aesthethic, CHF) * = precipitate by acid - can detect albumin * (+) presence of white zone (Sulfosalicylic Acid Test) * Turbidity or precipitate denotes the presence of albumin * **Test for Urea** * Urea is the main end product of protein metabolism in man and other animals. The ammonia released from the deamination of amino acids is removed through the urea cycle which occurs in the liver. The urea formed is then carried by blood to kidneys where it is excreted. * **Test for Uric Acid** * Uric acid is the final product of purine oxidation in the body. It is derived from the degradation of nucleoproteins in food and of body cells. * **Test for Creatinine** * Urine creatinine comes from creatine of the muscles. An abnormal concentration of urine creatinine is related to muscle wasting diseases. * The test for creatinine depends upon the reaction of creatinine with picric acid forming an orange compound. ## Multiple Choice Questions 1. A total lack of urine excretion indicative of excessive kidney damage. * * Oliguria * Polyuria * **Anuria** * Ketonuria 2. Normal yellow is bright yellow. * * True * **False** 3. Is the main organ for excretion. * * skin * **kidney** * liver * ureter 4. What is the final product of purine oxidation in the body. * * albumin * urea * **uric acid** * creatinine 5. What is the main product of protein metabolism? * * albumin * **urea** * uric acid * creatinine 6. What is the positive reaction for Fehling's test? * * brown precipitate * white precipitate * yellow precipitate * **brick-red precipitate** 7. What is the positive reaction to Heller's Test using concentrated nitric acid. * * **presence of white zone** * presence of grey ring * presence of white precipitate * presence of bubbles 8. Is a condition in which the amount of urine excreted is greater than the normal. * * Oliguria * **Polyuria** * Anuria * Ketonuria 9. This test denotes the presence of albumin in urine. * * Heller's test * **Exton's test** 10. This test is used to test for sugar in urine (galactose, lactose) which is a sign of pregnancy. * Heller's test * Exton's test * Nylander's test * **Benedict's test** 11. This condition is characterized by a decreased flow of urine. * * **Oliguria** * Polyuria * Anuria * Ketonuria 12. The density of urine varies between _______ g/mL. * * 1.001-1,010 * **1.002-1.020** * 1.003-1.030 13. The pH of urine range between ______ to ______. * * 3.0 to 7.0 * **4.0 to 8.0** * 5.0 to 9.0 * 6.0 to 10.0 14. How do enzymes speed up chemical reaction? * * Increasing activation energy * Increasing deactivation energy * **Decreasing activation energy** * Decreasing deactivation energy 15. What is a substance called if it speeds up a chemical reaction? * * reusable * specific * **catalysts** * fragile 16. Each enzyme only has 1 substrate that will fit its active site. What is this called? * * **specificity** * reusability 17. With what 3 letters do enzymes typically end? * * -ose * -ese * **-ase** * -tic 18. Any substance that is acted upon by an enzyme is called a(n)? * * coenzyme * apoenzyme * **substrate** * inhibitor 19. Compounds that increase the activity of an enzyme are called ______. * * coenzyme * apoenzyme * **inhibitors** 20. Some enzymes require the presence of a substance called a ______ before they can act effectively. * coenzyme * apoenzyme * inhibitors * **activators** 21. The protein part of an enzyme is called the * * coenzyme * **apoenzyme** * inhibitors * activators 22. ____ are the precursors of enzymes. * * Isozymes * **Zymogens** * Apoenzymes * Allosteric enzymes 23. Positive reaction for BIURET Test. * * red solution * yellow solution * green solution * **blue-violet solution** 24. Human enzymes has pH ______. * * 3 * 5 * **7** 25. Positive reaction for BENZIDINE Test (test for dissolved oxygen) * red solution * yellow solution * **blue-green solution** * blue-violet solution 26. ____ are enzymes with the same functions but slightly different structural features. * * Isozymes * Zymogens * Apoenzymes * Allosteric enzymes 27. ___ are key metabolic enzymes whose activity can be changed by molecules other than the substrate. * Isozymes * Zymogens * Apoenzymes * **Allosteric enzymes**