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Biochemistry Journal

Biochemistry Journal

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Subcellular frationation, cell membrane permeability and Osmosis
Subcellular frationation, cell membrane permeability and Osmosis

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Published by: Gian Angelo Arcellana Villalon on Mar 10, 2013
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Subcellular Fractionation

I. Background of the experiment
Organelles are membrane-enclosed vesicles inside all eukaryotic cells that function in a specific way and a variety of important cellular processes. The basic principle for all microscopes is that the cell is composed of smaller physical units, the organelles. Definition of the organelles is possible with microscopy, but the function of individual organelles is often beyond the ability of observations through a microscope. We are able to increase our chemical knowledge of organelle function by isolating organelles into reasonably pure fractions. A host of fractionation procedures are employed by cell biologists. Each organelle has characteristics (size, shape and density for example) which make it different from other organelles within the same cell. If the cell is broken open in a gentle manner, each of its organelles can be subsequently isolated. The process of breaking open cells is homogenization and the subsequent isolation of organelles is fractionation. Isolating the organelles requires the use of physical chemistry techniques, and those techniques can range from the use of simple sieves, gravity sedimentation or differential precipitation, to ultracentrifugation of fluorescent labeled organelles in computer generated density gradients.

Subcellular Fractionation involves the separation of different cell components from homogenous sets. usually organelles done by subjecting the cells to differential centrifugation The arrangement of macromolecules within a cell is as important to cellular function as their catalytic activities. With this technique. In this lab module. we will isolate several subcellular organelle fractions from liver cells. The supernatant is then transferred to another tube and centrifuged longer to pellet the lighter mitochondria. . Cellular compartmentalization is accomplished in part by various subcellular organelles. and will examine their various properties. a cell homogenate is made by rupturing the cell membranes in the tissue.e. the heaviest or most dense organelles. Cellular compartmentalization provides efficiency by bringing together related compounds that can interfere with each other (i. First. The homogenate is then centrifuged for a short period of time to remove cell debris and nuclei. nuclei pellet in less time and with less force than is required to pellet lighter organelles such as mitochondria. The method we will use to separate the various organelles utilizes differential centrifugation to isolate components of different densities. lysosomal hydrolytic enzymes).

 . keeping them intact. Some tissues. It is very difficult to get pure unbroken preparations of any organelle. making their use uncertain during winter months. Techniques providing optimal isolation of one organelle may completely rupture another organelle. The homogenization buffer is a solution which often includes sucrose to partially dehydrate the organelles. so are also well suited. Thus methods are often used to measure the contamination of one organelle fraction by another. and the method of homogenization are dictated by the biological system. but recently-harvested plant tissues are usually required. Homogenizers have a precise clearance between the glass tube and pestle. like those in the liver also have one cell type that predominates. No technique used to isolate organelles is perfect. it is important to obtain the organelles in a biochemically active. which tissue we use. Most chlorophyll-free plant tissues are acceptable for preparing mitochondria. This can be done by analyzing each organelle fraction for organelle-specific marker enzymes. Homogeneous cell populations from cell culture are well suited for cell fractionation. For this type of fractionation. Once a cell type is chose (we will work with liver). which breaks the cell membrane leaving the smaller organelle membranes intact. Homogenizers are used to break open the cells without damaging the organelles. morphologically whole state.

proteins.PROCEDURE FOR SUBCELLULAR FRACTIONATION: Cell lysis or extraction of the cell components Cell lysis or cellular disruption is a cell biology method for the release of biological molecules including organelles. Mild surfactants are often used in addition to mechanical lysis methods. RNA and lipids from inside a cell For most cells. DNA. . This is done by lowering the ionic strength of the surrounding solution. mild osmosis is usually enough to lyze the cells. causing the cells to swell and burst releasing their contents.

Cell Homogenization Homogenization involves in breaking open the cells and releases the cellular components into the resultant homogenate. It is also a process in which a mixture is made uniform throughout. Generally this procedure involves reducing the size of the particles of one component of the mixture and dispersing them evenly throughout the other component.PROCESS: Cut 10 g of fresh chicken liver and lyse by soaking in distilled water or tap water for at least one hour. PROCESS: Homogenize the cells using normal saline solution in an approximately 1:3 ratio .

The homogenate is then subjected to repeated centrifugations.Purification or Differential centrifugation Differential centrifugation is a common procedure in microbiology and cytology used to separate certain organelles from whole cells for further analysis of specific parts of cells. each time removing the pellet and increasing the centrifugal force. purification may be done through equilibrium sedimentation. Finally. In the process. size and shape. a tissue sample is first homogenized to break the cell membranes and mix up the cell contents. and the desired layer is extracted for further analysis. . It also separates the individual components within the homogenate according to density.

Golgi and plasma membrane fragments .PROCESS: Supernatant 1: nuclear fraction contains the nuclei and any unbroken cells Supernatant 2: mitochondrial fraction contains the mitochondria. lysosomes and microbodies Supernatant 3: ribosomal fraction contains the ribosomes and microsomes consisting of endoplasmic reticulum.

II. CHEMICAL PRICIPLE OF THE DIFFERENT QUALITATIVE TEST TEST FOR CARBOHYDRATES Molisch Test PRINCIPLE: Molisch test is a sensitive chemical test for the presence of carbohydrates, based on the dehydration of the carbohydrate by sulfuric acid to produce an aldehyde, which condenses with two molecules of phenol (usually α-naphthol, though other phenols (e.g. resorcinol, thymol) also give colored products) resulting in a red- or purple-colored compound.

TEST FOR LIPIDS Sudan Test PRINCIPLE: Sudan test is a test for lipids such as fats and oils based on their ability to selectively absorb pigments in fat-dyes such as the Sudan IV. The test will make red globs appear if it is indeed a lipid. To test for the presence of lipids in a solution you will use a Sudan IV Test. In this test dark red Sudan IV is added to a solution along with ethanol to dissolve any possible lipids. If lipids are present the Sudan IV will stain them reddish-orange, giving a positive test.

TEST FOR NUCLEIC ACIDS Bial’s test PRINCIPLE: Bial’s test is a chemical test for the presence of pentoses. The components include orcinol, hydrochloric acid, and ferric chloride. In the presence of a pentose, the pentose will be dehydrated to form furfural. The solution will turn bluish and a precipitate may form.

The test reagent dehydrates pentoses to form furfural. Furfural further reacts with orcinol and the iron ion present in the test reagent to produce a bluish product.

POSITIVE RESULT: violet in color . POSITIVE RESULTS MOLISCH TEST Adding 3 ml of Molisch reagent and 3ml of freshly prepared H2S04 POSITIVE RESULT: appearance of violet ring BIURET TEST 0.5% Cu S04.5 ml of 0.5 ml cell suspension add 0.5 ml of 10% NaOH and 0.III.

5 ml of cell suspension add 0. POSITIVE RESULT: blue green in color .5 ml of cell suspension then heat in water bath for 10 mins.SUDAN TEST 0.5 ml of chloroform and a pinch of Sudan IV crystals POSITIVE RESULT: red in color BIAL’S TEST Add 0.5 ml of Bial’s reagent to 0.

Cell fractionation also allows researchers to prepare specific cell components in bulk for research in the functions and structures of cell organelles. by centrifugation a specific cell fraction was determined to have enzymes that function in cellular respiration. SIGNIFICANCE AND APLLICATION OF THE EXPERIMENT Scientists use this tool to increase their knowledge of organelle functions. This unknown cell fraction was rich in mitochondrias. To be able to do so they isolate organelles into pure groups. .IV. For example. This method has already resulted in the knowledge several cell organelles' functions. Therefore there researchers obtained evidence that helped determine mitochondrias was the site of cellular respiration. such as isolating the mitochondria or the nucleus.

Cell Membrane Permeability and Osmosis .

I.Background of the Experiment The cell membrane is a biological membrane that separates the interior of all cells from the outside environment. The cell membrane is selectivelypermeable to ions and organic molecules and controls the movement of substances in and out of cells. It consists of the phospholipid bilayer with embedded proteins. Cell membranes are involved in a variety of cellular processes such as cell adhesion, ion conductivity and cell signaling and serve as the attachment surface for the extracellular glycocalyx and cell wall and intracellular cytoskeleton. Fluid mosaic model According to the fluid mosaic model of S. J. Singer and Garth Nicolson, the biological membranes can be considered as a two-dimensional liquid where all lipid and protein molecules diffuse more or less easily. This picture may be valid in the space scale of 10 nm. However, the plasma membranes contain different structures or domains that can be classified as: (a) proteinprotein complexes; (b) lipid rafts, and (c) pickets and fences formed by the actin-based cytoskeleton.

Lipid bilayer Lipid bilayers go through a self assembly process in the formation of membranes. The cell membrane consists primarily of a thin layer of amphipathic phospholipids which spontaneously arrange so that the hydrophobic "tail" regions are shielded from the surrounding polar fluid, causing the more hydrophilic "head" regions to associate with the cytosolic and extracellular faces of the resulting bilayer. This forms a continuous, spherical lipid bilayer. Forces such as Van der Waal, electrostatic, hyrdogen bonds, and noncovalent interactions, are all forces that contribute to the formation of the lipid bilayer. Overall, hydrophobic interactions are the major driving force in the formation of lipid bilayers

Membrane Transport Passive transport involves carriers, channels, or direct diffusion through a membrane. This type of transport always operates from regions of greater concentration to regions of lesser concentration. No external source of energy is required. Types: · Simple diffusion - a substance passes through a membrane without the aid of an intermediary such as a integral membrane protein. · Channel diffusion · Facilitated diffusion - it involves proteins known as carriers, which, as in the case of ion channels, are specific for a certain type of solute and can transport substances in either direction across the membrane. However, unlike channels, they facilitate the movements of solutes across the membrane by physically binding to them on one side of the membrane and releasing them on the other side.

It takes advantage of a previously existing concentration gradient (via carriers). across a semi-permeable membrane (permeable to the solvent. without input of energy. like diffusion. but is a passive process.Active transport involves the use of proteins that don't just passively facilitate the transport of substances across the cell membrane. Osmosis releases energy. Sodium pump · Secondary active transport– does not directly use ATP. but not the solute) separating two solutions of different concentrations. More specifically. There are two types of active transport: · Primary active transport – directly uses ATP ex. . it is the movement of water across a selectively permeable membrane from an area of high water potential (low solute concentration) to an area of low water potential (high solute concentration). but require the use of cellular energy (usually ATP) to actively pump substances into or out of the cell. It may also be used to describe a physical process in which any solvent moves. and can be made to do work. Osmosis Osmosis is the movement of water molecules through a selectively-permeable membrane down a water potential gradient. This type of transport operates from region of lesser concentration to regions of greater concentration.

Chemical Principle of Each Test Osmosis A.. Hemolysis may be produced in the laboratory by various physical agents (heat. sound). by chemicals. a condition called erythroblastosis fetalis. freezing. or Rh incompatibility of fetal and maternal blood. exposure to cold causes self-produced hemolyzing agents to destroy the individual’s own red cells . transfusion of the wrong blood type. in certain situations it is used as a specific laboratory test for antigen–antibody reactions. Apart from normal breakdown of aged red blood cells. thalassemia). Hemolysis caused by physical agents is rare in the living because of body buffering systems.II. breakdown or destruction of red blood cells so that the contained hemoglobin is freed into the surrounding medium. flooding with water. hemolysis is abnormal in the living but may be caused by inherited defects in the blood cells (e. venoms.g. hereditary spherocytosis. the toxic products of microorganisms. Antibody (lysin) attaches to the red cell but cannot cause bursting in the absence of a normal blood component called complement. but in the disease paroxysmal cold hemoglobinuria. Hemolysis Hemolysis. It is a major finding in hemolytic anemia.

or crenate.A condition or property of a solution in which its solute concentration is the same as the solute concentration of another solution with which it is compared. If water molecules continue to diffuse into the cell. If water molecules continue to diffuse out of the cell. it will cause the cell to shrink.is a solution having a lesser concentration of impermeable solutes on the external side of the membrane.a solution having a greater concentration of impermeable solutes on the external side of the membrane. When a cell’s cytoplasm is bathed in a hypotonic solution the water will be drawn out of the solution and into the cell by osmosis. When a cell’s cytoplasm is bathed in a hypertonic solution the water will be drawn into the solution and out of the cell by osmosis.Osmotic Pressure Osmotic pressure is the pressure which needs to be applied to a solution to prevent the inward flow of water across a semipermeable membrane. .  Hypotonicity . up to the point that cytolysis (rupture) may occur  Isotonicity . it will cause the cell to swell. Tonicity:  Hypertonicity .

and temperature of the molecules or solutes on either side. . How the membrane is constructed to be selective in its permeability will determine the rate and the permeability. Depending on the membrane and the solute. permeability may depend on solute size. as well as the permeability of the membrane to each solute. solubility. or chemistry.Permeability The rate of passage depends on the pressure. properties. concentration.

The membrane is constructed such that it allows the passage of certain charged components(ions) of the solutions. Willard Gibbshad predicted the effect some 30years before. The concentration of those ions that can pass freely though the membrane is the same on both sides of the membrane. As well. As a result.who proved its existence in biological cells. the total number of charged molecules on either side of the membrane is equal. however.Gibbs Donnan Equilibrium Donnan equilibrium (which ca also be referred to as the Gibbs-Donnan equilibrium) describes the equilibrium that exists between two solutions that are separated by a membrane. Donnan equilibrium is named after Frederick George Donnan . The impermeability of the membrane is typically related to the size of the particular ion. The two solutions vary in osmotic pressure. The membrane. A consequence of the selective permeability of the membrane barrier is the development of an electrical potential between the two sides of the membrane. the passage of some ions across the membrane will be promoted . with one solutionhaving more of a certain type(species) or types of ion that does the other solution. Anion can be too large to pass through the pores of the membrane to the other side. does not allow the passage of all the ions present in the solutions and is thus a selectively permeable membrane. J.

Osmotic Pressure TEST TUBE NUMBER #1 whole blood + 0. Positive Results Osmosis Hemolysis Hemolysis occured after 5 more drops of distilled water.9% NaCl Isotonic TEST TUBE NUMBER #3 whole blood + 10% NaCl Hypertonic         .III.25% NaCl Hypotonic TEST TUBE NUMBER #2 whole blood + 0. The supertanant becomes clear red.

2g sucrose+ 2ml whole blood sucrose cant enter the cell .Permeability   Test tube 1: 5 ml NSS + 2ml whole blood Physiological concentration   Test tube 2: 5 ml NSS +0.2g urea/glucose+ 2ml whole blood Glucose enters cell    Test tube 3: : 5 ml NSS +0.

Gibbs Donnan Equilibrium     Gelatin – impermeable substance Colloidal bag – semipermeable membrane Phenolphthalein – indicator NaOH – permeable substance Cl concentration gradient electrochemical gradient concentration gradient K electrochemical gradient .

it doesn't control what goes in and out (the nucleus does that) but it can see everything which goes in and out. Osmolsis  In Plants: 1. This means pH and other ion concentrations need to be tightly regulated. so a semi permeable membrane allows for regulation of these concentrations via protein channels. Without the cell membrane the cell would distort and probably mix in with other cells. 2. Cells need to maintain a very specific environment to function properly. Opening and closing of stomata.Absorbption of water from the soil.Fresh water animals have to maintain their osmotic pressure with the external environment. Significance and Application of the Experiment Cell membrane Cell membrane is what keeps the shape and size of the cell. Remember that even a small change in pH can cause proteins to lose their secondary and tertiary structure. Thus they are either having a contractile vacuole (as in amoeba) .  In Animals: 1.IV.

. In addition to the effect of Gibbs-Donnan equilibrium on Na+ distribution between plasma and ISF. our model predicts that the altered distribution of osmotically active nonNa+ ions will also have a modulating effect on the [Na+]pw by affecting the distribution of H2O between the plasma and ISF. The new physiological insights provided by this model can for the first time provide a basis for understanding quantitatively how changes in the plasma protein concentration modulate the [Na+]pw. this model defines all known physiological factors that may modulate the [Na+]pw and is especially helpful in conceptually understanding the pathophysiological basis of the dysnatremias. and the plasma water Na+ concentration ([Na+]pw) and validated the model using empirical data from the literature. The new model can account for the alterations in all ionic concentrations (Na+ and non-Na+ ions) between the plasma and ISF due to Gibbs-Donnan equilibrium. We have derived a new mathematical model to define the quantitative interrelationship between the Gibbs-Donnan equilibrium. impermeant proteins in the plasma space alters the distribution of diffusible ions in the plasma and interstitial fluid (ISF) compartments to preserve electroneutrality.Gibbs donnan equilibrium The presence of negatively charged. Moreover. the osmolality of body fluid compartments.

DNA Extraction From Kiwi Fruit .

A single subunit of DNA is called a nucleotide and consists of a nitrogen-containing base. a sugar. Background of the Experiment: DNA is in the cells of all living organisms. It determines the structure. function and behavior of the cell/organism. and two chains are paired together and twisted into a double helix to form the DNA finished molecule. DNA is essentially the blueprint for an organism. Hundreds of thousands of nucleotides are hooked together to form a chain.I. It encodes genetic information in the nucleus of cells. and a phosphate group. This procedure is designed to extract DNA from kiwi fruit in sufficient quantity to be seen and spooled. from bacteria to humans. . Deoxyribonucleic acid is the genetic material present in all organisms.

Chemical Principle of Each Test: Test for Ribose (Molisch Test) The Molisch test uses concentrated sulfuric acid as the dehydrating acid. Carbohydrate  dehydration product  purple product Test for Phosphates Phosphate Test gave out a yellow result for the precipitate which simply means that a phosphodiester bond exists between DNA and RNA between the 3' Carbon atom and the 5' Carbon of the ribose sugar. . furfural or 5-hydroxymethylfurfural. The dehydration products of carbohydrates.II. result from the reaction of the sulfuric acid with pentoses and/or hexoses. so the test is used to distinguish between carbohydrates and non-carbohydrates. This acid dehydrates all carbohydrates. These products condense with α-naphthol to yield a purple condensation product.

. which produces a green coloration when the sample is treated with bromine water. The addition of barium hydroxide will turn the liquid purple.Test for Purines (Murexide Test) A reaction giving rise to murexide when uric acid or a related compound is heated with nitric acid and the product is treated with ammonia—called also murexide reaction Test for Pyrimidines (Wheeler-Johnson Test) Wheeler-Johnson is a qualitative test for the pyrimidine bases cytosine and uracil.

. Positive Results: Test for Ribose (Molisch Test) The positive reaction of Molisch test is the formation of violet ring on the solution which indicates the presence of carbohydrates.III. Test for Phosphates The positive reaction of this test is the formation of yellow precipitate on the solution which indicates the presence of phosphate on the solution.

. Wheeler-Johnson Test The positive reaction of Wheeler-Johnson test is the formation of violet precipitate which indicates the presence of pyrimidine on the solution.Murexide Test The positive reaction of Murexide test is the formation of red to brown residue which indicates the presence of purines on the solution.

Understanding how nucleic acids store and deliver genetic information within the cells is necessary to understand diseases and to devise strategies for disease treatment. RNA is made when the complex biochemical decodification machinery of the cell acts on the DNA to extract the information needed for a particular function. There is also regulatory RNA. RNA is a key factor for protein synthesis. but recognizing the importance of nucleic acids in these diseases may be the key that eventually unlocks a cure.IV. Role of Nucleic Acids in Diseases When an error occurs in any of the steps involved in expressing the genetic information contained in DNA a genetic disease may occur. Many genetic diseases cannot be cured at the moment. Everything that the cells has to do. that is RNA molecules capable of regulating gene expression by different mechanisms such as interference or blocking. and transfer RNA (tRNA) is responsible for transporting aminoacids to the ribosomes to make the required proteins. Messenger RNA (mRNA) is the nucleic acid that brings information (from the nucleus to the cytoplasm) about which protein to make. DNA functions as the molecule that carries on the genetic information from parent to offspring. and how it has to do it is determined by the information contained in the DNA molecule. Significance and Application The DNA is the biological molecule that stores all the genetic information of the cell (in some viruses RNA may function as the molecule that stores the genetic information). In addition. . RNA is responsible for transferring the information contained in the DNA to make a particular protein needed in a specific process for a specific function. at what time in its life cycle.

Isolation of Casein From Milk Chemical Characterization and Denaturation of Proteins .

These are attached mainly to the hydroxyl groups of the serine and threonine moieties. potassium. proteins (mostly casein). and trace metals). Casein. The only important nutrients lacking in milk are iron and vitamin C. 3.9% carbohydrates. . phosphorus. Introduction Milk is probably the most nutritionally-complete food that can be found in nature. All three are globular proteins which tend to fold back on themselves into compact. All kinds of milk.7% minerals. B12. There are three kinds of proteins in milk: casein. and vitamins A. nearly spheroidal units. pantothenic acid. 4. lactalbumins. is a phosphoprotein which has phosphate groups that are attached to some of the amino acid side chains. Background of Experiment I. sodium. 3.4% protein. riboflavin. and are more easily solubilized in water as colloidal suspensions than fibrous proteins. The average composition of whole cow’s milk is 87. the main protein in milk.1% water. and D). and lactoglobulins.9% fats.I. and 0.I. minerals (calcium. contain vitamins (principally thiamine. human or animal. carbohydrates (principally lactose). and lipids (fats).

it is insoluble in solutions of pH less than 4.Casein exists in milk as the calcium salt.6. Denaturation of proteins involves the disruption and possible destruction of both the secondary and tertiary structures. therefore. If acid is added to milk. the primary structure (sequence of amino acids) remains the same after a denaturation process. lactic acid is produced by bacterial action.6. The isolation of casein from milk will be carried out in this experiment. Since denaturation reactions are not strong enough to break the peptide bonds. casein has a negative charge at this pH and is solubilized as a salt.6. By the isolation of casein from milk. and the neutral protein precipitates with the calcium ions remaining in the solution. and the consequent lowering of the pH causes the same clotting reaction. the protein is denatured. Calcium caseinate has an isoelectric (neutrally) point at pH 4. Denaturation disrupts the normal alpha-helix and beta sheets in a protein and uncoils it into a random shape. The pH of milk is about 6. calcium caseinate. the negative charges on the outer surface of the casein micelles are neutralized (the phosphate groups are protonated). Ca2+Caseinate + 2HCl → Casein ↓ + CaCl2 When milk sours. . Therefore.

Continue the acid addition (lightly less than 2ml will be required).      . then remove it with a stirring rod and place it in another beaker.5ml of 1M NaOH. keeping the beaker on the water bath.I. Procedure    Weigh out 5g of powdered non-fat dry milk and dissolve it in 20ml of warm water. stopper the mixture. and let it stand in the air for 10 minutes. and weigh the two portions. Place one portion in a 125ml Erlenmeyer flask with 35ml of water and 0. Then add dropwise a solution of 10% HOAc while stirring with a stirring rod. and save it for use in the chemical tests to be performed next. Press the solid with a spatula. Do not add the acid too rapidly. Place the casein between several layers of paper towels to help dry the product. Divide the wet product in half. and shake it to ensure solution of as much of the casein as possible. Do not add too much acid. and filter the product. Collect the casein by filtration to remove as much water as possible. until the liquid changes from milky to almost clear and the casein no longer separates. Place the casein in a 100ml beaker and add 5ml of a mixture of 1:1 ethyl ether and ethanol (CAUTION: HIGHLY FLAMMABLE – NO FLAMES). decant the ether.II. Stir the casein in the ether for a few minutes. Bring the temperature of the solution to 55°C (do not exceed 60°C). Stir the precipitated casein until it forms a large amorphous mass.

Principle: It is a chemical test used for detecting the presence of peptide bonds. Copper (II) ions. in basic solution. . Color Reaction Tests Using 0.5ml each of 10% NaOH and 0. Chemical Principle of each Test II.II. then perform the following tests: Biuret Test Add 0.2g of casein for each test tube. Any four peptide bonds can coordinate with the copper ion to form the color. will complex with the nitrogen atoms of four peptide (amide) bonds to form violet colored complex products.5% CuSO4 to the test sample. The general illustration of the reaction is: These peptide bonds do not have to be in any order or any sequence in the protein.I.

therefore many proteins that are not glycoproteins give a negative result to Molisch’s test. and then slowly add. Therefore.Molisch Test To 1ml of cell suspension. 40 drops of concentrated H2SO4. allowing to run on the side. glycoproteins give positive results to Molisch’s test because they are proteins in complex with carbohydrates. Glycoproteins are very common. . Mix thoroughly and tilt the tube. Principle: It is a test for the presence of carbohydrates. add 2 drops of Molisch reagent.

the phenol group of tyrosine is first nitrated by nitric acid in the test solution. .5ml of 1% Ninhydrin solution to the sample and heat in water bath for 5 minutes. Therefore.” as amines left over from proteins sloughed off in fingerprints react with Ninhydrin giving a characteristic purple color.Ninhydrin is most commonly used as a forensic chemical to detect “fingerprints. In Millon’s test.Millon’s Test Add 0. Principle: It is test for detecting free amino groups. molecules with a free –NH2 group (a free amino group) will form a purple-blue color with the Ninhydrin reagent. no color will result. hydroxyl group attached to a benzene ring. the only amino acid containing a phenol group. If all amino groups are part of a peptide bond.5ml of Millon’s reagent to the sample and note the result. Then the nitrated tyrosine complexes mercury (I) and mercury (II) ions in the solution to form red precipitate or a red solution. Principle: It is a test specific for tyrosine. Ninhydrin Test Add 0. both positive results.

Other foods are cooked to denature the proteins to make it easier for enzymes to digest them. NaOH → S2 Pb2+ → PbS . Protein Denaturation Using 1ml of albumin (egg white) for each test tube. Principle: It is a test for proteins containing sulfur (in cysteine and cystine) which give a positive result when heated with lead acetate in alkaline medium. Heat the test sample in water bath for 10 minutes. This occurs because heat increases the kinetic energy and causes the molecules to vibrate so rapidly and violently that the bonds are disrupted. Sulfur-containing protein II. perform the following: By Heat Heat the sample in water bath for 5 minutes and observe for changes. The proteins in eggs denature and coagulate during cooking.5ml each of 20% NaOH and 10% Lead acetate to the sample.Sulfur Test Add 0. Principle: Heat can be used to disrupt hydrogen bonds and non-polar hydrophobic interactions.II.

The like charges will repel each other and prevent the protein from aggregating as readily. In some cases the unfolding may be extensive enough to expose hydrophobic groups and cause irreversible aggregation. Principle: If the pH is lowered far below the isoelectric point. the protein will lose its negative and contain only positive charges. the intramolecular repulsion may be great enough to cause unfolding of the protein. Alcohol denatures proteins by disrupting the side chain intramolecular hydrogen bonding. Until this occurs such unfolding will be largely reversible. In areas of large charge density. This will have an effect similar to that of mild heat treatment on the protein structure. New hydrogen bonds are formed instead between the new alcohol molecule and the protein side chains. Hydrogen bonding between “side chains” occurs in tertiary protein structure in a variety of amino acid combinations. Principle: Hydrogen bonding occurs between amide groups in the secondary protein structure. All of these are disruptedby the addition of another alcohol.By Alcohol Add 1ml of ethyl alcohol to egg albumin and observe for precipitation. By Inorganic Acids To the sample. add 3 drops of concentrated H2SO4. .

Pb+2. By Alkaloidal Reagents Add 2 drops of 5% Tannic acid to the egg albumin. Principle: Alkaloidal reagents (e. Heavy metal salts usually contain Hg+2.g. Since salts are ionic they disrupt salt bridges in proteins. . Principle: Heavy metal salts act to denature proteins in much the same manner as acids and bases. Cd+2 and other metals with high atomic weights.By Heavy Metal Salts Add 2 drops of 5% FeCl3 to the egg albumin. You may perform the same procedure using 1ml of 5% Picric acid. tannate and trichloroacetate) are high molecular weight anions. You may do the same procedure using 10% Lead acetate. The reaction of a heavy metal salt with a protein usually leads to an insoluble metal protein salt. Ag+1 Tl+1. The negative charge of these anions counteracts the positive charge of the amino group in proteins giving a precipitate.

Molisch Test Positive result: The positive reaction for the Molisch test is the formation of violet ring that indicates the presence of carbohydrates. . Color Reaction Tests Biuret Test Positive result: The positive reaction for the Biuret test is the formation of the violet color that indicates the presence of peptide bonds. Positive Results III.III.I. Solutions without peptide bonds (such as free amino acids) will not form the violet color and will test negative in the biuret test for the presence of peptide bonds.

Protein chains have a free amino group at the beginning of the protein. the Ninhydrin test is interpreted as being negative for the presence of free amino groups. Only FREE amino groups (like those in free amino acids and protein side chains) will give a positive reaction with the Ninhydrin reagent. but this may not be enough to produce a positive reaction. Solutions with no (or very few) free amino groups will not produce the purpleblue color. . The red color is probably due to a mercury salt of nitrated tyrosine. and hence.Millon’sTest Positive result: Proteins that contain tyrosine will therefore yield a positive result of pink to dark-red color. The Millon’s reagent is a solution of mercuric and mercurous ions in nitric and nitrous acids. which contains a secondary amino group (instead of the usual primary amino group found in most amino acids and amino acid side chains) and will produce an orange color instead of the purple-blue. Ninhydrin Test Positive result: A positive Ninhydrin reaction is the purple-blue color (lavender solution) which indicates the presence of free amino groups. An exception is the amino acid proline.

II. . Protein Denaturation By Heat Result: When the egg albumin is heated in the water bath for 5 minutes. coagulation occured turned out like a hardboiled egg. III.Reduced Sulfur Test Positive result: The positive reaction for the Reduced Sulfur test is the black solution/ deposit of lead sulfide (PbS) when heated with lead acetate in alkaline medium which indicates the presence of proteins containing sulfur.

By Alcohol Result: When 1ml of ethyl alcohol was added to egg albumin. By Inorganic Acids Result: When 3 drops of concentrated H2SO4 was added to the egg albumin sample. coagulation occurred but not all proteins. . some liquid portion of the egg albumin did not coagulate. the H2SO4 settled at the bottom part of the test tube and coagulation occurred.

coagulation occurred and the egg albumin became brown solution. coagulation occurred and the egg albumin became yellow solution with a formation of darker yellow precipitate.By Heavy Metal Salts Result: When 2 drops of 5% FeCl3 or 2 drops of 10% Lead Acetate was added to 1ml egg albumin. By Alkaloidal Reagents Result: When 2 drops of 5% Tannic acid or 1ml of 5% Picric acid was added to 1ml of egg albumin. therefore the proteins were denatured. .

Significance and Application of the Experiments Casein is the principal protein found in milk from which it has been extracted commercially for most of the 20th century. emulsification and texture. buckles etc. The major uses of casein until the 1960s were in technical. It is responsible for the white. . During the past 30 years. in paper coating. and to improve their nutrition. leather finishing and in synthetic fibers. as well as plastics for buttons. non-food applications such as adhesives for wood. called micelles. the principal use of casein products has been as an ingredient in foods to enhance their physical (so-called functional) properties.IV. water binding and thickening. opaque appearance of milk in which it is combined with calcium and phosphorus as clusters of casein molecules. such as whipping and foaming. however.

In New Zealand. Casein products are used mainly as ingredients in foods for the purpose of either modifying the physical properties of that food product or providing nutritional supplementation to it. are called caseinates. Casein is generally not consumed as a food on its own. . casein is precipitated from the skim milk that is produced after centrifugal separation of whole milk. initially in adhesives and waterbased paints. The precipitated casein in curd is separated from the whey. washed and dried. resulting in the so-called rennet casein. they usually form a relatively minor proportion of the food. produced by reaction with alkalis. As a consequence. Water-soluble derivatives of acid caseins. The skim milk may be acidified to produce acid casein or treated with an enzyme. These applications have multiplied during the 20th century. Casein has been used commercially in non-food technical applications since the mid-19th century.

Carbohydrate Chemistry .

The empirical formula of carbohydrates is (CH2O) n. . Ex: Glucose. Background of the Experiment Carbohydrates are the most abundant biomolecules on earth. Ex: Maltotriose which is a trisaccharide. Polysaccharides consist of more than twelve monosaccharides. Ex: Maltose. They have either an aldehyde or a keto group in addition to many hydroxyl groups. Dihydroxyacetone is the simplest carbohydrate with a keto group. Carbohydrates are defined as the polyhydroxy aldehydes or polyhydroxy ketones or substances that yield such compounds on hydrolysis. Carbohydrates serve as energy stores. Oligosaccharides consist of three to twelve monosaccharides. Ex: Cellulose. structural elements and they are precursors for many organic compounds like fats and amino acids. Disaccharides consist of two monosaccharides linked by glycosidic linkage. Carbohydrates are classified into four categories.I. made up of three glucose units. Monosaccharides (simple sugars) containing a single polyhydroxy aldehyde or ketone unit.

Classification Monosaccharides: They are the simplest Carbohydrates. crystalline substances which have a sweet taste and are soluble in water. Some common disaccharides are: Glucose + Glucose = Maltose Glucose + Galactose = Lactose Glucose + Fructose = Sucrose . Fructose (keto.hexose). The monosaccharides are connected by a glycosidic linkage. doesn’t possess the characteristics carbohydrates. if they are further broken. Monosaccharides are colorless. Disaccharides: They are formed by the condensation of two monosaccharide molecules. They have a single carbon chain having a free aldehyde or ketone group. Examples: Glucose (aldo-hexose).

E. Homopolysaccharides . They form a colloidal solution when heated with water. They are further divided into: I. II. . Although it is present in the human diet. b) Plant origin: The most common example is the mucilage group which includes agar.g. blood group. Starch is stored in the form of glucose in plants.Polysaccharides: They are formed by the condensation of a large number of monosaccharide molecules and have a high molecular weight. mucoplysaccharide group which includes hyaluronic acid.They are made up of the same kind of monosaccharide units. chondriotin sulphate. vegetable gums and Dectins. heparin. Heteroploysaccharides . In plant cell walls.: starch. They are polymers of glucose and inulin (polymer of fructose). glycogen. Glycogen is stored in the form of glucose in Animals. hence are slightly soluble in cold water. but it cannot be digested because humans lack the enzyme cellulase. Cellulose is a structural polysaccharide of plant cells.They are made up of different kinds of monosaccharide units.g. and cellulose. and are further divide into two main groups: a) Animal origin: E. cellulose occurs in densely packed fibrils called hemicelluloses. and serum mucoids. They are not sweet and don’t exhibit any of the properties of the aldehyde or ketone groups as in case of monosaccharides and disaccharides.

therefore. II. and thereby cannot reduce other substances. and an enzyme. by virtue of this. these sugars have a free anumeric carbon atom. In other words. Non-reducing sugars: Their anumeric carbon atoms are engaged in making bonds with each other.e. on hydrolysis with an acid. . Reducing Sugars: Reducing sugars are those which have potentially an aldehyde and ketone group in their structure. they don’t have a free aldehyde or ketone group in their structure.i. they have the ability to give a H atom to other substances . hey have the ability to reduce other substances and themselves get oxidized. glucose. base.Chemical types of Carbohydrates I. and galactose are reducing sugars because they have a free carbon atom. All monosaccharides i. Disaccharides as maltose and lactose are also reducing sugars. fructose.e. Some disaccharides like sucrose and all polysaccharides are non-reducing sugars. However. they yield smaller units which have a reducing capacity.

These products condense with α-naphthol to yield a purple condensation product. Carbohydrate  dehydration product -> purple product Furfural . The dehydration products of carbohydrates.Chemical Principle of Test Color Reaction Tests for Carbohydrates Molisch Test The Molisch test uses concentrated sulfuric acid as the dehydrating acid. This acid dehydrates all carbohydrates. result from the reaction of the sulfuric acid with pentoses and/or hexoses. furfural or 5hydroxymethylfurfural. so the test is used to distinguish between carbohydrates and non-carbohydrates.

Pentoses subjected to the test yield a blue or green condensation product. pentose  dehydrating product  blue or green condensation product furfural hexose  dehydrating product muddy brown-gray condensation product 5-hydroxymethylfurfural .Bial’s Test A test uses concentrated hydrochloric acid as the dehydrating acid and orcinol with a iron (III) chloride as the condensation reagent. while hexoses yield a muddy brown-to-gray condensation product. Bial's test is used to distinguished between pentose and hexoses.

and galactose are reducing sugar having free carbon atom. which may further change to a peach color. Seliwanoff’s Test Seliwanoff’s test is used to identify ketohexoses like fructose. Aldopentoses react after 2 minutes to form a bluegreen condensation product. Monosaccharide such as glucose. Disaccharides as maltose and lactose are also reducing sugars. Seliwanoff’s test uses 6M hydrochloric acid as the dehydrating agent and resorcinol as the condensation reagent.Moore’s Test Moore’s test is used to identify the presence of reducing sugars. Ketopentoses and ketohexoses react within 2 minutes to form a cherry red solution when mixed with Seliwanoff’s reagent. This test gives the positive result of brown solution with caramel scent. Ketose  dehydration product  cherry-red product  . fructose.

and amylopectin. . Thus. a helical saccharide polymer. Iodine forms a large complex polysaccharide with the αamylose helix. producing the blue-black color. Simpler oligosaccharides and monosaccharides do not form this complex with iodine. Starches contain α -amylose.Iodine Test Starches form deeply colored blue-black complexes with iodine. the I2/KI test can be used to distinguish starches from other carbohydrates.

Cupric ion complexed with tartrate ion is reduced to cuprous oxide. . sodium citrate. A positive result is indicated by the formation of a brick red precipitate. it will reduce the copper (II) ions to copper (I) oxide.R-CO2 (carbohydrate ion) + Cu2O (s. Like other aldehydes.red) + 3H2O Fehling’s Test Fehling's tests for aldehydes are used extensively in carbohydrate chemistry. and sodium carbonate in a mildly basic solution.Test for Reducing Sugar Benedict’s Test Benedict's test uses a mixture of copper (II) sulfate. aldoses are easily oxidized to yield carboxylic acids. a red precipitate. R-CHO ( reducing carbohydrates) + 2Cu + 5OH. This reagent is used as a general test for detecting reducing sugars. If the saccharide is reducing sugar.

so slow that solutions of sucrose can sit for years with negligible change.Hydrolysis of Oligosaccharides and polysaccharides Sucrose Hydrolysis Hydrolysis breaks the glycosidic bond. are positive. glucose and fructose. Sucrose has been hydrolyzed in the presence of concentrated HCL into its reducing monosaccharide constituents. Hydrolysis is. and to create an alkaline medium which provides the optimum pH for the reducing ability of reducing sugars. Sodium hydroxide is added for the purpose of neutralizing excessive HCL. converting sucrose into glucose and fructose. Hence the test for reducing sugars. Hydrolysis can be accelerated with acids. . however. such as hydrochloric acid.

Starch is a non reducing polysaccharide therefore it does not give positive result with Benedict’s. Its further hydrolysis produces achrodextrins. and hence Benedict’s. . test. However after hydrolysis into monosaccharide by the actions of strong acid.Starch Hydrolysis Hydrolysis of Starch into Glucose: Polymers are broken down by hydrolysis which is essentially the reverse of condensation. Sodium hydroxide is added to neutralize excessive HCl. which gives negative iodine. because the reducing ability of reducing sugars is high in alkaline medium. give positive iodine test. Polysaccharides such as starch. Erythrodextrin give red colour. This is a hydrolysis reaction because water (hydro) is used to break (lyse) a bond. The reason being is to provide time to complete hydrolysis of achrodextrin into maltose and maltose into glucose.Selivanoff’s and Osazone tests. we heat test tubes for two minutes more. An –OH group from water attaches to one monometer and a H attaches to the other. and Fehling’s reagents. and hence gives good results of Benedicts . Because glucose have free Aldehyde group. energy is released. Selivanoff’s and Osazone tests become positive. When the iodine test becomes negative. When a bond is broken. HCl causes its hydrolysis into glucose. its components (glucose molecules) give all the test positive. PRINCIPLE: Heating of starch in the presence of conc. therefore it is a strongly reducing monosaccharide. dextrin and glycogen.

RESULT: The positive reaction for Molisch test is the formation of violet ring which indicates the presence of carbohydrates on the sample solution. lactose. 3 mL of freshly prepared concentrated H2SO4. add 3ml of Molisch reagent. then slowly add. sucrose. perform the following tests: Molisch Test To 1 ml of cell suspension. fructose. galactose.III. Tilt the tube. . Mix thouroughly. allowing to run on the side. Positive Result Color Reaction Tests for Carbohydrates To 10% solution of the following sugars: glucose.

. RESULT: The positive reaction for Moore’s test is the formation of brown solution with caramel scent which indicates the presence of reducing sugar in the sample solution.5mL sugar solution then heat in water bath for 10 minutes. RESULT: The positive reaction for Bial’s test is the formation of blue-bluegreen color in the solution which indicates the presence of peptose sugar on the sample solution.5mL of Bial’s reagent to 0. add 1 mL of 30% NaOH then heat in water bath for 10 minutes.Bial’s Test Add 0. Moores’s Test To 1mL of sugar solution.

5 mL of sugar solution then heat in water bath for 10 minutes. RESULT: The positive reaction for Iodine test is the formation of dark blue to black solution which indicates the presence of polysaccharide.5 mL of Seliwanoff’s reagent to 0.Seliwanoff’s Test Add 0. particularly starch on the sample solution. . Iodine Test To 1 mL of sugar solution add 3 drops of iodine solution. RESULT: The positive reaction for Seliwanoff’s test is the formation of red solution with brown precipitate which indicates the presence of ketose sugar on the sample solution.

5 mL each of Fehling’s A and Fehling’s B then heat in water bath for 5 minutes.5 mL of Benedict’s reagent and heat in water bath for 5 minutes. RESULT: The positive reaction for Benedicts’s test is the formation of brick red precipitate on the solutionwhich indicates the presence of a reducing sugar.Test for Reducing Sugars Benedict’s Test Add 0. Fehling’s Test Add 0. RESULT: The positive reaction for Fehling’s test is the formation of brick red precipitate on the solution which indicates the presence of a reducing sugar. .

perform Fehling’s Test and Benedict’s Test.Hydrolysis of Oligosaccharides and Polysaccharides Sucrose Hydrolysis To 3mL of sucrose solution. .5 mL each of the hydrolyzed solution. RESULT: The positive result for Benedicts test and Fehlings test is the formation of brick red precipitate on the solution which indicates the presence of reducing sugar. glucose and fructose which was the hydrolyzed form of sucrose. To 0. add 2 drops of concentrated HCl then heat in water bath for 20 minutes.

Upon treating with concentrated HCl the solution gives positive result to Fehlings and Benedict’s test with a positive result of brick red precipitate on the solution which indicates the presence of glucose that is the product of hydrolyzed starch. .Starch Hydrolysis RESULT: The positive reaction of starch in Iodine test is the formation of dark blue to black solution.

Seliwanoff’s test produced cherry red color due to condensation in its mechanism. Significance and Application of the Experiment Each group of carbohydrates can be observed through different qualitative test for carbohydrates that exhibits changes in color base on certain reactions after reacting on specific functional groups in them. Iodine test exhibits blue-black complex that involves complexation where insertion of iodine into the helical structures of starch and glycogen occurred. Molisch test resulted in purple color involves dehydration by H2SO4 and condensation with α-naphthol. Seliwanoff’s test. Benedict’s test resulted in red precipitate is a test for reducing sugars. . and Bial’s test. Six qualitative tests for carbohydrates are the Molisch’s test. Iodine test. Two unknown carbohydrate samples were identified as fructose and lactose base on the color change observed in their reactons on certain reagents. Six different test were conducted in this experiment to identify the given unknown carbohydrate samples and at the same time. and the Bial’s test that gave out blue precipitate due to condensation with Orcinol. Specific nature of condensation reaction varies with the carbohydrate under investigation (3). Barfoed’s test that forms rusty color distinguished reducing monosaccharides from reducing disaccharides. Barfoed’s test. which react with the carbohydrates to produce highly colored products.IV. Many carbohydrates can be identified using condensation reagents. Benedict’s test. to understand the mechanisms that undergone on certain reactions occurred.

•Structure and component linkage. •Invert Sugar. •Dissacharidase deficiency. .venous.Benedict’s Test CLINICAL APPLICATIONS: •This is widely employed test for detection of glucose in urine. •Sources of sucrose. •Role of Sucrose when given as intra. •It is commonly used for preliminary screening for hyperglycemia and for monitoring the effect of Treatment Sucrose Hydrolysis CLINICAL CORRELATION •Digestion of sucrose in the body.

Lipid Chemistry .

they contain both polar and non-polar parts. sphingolipids.I. The simplest unit of lipid is fatty acids which are classified as either saturated or unsaturated based on the presence or absence of double bonds. except for waxes which is completely hydrophobic due to its long hydrocarbon tail. Classification is basically based on the back bone unit present. chloroform. Background of the Experiment: Lipids are generally characterized as being insoluble in water but soluble in organic non-polar solvents such as ether. and carbonic tetrachloride to name a few. Most lipids are amphipathic. Aside from waxes. that is. lipids can also be classified as triacylglycerol. glycolipids. and steroids. .

chloroform. Test for Unsaturation There are two way to test the unsaturation of lipids. It maybe defined as Miscible or Immiscible. Na2CO3 or also known as washing soda. Chemical Principle of each test: Lipid Solubility Test This indicates that the coconut oil is soluble only in organic compounds such as ether. you need solutions such as: dishwashing liquid. . it means more drops are needed. and carbonic tetrachloride. II. these are: Iodine Absorption Test and Bromine Absorption Test. Note: in order to be miscible. the more drops will be needed for color reaction. Note: High Carbon = High Unsaturation. Note: the more double bonds. Lipid Emulsification Emulsify means it allows the substance to mix.

> Potassium Sulfate = Unsaturated Aldehyde >> Acrolein Test (test for the presence of fatty acids/glycerin) . The term saponification is the name given to the chemical reaction that occurs when a vegetable oil or animal fat is mixed with a strong alkali. The water is only a vehicle for the alkali. Acrolein Test It is also known as Fat Denaturation (Transidity of Lipids). which is otherwise a dry powder. It is the production of glycerol/sodium soap or sodium salt. Glycerol .Saponification * This is simply means soap making. but it does not enter into the chemical reaction. The products of the reaction are two: soap and glycerin. Water is also present.

Positive Results: Lipid Solubility Test TEST TUBE NUMBER #1 Coconut oil + H2O Result: Insoluble TEST TUBE NUMBER #2 Coconut oil + CHCl3 Result: Soluble .III.

TEST TUBE NUMBER #3 Coconut oil + ethanol at room temperature) Result: Insoluble TEST TUBE NUMBER #4 Coconut oil + hot ethanol Result: Insoluble .

Test for Unsaturation TEST TUBE NUMBER #1 Coconut oil Result: Saturated TEST TUBE NUMBER #2 Olive oil Result: Most unsaturated TEST TUBE NUMBER #3 Palmitic acid Result: More saturated .

Lipid Emulsification TEST TUBE NUMBER #1 H2O + coconut oil Result: Immiscible TEST TUBE NUMBER #2 H2O + coconut oil + dishwashing liquid Result: Miscible TEST TUBE NUMBER #3 H2O + coconut oil + Na2CO3 Result: Miscible .

Saponification This results from the formation of soap. Acrolein Test The positive reaction of Acrolein test is the formation of colorless or yellow liquid with a disagreeable solution which indicates the presence of glycerol and fatty acid. .

the dishwashing liquid that is normally made from fats. cleaning house or even during bathing .IV. Emulsifier such as dishwashing liquid can help mix two solution that don’t normally mix together. the more drops of iodine needed for absorption. Like in washing the dishes. Saponification is the process used to make our soaps which we used on our daily activities such as cleaning dishes. Significance and Application of the Experiment: The lipid solubility test is used to determine the presence of saturated and unsaturated fatty acids. In the experiment if theres a formation of layer on the solution. it indicates that the more unsaturated or the more the double bond the sample has. washing clothes. Saturated food are more harmful for people while unsaturated are less harmful for people. meaning they are soluble mixture. For the test for unsaturation. We can use it to test the saturation. uses to clean our dishes only because the fats on the liquid and the stains on our dishes mix together making them easy to remove and wash. it means that the mixture is insoluble with one another while if the solution doesn’t form any layer.

Catalase Activity .

Since enzymes are selective for their substrates and speed up only a few reactions from among many possibilities. we have studied the activity of the enzyme catalase. During a reaction. Enzymes catalyze chemical reactions involving the substrates. an enzyme that functions to catalyze the decomposition of hydrogen peroxide to oxygen and water. the set of enzymes made in a cell determines which metabolic pathways occur in that cell. which are then released from the active site. Background of the Experiment Enzymes are globular proteins. the substrate binds with the enzyme’s active site. and an enzyme-substrate complex is formed. One enzyme may catalyze thousands of reactions every second. decomposition of the concentrations of the harmful hydrogen peroxide in our body to water and oxygen would not be possible. The substrate is transformed into one or more products. In this experiment. Almost all processes in a biological cell need enzymes to occur at significant rates.I. . responsible for most of the chemical activities of living organisms. Hydrogen peroxide is naturally produced in organisms as a by-product of oxidative metabolism. Without the presence of catalases. They act as catalysts. Substrates are molecules upon which enzymes act. substances that speed up chemical reactions without being destroyed or altered during the process.

Almost every significant life process is dependent on enzyme activity. As catalysts. Intracellular enzymes catalyze the reactions of metabolic pathway. enzymes excel all other chemicals in their power and specificity.Almost all enzymes are composed of proteins and are found in all tissues and fluids of the body. . Plasma membrane enzymes regulate catalysis within cells in response to extracellular signals. and enzymes of the circulatory system responsible for regulating the clotting of blood.

NORMAL CATALASE ACTIVITY PRINCIPLE: Catalase is a common enzyme found in nearly all living organisms that are exposed to oxygen. then seeing how long it takes to rise in a tube of hydrogen peroxide . where it functions to catalyze the decomposition of hydrogen peroxide to water and oxygen.II.Chemical principle of each test A. The reaction of catalase in the decomposition of hydrogen peroxide is: 2 H2O2 → 2 H2O + O2 This can be tested by taking a solution of the enzyme and dipping a piece of paper into it.

OCCURRENCE OF CATALASE PRINCIPLE: In living systems the optimal ranges of temperature.IS CATALASE REUSABLE? PRINCIPLE: Catalase is reusable because during the chemical reaction. they are not permanently changed or destroyed which makes them able to still have an end product of hydrogen peroxide or bubble formation. pH and salt concentration for a given enzyme are the ranges found. those who exhibit the characteristic with the most optimum value of the factors that affect catalase activity will have the fastest rate of reaction. . Meaning.

more of the reacting molecules have the kinetic energy required to undergo the reaction. When the temperature increases. In both cases the changes produced in the chemical bonds of the enzyme molecule result in a change in conformation that decreases enzyme activity. values above 7 being basic and a value around 7 is neutral. As the pH moves into the basic range the enzyme tends to lose hydrogen ions to the solution. chemical reactions speed up as the temperature is raised.EFFECTS OF TEMPERATURE ON CATALASE ACTIVITY PRINCIPLE: In general. . Enzyme catalyzed reactions also tend to go faster with increasing temperature until a temperature optimum is reached. Temperatures above 40-50°C denature many enzymes. Above this value the conformation of the enzyme molecule is disrupted. It is measured on a scale of 0-14 with pH values below 7 being acidic. EFFECTS OF PH ON CATALASE ACTIVITY PRINCIPLE: pH is a measure of the acidity or hydrogen ion concentration of a solution. As the pH drops into the acidic range an enzyme tends to gain hydrogen ions from the solution. Changing the conformation of the enzyme results in less efficient binding of the substrate.

estimate the time passed (how rapidly the solution bubbles) before the bubbles appear. Feel the temperature of the test tube with your hand. . cut a small piece of liver and add it to the test tube.III. (Note if it is warm or not) RESULTS: The estimated time before bubbles formed when the liver came into contact with hydrogen peroxide at room temperature. and DATA TABLE 2 as the rate room temperature. in seconds. RESULTS WITH ILLUSTRATIONS NORMAL CATALASE ACTIVITY PROCESS:      Place 2mL of 3% hydrogen peroxide solution into a clean test tube. Using forceps and scissors. was 2 seconds. Record the time in DATA TABLE 1. Through this investigation. Push it into the hydrogen peroxide.

therefore enzymes. RESULTS: The reusability of the enzyme Catalase was tested by pouring unused H2O2 over the chicken liver used for previous part. particularly Catalase in this experiment. are/is reusable . Results agreed with the established biological fact that enzymes don't change.  Pour off the liquid into a second test tube.  Observe.  Add another 2mL of H2O2 to the liver remaining in the first test tube.IS CATALASE REUSABLE? PROCESS:  Place 2mL of 3% H2O2 solution into a clean test tube and add a small piece of liver.

OCCURRENCE OF CATALASE PROCESS:  Place 2mL of H2O2 in each of 3 clean test tubes. we can infer that catalase is more concentrated in animal cells and bacteria because their reaction time to form bubbles or froth is faster (1 second for the bacteria and 2 seconds for the liver) than the potato (65 seconds) and apple (112 seconds) samples which contain plant cells. RESULTS: After performing the experiments.     Tube 1: add a small piece of potato Tube 2: add a loopwhole of Staphylococcus aureus Tube 3: add a small piece of apple As you add each test substance. record the reaction rate (in seconds) for each tube in TABLE 1. .

 Remove the test tube from the water bath. 37 degrees Celcius 100 degrees Celcius . allow it to cool. Add 3mL of H2O2. You recorded the time for ROOM TEMPERATURE earlier. pour each tube of H2O2 into the corresponding tube of liver and observe the reaction. then pour out the water.EFFECTS OF TEMPERATURE ON CATALASE ACTIVITY PROCESS:  Put a piece of clean liver in a clean test tube and soak it in 3mL water. the enzymes react faster as it gets near the optimum temperature. Record the time in DATA TABLE 2.  Put equal quantities of liver into 2 clean test tubs and 2ML H2O2 into 2 other test tubes. RESULTS: SAMPLE SPEED OF ENZYME ACTIVITY 6 seconds 2 seconds 1second 60 seconds 0 degrees Celsius Room tempertaure The reaction rate of liver stored at room temperature compared to iced liver is faster.  Put one test tube of liver and one of H2O2 into each of the following water baths: Ice bath: 0 degree Celsius  Warm bath: 37 degrees Celsius  After 3 minutes. Note the time it takes before the bubbles appear. This was so because as long as the enzymes don't get denatured. Place this tube in a boiling water bath for 5 minutes. Record the reaction time in DATA TABLE 2.

RESULTS: SAMPLE SPEED OF ENZYME ACTIVITY 10 seconds 2 seconds 1 seconds acid pH basic pH neutral pH RESULT: Reaction rate was fastest at Neutral pH. which formed froth after 10 seconds. that had occurred after 2 seconds and the reaction on acid pH took the slowest enzyme activity rate. Treat each tubes as follows:  Tube 1: 5 drops 1M HCl at a time until acid Ph  Tube 2: 5 drops 1M NaOH at a time until basic pH  Tube 3: 1 drop H2O  Add a small piece of liver to each test tube. around 7. Estimate the reaction (in seconds) and record in DATA TABLE 3. This was so because the optimum pH for the enzyme Catalase is usually. Preceded by the reaction on basic solution.EFFECTS OF PH ON CATALASE ACTIVITY PROCESS:  Add 2mL H2O2 to each of 3 clean test tubes. .

Human catalase works at an optimum temperature of 37°C. A Catalase deficiency may increase the likelihood of developing Type II Diabetes. Significance and Application of the Experiment Hydrogen peroxide is a harmful by-product of many normal metabolic processes: to prevent damage. Some human beings have very low levels of catalase (acatalasia). In contrast.IV. indicating that this enzyme is dispensable in animals under some conditions. which is approximately the temperature of the human body. The true biological significance of catalase is not always straightforward to assess: Mice genetically engineered to lack catalase are phenotypically normal. . yet show few ill effects. To this end. It is likely that the predominant scavengers of H2O2 in normal mammalian cells areperoxiredoxins rather than catalase. catalase isolated from thehyperthermophile archaea Pyrobaculum calidifontis has a temperature optimum of 90°C. less dangerous substances. it must be quickly converted into other. catalase is frequently used by cells to rapidly catalyze the decomposition of hydrogen peroxide into less reactive gaseous oxygen and water molecules.

catalase has also begun to be used in the aesthetics industry. Hydrogen peroxide is used as a potent antimicrobial agent when cells are infected with a pathogen. . and Campylobacter jejuni. Catalase is also used in the textile industry. Peroxisomes in plant cells are involved in photorespiration (the use of oxygen and production of carbon dioxide) and symbiotic nitrogen fixation (the breaking apart of diatomic nitrogen (N2) to reactive nitrogen atoms). Recently. Legionella pneumophila. Catalase is used in the food industry for removing hydrogen peroxide from milk prior to cheeseproduction.Catalase is usually located in a cellular organelle called the peroxisome. make catalase in order to deactivate the peroxide radicals. Another use is in food wrappers where it prevents food from oxidizing. a solution containing catalase is then used to decompose the hydrogen peroxide before the lens is used again. thus allowing them to survive unharmed within the host. Pathogens that are catalase-positive. removing hydrogen peroxide from fabrics to make sure the material is peroxide-free. such as Mycobacterium tuberculosis.a few lens-cleaning products disinfect the lens using a hydrogen peroxide solution. Several mask treatments combine the enzyme with hydrogen peroxide on the face with the intent of increasing cellular oxygenation in the upper layers of the epidermis. A minor use is in contact lens hygiene .

Digestion of Proteins and Carbohydrates .

Pepsin is a material that has its own unique function in the digestion of food. Pepsin is made out of hydro chlorine acid and pepsi.  Pepsin is a digestive enzyme that is released in the stomach as pepsinogen. so the protein is not digested completely to the amino acid level. Background of the Experiment  Digestion occurs in all mammals. In order for that to occur. the food has to pass into the intestines.I. . each of which is related to specific digestive enzymes. where other enzymes complete the digestion process. including human beings. When pepsinogen is exposed to the hydrochloric acid in the stomach. Some of these are found in the mouth. while others are found in the intestines. It breaks down proteins only at certain points. The release of hydrochloric acid stimulates the release of this basic form of pepsin.  The main function of pepsin is to break down proteins that are found in foods such as meat and eggs into smaller pieces (polypeptides). the pepsinogen unfolds and breaks into pepsin. There are multiple steps involved in the breaking down of food.

. where it begins the chemical process of digestion. Amylase is also synthesized in the fruit of plants during ripening. Plants and some bacteria also produce amylase. The primary function of the enzyme amylase is to break down starches in food so that they can be used by the body. Amylase is present in human saliva. needed for the breakdown of long-chain carbohydrates (such as starch) into smaller units. The pancreas also makes amylase (alpha amylase) to hydrolyse dietary starch into disaccharides and trisaccharides which are converted by other enzymes to glucose to supply the body with energy. causing them to become sweeter. Amylase helps you digest carbohydrates into simple sugar so that our cells can use them for energy pepsin is used to digest protein and they are absorbed into the bloodstream from the small intestine. Amylase is a digestive enzyme classified as a saccharidase (an enzyme that cleaves polysaccharides). It is mainly a constituent of pancreatic juice and saliva.Amylase is an enzyme that catalyses the breakdown of starch into sugar.

7. Amylase is present in human saliva. a copper(II) ion forms a violet-colored complex in an alkaline solution. Carbohydrates Digestion (Digestion in the Mouth) Amylase is an enzyme that catalyses the breakdown of starch into sugar.II. It has an optimum pH of 6. In the presence of peptides. . Protein Digestion The Action of Pepsin Pepsin is an enzyme that catalyses the breakdown of protein into smaller pieces (polypeptides). Chemical Principle of Each Test A. Biuret Test The Biuret test is a chemical test used for detecting the presence of peptide bonds.8 . where it begins the chemical process of digestion.0. Protein has an optimum pH of 1.5 – 1.6. B.

Starches contain α -amylose. Simpler oligosaccharides and monosaccharides do not form this complex with iodine. sodium citrate. R-CHO (reducing carbohydrates) + 2Cu + 5OH.red) + 3H2O . the I2/KI test can be used to distinguish starches from other carbohydrates. If the saccharide is reducing sugar. Iodine forms a large complex polysaccharide with the αamylose helix. it will reduce the copper (II) ions to copper (I) oxide. This reagent is used as a general test for detecting reducing sugars.Iodine Test Starches form deeply colored blue-black complexes with iodine. and sodium carbonate in a mildly basic solution. producing the blue-black color. Thus. a red precipitate. a helical saccharide polymer. and amylopectin. Benedict’s Test Benedict's test uses a mixture of copper (II) sulfate.--- R-CO2 (carbohydrate ion) + Cu2O (s.

Positive Result: Protein Digestion The Action of Pepsin TEST TUBE NUMBER # 1 Pepsin + Egg Albumin solution Positive: violet solution .III.

TEST TUBE NUMBER # 2 Pepsin + Glacial Acetic Acid + Egg Albumin Solution Positive: light blue solution TEST TUBE NUMBER # 3 Egg Albumin Positive: blue-violet solution .

TEST TUBE NUMBER # 4 Pepsin + milk solution Positive: violet solution TEST TUBE NUMBER #5 Pepsin + Glacial Acetic Acid + Milk Solution Positive: whitish solution w/ precipitate .

TEST TUBE NUMBER # 6 Milk Solution Positive: blue-violet solution .

. .After 2 test the second part becomes negative. Benedict’s Test The positive reaction for Benedicts’s test is the formation of brick red precipitate on the solution which indicates the presence of a reducing sugar.The positive reaction for Iodine test is the formation of dark blue to black solution which indicates the presence of polysaccharide on the sample solution. Iodine test is positive .On the first test.Carbohydrates Digestion (Digestion in the Mouth) Iodine Test .

As we chew the food. Carbohydrate Digestion: Digestion of carbohydrate involves conversion of the large molecules of carbohydrates like disaccharides and polysaccharides into simple monosaccharide molecules which can be easily absorbed by the body. which are required by the body. The initial phase of protein digestion and absorption occurs in the stomach and the latter takes place in the small intestine. This is possible because of the presence of a special enzyme named amylase in the saliva. the protein digestion process basically consists of the breakdown of proteins into amino acids. the body is incapable of producing them by its own.IV. Then we swallow the food and it goes to the stomach. which is the absorbable form of protein molecules. the saliva released by the salivary glands of the mouth starts its work of breaking up of the carbohydrates. but unfortunately. Significance and Application of the Experiment Protein Digestion: Digestion of proteins takes place in different stages. Well. The first step of carbohydrate digestion will start the moment we put the food in mouth. Here the digestive acids secreted by the glands of the stomach play a major role in processing the carbohydrate molecules further. . Proteins provide the body with amino acids. The digestive enzymes of the stomach also helps in this work but they do not have much significant role in carbohydrate digestion in the stomach.

Group 1 sec:2MLS-K Members: Ilarde. Hiyas Mina . Emelyn Therese Lagat. Janine Joy Reyes.Queenie Tan. Queenie Jose.

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