ACTIVITY 1A SUBCELLULAR COMPONENTS OF THE LIVING CELL Procedure: Separation scheme: 1.

Wash off the blood from the chicken liver using a few drops of the suspending medium assigned to your group. Blot with Filter paper.

2. Minced and keep frozen.

3. Weigh the liver. Add 5 ml. of the suspending medium per gram of liver.

4. Homogenized at low speed 5-10 min.

5. Centrifuge for 10 min. and decant. Label the liquid as supernate I and the solid as sediment I.

ACTIVITY 1B QUALITATIVE TEST FOR THE CHEMICAL COMPOSITION OF ORGANIZATION Objectives: To determine the composition of organelles To understand and appreciate the different biochemical systems through separating the subcellular components of a cell To be able to perform different qualitative tests specific for carbohydrates, proteins, and lipids.

Procedure: Qualitative tests: Make test as qualitative as possible, perform blank test on sediment/supernate 1. Carbohydrates Molisch test: a. To 5 drops of the sediment/supernate

b. Add 5 drops of Molisch reagent

c. Shake

d. Layer with 1 ml. of conc. H2SO4

CONTROL TEST: a. 5 drops of 1% Ribose Solution

b. Add 5 drops of Molisch Reagent

d. Layer with 1 ml. of conc. H2SO4

c. Shake

2. Proteins Biuret test: a. To 5 drops of the sediment/supernate

b. Add 5 drops of 10% NaOH

c. Add 1 drop of .5% CuSO4

CONTROL TEST: a. 5 drops of 1% albumin b. Add 5 drops of 10% NaOH c. 1 drop of .5% CuSO4

3. Lipids Sudan Test: b. Add 5 drops of Sudan IV a. To 5 drops of cell sediment/supernate

CONTROL TEST: a. 5 drops of 1% lecithin b. add 5 drops of Sudan IV

RESULTS AND OBSERVATIONS: 1. Carbohydrates Molisch Test: 2. LIPID Sudan Test:

3. Protein Biurest test: -Using Supernate-Using Sediment-

Tests

Control

Sediment I +

Supernate I +

CARBOHYDRATES 1% RIBOSE 1. Molisch Purple Ring Test PROTEINS 2. Biuret Test LIPIDS 3. Sudan Test 1% ALBUMIN Purple 1% LECITHIN Red

-In Biuret test Supernate I result is negative because there is no purple coloration appear and it is also because the Cu ions in the biuret solution react with peptide bonds, a completely hydrolyzed protein will have no peptide bonds and thus will display as negative. A peptide bond is a C-N bond between a carboxylic acid group and an amine group. Guide Questions: 1. Give the principle involved in each of the test used to determine the chemical components of the organelles. Molisch Test: Dehydration of the carbohydrate by sulfuric acid to produce an aldehyde. When sugar solution is mixed with alpha-naphthol is brought in contact with conc. H2SO4, a violet ring is formed at the junction of the 2 liquids. Biuret Test: Reduction of copper ions which then complexes with nitrogen atoms on peptide bonds at high pH. Alkaline solution of proteins treated with copper sulfate results in the production of rose-pink to violet, then purple. Sudan test: Ability of fat cells to selectively absorb pigments in fat dyes such as Sudan IV The chemical Sudan IV is not soluble in water, it is, however, soluble in lipids. Therefore to test for the presence of lipids in a solution you will use a Sudan IV Test. In this test Sudan IV is added to a solution to dissolve any possible lipids. If lipids are present the Sudan IV will stain them red, giving a positive result.

2. Explain the principle involved in the separation scheme for the subcellular components. The principle involved in the separation scheme for the subcellular components is Differential Centrifugation. Tissues or cells are first disrupted to release their internal contents. Larger particles sediment faster than smaller ones and this provides the basis for obtaining crude organelle fractions by differential centrifugation.

ACTIVITY 2A QUALITATIVE TEST FOR PREOTEINS Objectives: To detect proteins through the different color reaction tests. To know the significance of peptide bonds and amino acids in detecting proteins.

Procedures: BIURET TEST 1. Mix 1 ml. of egg albumin solution and 1 ml. of 10% Sodium hydroxide. 2. Add .5% Copper Sulfate drop by drop mixing thoroughly sfter each addition. NINHYDRIN TEST 1. 2. 3. 4. Place 1 ml, of egg albumin solution in a test tube. Add 1 ml. of freshly prepared 0.1% aqueous solution of Ninhydrin Heat to boiling Allow to cool and observe the color produced SAKAGUCHI REACTION FOR ARGININE 1. 2. 3. 4. To 1 ml. of test solution, add 1 ml of 10% Sodium Hydroxide Add 6 drops of dilute alcoholic a-naphthol solution Mix well and add 1 ml. of 10 drops of Sodium Hypobromite solution Note the results ACTIVITY 2B PRECIPITATION TEST FOR PROTEINS Objectives: To observe the reactivity of reagents & solutions in each test To Identify and observe the precipitate in each test which yield a positive result

Procedures: PRECIPITATION BY METALLIC SALTS 1. Place 1 ml. of dilute egg albumin in a test tube 2. Add 1 ml of Copper Sulfate 3. Note the changes PRECIPITATION BY ALKALOIDAL REAGENTS 1. 2. 3. 4. Place 1 ml. of dilute egg albumin solution in a test tube. Add 1 ml. of picric acid solution. Heat. Observe the changes produced. Do the same using Trichloroacetic acid.

PRECIPITATION BY ALCOHOL 1. 2. 3. 4. 5. Place in each of the three test tube 5 ml of 95% alcohol To the first add 1 drop of dilute HCL To the second tube add 2 drops of 10% NaOH Leave the third neutral Add to each tube 3 drops of egg albumin solution

COAGULATION BY HEAT 1. Heat to boiling 5 ml. of albumin solution and add 2 drops of acetic acid TEST FOR DENATURED/COAGULATED PROTEINS 1. Suspend each of the precipitate in 10 ml. of distilled water 2. Place 3 ml. of each in test tube 3. Perform Biuret test RESULTS AND OBSERVATIONS BIURET TEST: NINHYDRIN TEST:

SAKAGUCHI REACTION FOR ARGININE:

Guide Questions: 1. Explain the principle involved in each of the test for proteins. Biuret Test: Reduction of Cu2+ which then complexes with the N atoms on the peptide bonds at high pH. Alkaline solution of proteins treated with copper sulfate results in the production of a rose-pink to violet, then purple. It is also a test used to detect the presence of peptide bonds by hydrolysis. A copper(II) ion is reduced to copper(I), which forms a complex with the nitrogens and carbons of the peptide bonds in an alkaline solution Ninhydrin Test: Amino Acid containing a free amino group and a free carboxylic acid group that react together with ninhydrin to produce colored products. Group on the alpha-carbon and can react with ninhydrin to produce blue purple product, Sakaguchi Test: In alkaline solution, protein containing Arginine gives red color with Alpha-Napthol and sodium hypochloride. Sakaguchi Reaction for Arginine 1 mL egg albumin + 1 mL 10% NaOH add 6 drops dil. alc. alpha-naphthol + 10 drops Na hypochlorite. It is a test for the detection of a specific type of protein with the amino acid containing the guanidinium group to form a red color and it is due to the presence of arginine. RESULTS AND OBSERVATIONS B. Precipitation by Metallic Salt Chemical Copper Sulfate Results and Observations (+) Blue Soln/Precipitate

C. Precipitation by Alkaloidal Reagents Chemicals 1 ml. of Picric Acid Trichloroacetic Acid Results and Observations (+) Yellowish Soln/Precipitate (+) Whitish Soln/Precipitate

D. Precipitation by Alcohol Chemicals 95% Ethyl Alcohol+egg Albumin+Dil. HCl 95% Ethyl Alcohol+egg Albumin+10%NaOH 95% Ethyl Alcohol+egg Albumin Results and Observations (+)Cloudy Soln/Precipitate (+)Clear Soln/Precipitate (+)Whitish, Cloudy Soln/Precipitate

E. Coagulation by Heat Chemical 5 ml. of Albumin+HAC Results and Observations (+) White Cloudy Soln/Precipitate

F. Test for denatured/Coagulated Proteins Suspended precipitate Acetic Acid Biuret Test (+)

GUIDE QUESTIONS: 1. Give the principles involved in each of the tests . B. Precipitation by Metallic Salts Proteins are precipitated by salts of heavy metals, such as mercuric chloride, zinc sulfate, etc. In weak alkaline solutions, protein molecules carry negative charges and combine with positively charged metal ions to form insoluble salts which precipitate from the solution. The precipitated proteins are denatured and this process in irreversible. C. Precipitation by alkaloidal reagents Alkaloidal reagents (e.g. Tannic acid and trichloroacetic acid ) are high molecular weight anions. The negative charge of these anions counteracts the positive charge of the amino group in proteins giving a precipitate. D. Precipitate by alcohol Alcohol denatures proteins by disrupting the side chain intramolecular hydrogen bonding. New hydrogen bonds are formed instead between the new alcohol molecule and the protein side chains. E. Coagulation by heat The result was the coagulation of the albumin solution. Egg-white is faintly alkaline. Complete precipitation takes place only in faintly acid solution. The temperature at which coagulation takes place depends to a large extent amount of acid and of salts present. F. Test for denatured/coagulated proteins The term denaturation is used more frequently than coagulation by scientific investigators at the present time to denote certain changes in proteins. Definite characteristics of the proteins are changed when they are coagulated, among which is loss solubility in water and dilute salt solutions. In some instances and under certain conditions the coagulation process may be reversible. Manner in which denaturation may be brought about. Coagulation of proteins may be brought about by a variety of processes. But in addition to heat the action of acids, alkali, salts, alcohol, mechanical agitation, radiation, and ultra-sonic vibrations may denature the protein and convert it from a soluble into insoluble from weight anions. The negative charge of these anions counteract the positive charge of the amino group in proteins giving a precipitate. Therefore, even a protein is denatured it will still give a positive result from the Qualitative of proteins.

2. Using milk as test solution, how will you prove that milk is a protein Biuret solution is used to identify the presence of protein. Biuret test reagent is a blue solution that, when it reacts with protein, will change color to purple.

3. Show a schematic diagram to arrive at your answer. To a test tube, add 40 drops of milk solution Add 3 drops of Biuret reagent (Sodium Hydroxide 10% copper sulfate 0.5%) Shake gently to mix Change in color of solution to Pink-purple confirms presence of protein