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:

2. Proteins

Biuret test:

a. To 5 drops of the sediment/supernate

b. Add 5 drops of 10% NaOH

a. 5 drops of 1%

Ribose Solution b. Add 5 drops of

Molisch Reagent

c. Shake

d. Layer with

1 ml. of conc.

H2SO4

c. Add 1 drop of .5% CuSO4

CONTROL TEST:

3. Lipids

Sudan Test:

a. To 5 drops of cell sediment/supernate b. Add 5 drops of Sudan IV

CONTROL TEST:

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

a. 5 drops

of 1% albumin

b. Add 5 drops of

10% NaOH

c. 1 drop of .5%

CuSO4

RESULTS AND OBSERVATIONS:

1. Carbohydrates 2. LIPID

Molisch Test: Sudan Test:

3. Protein

Biurest test:

-Using Supernate- -Using Sediment-

Tests Control Sediment

I

Supernate

I

CARBOHYDRATES

1. Molisch

Test

1% RIBOSE

Purple Ring

+

+

PROTEINS

2. Biuret Test

1% ALBUMIN

Purple

+

-

LIPIDS

3. Sudan Test

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. Place 1 ml, of egg albumin solution in a test tube. 2. Add 1 ml. of freshly prepared 0.1% aqueous solution of Ninhydrin

3. Heat to boiling

4. Allow to cool and observe the color produced

SAKAGUCHI REACTION FOR ARGININE

1. To 1 ml. of test solution, add 1 ml of 10% Sodium Hydroxide

2. Add 6 drops of dilute alcoholic a-naphthol solution 3. Mix well and add 1 ml. of 10 drops of Sodium Hypobromite solution

4. 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. Place 1 ml. of dilute egg albumin solution in a test tube.

2. Add 1 ml. of picric acid solution. Heat.

3. Observe the changes produced.

4. Do the same using Trichloroacetic acid.

PRECIPITATION BY ALCOHOL

1. Place in each of the three test tube 5 ml of 95% alcohol 2. To the first add 1 drop of dilute HCL

3. To the second tube add 2 drops of 10% NaOH

4. Leave the third neutral

5. 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 Results and Observations

Copper Sulfate

(+) Blue Sol’n/Precipitate

C. Precipitation by Alkaloidal Reagents

Chemicals Results and Observations

1 ml. of Picric Acid

Trichloroacetic Acid

(+) Yellowish Sol’n/Precipitate

(+) Whitish Sol’n/Precipitate

D. Precipitation by Alcohol

Chemicals Results and Observations

95% Ethyl Alcohol+egg Albumin+Dil.

HCl

95% Ethyl Alcohol+egg

Albumin+10%NaOH 95% Ethyl Alcohol+egg Albumin

(+)Cloudy Sol’n/Precipitate

(+)Clear Sol’n/Precipitate

(+)Whitish, Cloudy Sol’n/Precipitate

E. Coagulation by Heat

Chemical Results and Observations

5 ml. of Albumin+HAC

(+) White Cloudy Sol’n/Precipitate

F. Test for denatured/Coagulated Proteins

Suspended precipitate Biuret Test

Acetic Acid

(+)

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