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Activity Report
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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