EXPERIMENT

4 - Glycogen is primarily found in animal

ISOLATION OF GLYCOGEN tissues especially in the LIVER and
MUSCLE as well as in mollusks such as
GLYCOGEN oysters (talaba), clams (tulya) and
- Store glucose as a polymer in the form scallop (halaan)
of GLYCOGEN, which is similar to
starches found in plants Low levels of glucose in the blood can
trigger release of glycogen in the liver.
- It has a branched structure like
amylopectin (present in starch) but is Liver glycogen breaks down to glucose-6-
MORE HIGHLY BRANCHED phosphate, which is hydrolyzed to glucose

- The branches occur at every 13 glucose The release of glucose from the liver by this
residues breakdown of glycogen replenishes the
supply of glucose in the blood.
- Sometimes called “animal starch”
because of this similarity In muscle, glucose-6-phosphate obtained
from glycogen breakdown enters the
In the degradation of glycogen GLYCOLYTIC PATHWAY rather than being
- several glucose residues can be released exported to the bloodstream
at one time, one from each end of a
branch, rather than one at a time as Glycogen can be EXTRACTED by boiling the
would be in the case of a linear polymer fresh minced tissue w/ water.

- this feature is useful to an organism in ADDING ALCOHOL to the resulting
meeting short-term demands for energy opalescent solution will precipitate the
by increasing the glucose supply as glycogen
quickly as possible
MATERIALS AND CHEMICALS
- the structure of glycogen shows its Materials
ability to store and deliver energy - As is
quickly for the longest amount of time
possible Chemicals
- 10% acetic acid
- this can be observed from the average
chain length of the branches (13 glucose
residues). If the average chain length
were much greater or much shorter,
GLYCOGEN WOULD NOT BE AS
EFFICIENT vehicle for energy storage
and release on demand



PROCEDURE B. Isolation of glycogen from chicken liver
A. Isolation of glycogen from clams 1. Rinse the chicken liver with tap water
1. Mince about 40 pcs. of tulya
2. Pat dry using ordinary filter paper.
2. Drop into 200mL vigorously boiling
water. 3. Weigh 5-gram sample of the dried
liver
3. Continue boiling for about 10
minutes. 4. Mince the 5 grams’ liver and drop
into 100mL of vigorously boiling
4. Remove the tissue from the boiling water.
water and grind with sand in a
mortar and pestle 5. Cover the boiling mixture with watch
glass and continue boiling for 20
5. Return grinded tissues to the boiling minutes
water and continue boiling for 5
minutes 6. The volume of the solution should
have been reduced to about 50mL
6. Acidify slightly with 10% acetic acid
to coagulate the proteins 7. Acidify slightly with 10% acetic acid
to coagulate the proteins
7. Filter the mixture while hot.
8. Filter the mixture while hot.
8. Describe the appearance of the
filtrate 9. Describe the appearance of the
filtrate


















EXPERIMENT 5 2. Disaccharide
CHEMISTRY OF CARBOHYDRATES - Oligosaccharides which can be
hydrolyzed to 2 simple sugars
Carbohydrates
- Energy-storage molecules of many - Can yield 2 monosaccharide if subjected
organisms to hydrolysis

- Formed by the process of - 2 monosaccharide linked together by
photosynthesis GLYCOSIDIC BOND

- Biomolecules simply defined as 3. Oligosaccharide
polyhydroxy aldehydes or polyhydroxy - Polymers consisting of 2 to 10
ketones and/or compounds that break monosaccharides that can be
down into theses substances hydrolyzed to their respective
monosaccharides
- Have the formula (CH2O)n; hydrates of
carbon however, not true for all known 4. Polysaccharide
molecules that belong to this group - High molecular weight polymers
composed of many monosaccharide
- Starch= contains fewer parts of water units
than its corresponding monosaccharide
Starch
Sugars - reserved food of plants
- Carbohydrates that are soluble in water
and are sweet to the tastes Glycogen
- reserved food of animals
CLASSIFICATION OF CARBOHYDRATES
1. Monosaccharide Chitin
- Simple sugars - polysaccharide rich in fungi

- Smallest carbohydrate units “that can Peptidoglycans
be hydrolyzed” - in bacteria

- These sugars are very soluble in water Cellulose
- a tough fibrous material that gives form
- Sparingly soluble in ethanol and and rigidity to plant walls
insoluble in ether

Reducing sugars
- All FREE monosaccharides
- Arabinose, ribose, 2-deoxy-ribose,
glucose, fructose and galactose


PHYSICAL PROPERTIES OF 3. Reducing and Non-Reducing Nature of
CARBOHYDRATES Carbohydrates
Carbohydrates exist as cyclic structures
Presence of numerous hydroxyl groups containing functional groups…
- makes it very soluble in water
- some polysaccharides are not soluble Reducing agent
- The end w/ hemiacetal
Carbohydrates have HIGH melting point
Non-reducing agent
SOME taste sweet - The end w/o hemiacetal

SOME are white and possess crystalline In OXIDATION PROCESS
structure - Reducing agents like carbs are
susceptible to attack by oxidizing agents
CHEMICAL PROPERTIES OF such as
CARBOHYDRATES Benedict’s
Fehling’s
1. Dehydration Barfoed’s
- Carbohydrates when mixed with non-
oxidizing acids NOT REDUCING AGENTS
1. Sucrose – no hemiacetal
Seliwanoff’s and Molisch reagents 2. Amylase and Amylopectin
- 2 common dehydrating reagents in the - Have hemiacetal groups which appear
lab for every 2,000-10,000 monosaccharic
units
Seliwanoff’s reagent
- differentiate reactions between aldoses
and ketoses

Molisch reagent
- simply detects presence of
carbohydrates

2. Hydrolysis
- Process by which polysaccharides or
disaccharides are broken down into
separate sugars either by inorganic acids
or enzymes

Disaccharides = full hydrolysis
Polysaccharides = partial hydrolysis

Starch = can yield DEXTRINS which are
many shortened starch molecules
GENERAL TEST FOR CARBOHYDRATES 4. Qualitative test for Sugars
1. Molisch Test a. Seliwanoff’s Test
- A general test for carbohydrates in free - Used to differentiate aldoses and
or combined form ketoses

- Glycosidic bonds are hydrolyzed using - Both will give CHERRY RED COLOR
concentrated sulfuric acid giving reaction, diff. reaction time
monosaccharides which are dehydrated
to furfural, hydroxymethylfurfural and Aldoses = slower
other decomposition products. Ketoses = faster

- These react with ALPHA-NAPTHOL b. Barfoed’s Test
forming PURPLE COLORED - Used to distinguish between reducing
condensation products monosaccharide and reducing
disaccharide
- Not specific test for carbohydrates…
however, negative result is a good reducing disaccharide = act very slowly and
evidence of the absence of carbs requires prolonged heating

reducing monosaccharide = reacts quickly
2. Anthrone Test
- another general test for carbs - Positive result is a BRICK-RED
PRECIPITATE formed within 5 minutes
- the sulfuric acid hydrolyses bound
sugars and reacts with sugars to give c. Fehling’s Test
furfural or furfural derivatives - Used to test reducing ability of sugar
- Forms RED PRECIPITATE
- these derivatives react with ANTHRONE
to form DARK BLUE-GREEN COLORED d. Benedict’s Test
compounds - Also a test for reducing ability of sugar
- Yields a RED PRECIPITATE product
3. Iodine Test for Polysaccharides
- Used to differentiate helical e. Bial’s or Orcinol Test
polysaccharides from non-helical - Used to detect the presence of a
polysaccharides pentose
- Formation of a GREEN to BLUE color
- Amylose is not branched and is helical… within 5 minutes

- IODINE form complexes with amylose to
produce BLUE COLOR




PROCEDURE B. REDUCING AND NON-REDUCING
A. Hydrolysis of Carbohydrates SUGAR
- Get 3 clean and dry test tubes 1. FEHLING’S REAGENT
a. Get 5 test tubes
1st test tube b. Add 1mL of the substances listed:
a. Mix 1mL of 3M HCL and
5mL of a 1% sucrose solution Test tube 1 – 1% glucose
Test tube 2 – 1% fructose
b. Stir and heat the sol’n in a boiling Test tube 3 – 1% lactose
water bath for 20 mins. Test tube 4 – 1% sucrose
Test tube 5 – 1% starch
c. Cool and add 1mL of 3M NaOH
dropwise to make the neutral c. Add 2mL of prepared Fehling’s sol’n

d. Transfer 10 drops of this solution to d. Mix each sol’n thoroughly and place
another test tube then add 2mL of them in a boiling water bath for 5
Fehling’s A and B sol’n minutes

e. Heat for a few minutes e. Record observation

f. Record observation C. GENERAL TEST FOR CARBOHYDRATES
1. MOLISCH TEST
2nd Test tube i. Get 5 clean and dry test tubes label:
a. Place 5mL of 1% starch sol’n 10 drops each
Test tube 1 – 1% glucose
3rd Test tube Test tube 2 – 1% fructose
a. Place 5mL 1 of 1% glycogen sol’n Test tube 3 – 1% lactose
(from isolation of glycogen, Test tube 4 – 1% sucrose
experiment 4) Test tube 5 – 1% starch
Test tube 6 – 1% glycogen (from exp4)
*Add 5 drops of concentrated HCl to each ii. Add 1 drop of Molisch reagent
test tube iii. Mix well
iv. Incline test tube carefully and add
*Cover each tube with a marble 1mL of concentrated sulfuric
(H2SO4) acid allowing the acid to
*Boil the samples in a water bath for 30 run gently down the side of the test
minutes tubes
v. DO NOT MIX
*After 30 minutes, the boiled samples will vi. The H2SO4 will form a layer at the
be called the HYDROLYSATE SAMPLES of bottom of the tube
starch and glycogen which will be used for vii. Note the color at the junction of the
the qualitative Benedict’s and Barfoed’s two liquids formed
test.

 2. ANTHRONE TEST v. Remove from the water bath if
a. Get 5 clean and dry test tubes label: formation of a brick red precipitate
10 drops each has observed.
Test tube 1 – 1% glucose vi. Note length of time
Test tube 2 – 1% fructose
Test tube 3 – 1% lactose b. BARFOED’S TEST
Test tube 4 – 1% sucrose i. Get 6 clean and dry test tubes label:
Test tube 5 – 1% starch 10 drops each
Test tube 6 – 1% glycogen (from exp4) Test tube 1 – 1% glucose
b. Add 2mL of Anthrone reagent in each Test tube 2 – 1% fructose
test tube Test tube 3 – 1% lactose
c. Mix thoroughly and carefully since it Test tube 4 – 1% sucrose
contains concentrated sulfuric acid Test tube 5 – 5 drops starch H.
d. Note color that develops IN 5 TO 30 Test tube 6 – 5 drops glycogen H.
MINS ONLY. ii. Add 2mL of Barfoed’s reagent to
each test tube
iii. Mix well
3. IODINE TEST FOR POLYSACCHARIDES iv. Boil the mixture in a water bath for 3
a. Get a clean and dry spot plate mins.
b. Place 3 drops of 1% sol’n of starch v. Note length of time and formation of
3 drops of 1% glycogen in diff precipitate
concaves
c. Make 3 trials for each sample c. SELIWANOFF’S TEST
d. Add 1 drop of iodine solution in each i. Get 3 clean and and dry test tube
sample
e. Note the color change w/ iodine sol’n Test tube 1 – 1mL of 1% glucose
Test tube 2 – 1mL of 1% fructose
Test tube 3 – 1mL of 1% sucrose
4. QUALITATIVE TEST FOR SUGARS
a. BENEDICT’S TEST ii. Add 2mL of Seliwanoff reagent
i. Get 6 clean and dry test tubes label: iii. Immerse the test tube at the
10 drops each same time in a water bath of
Test tube 1 – 1% glucose boiling water
Test tube 2 – 1% fructose iv. Note length of time and
Test tube 3 – 1% lactose formation of ppt.
Test tube 4 – 1% sucrose
Test tube 5 – 5 drops starch H.
Test tube 6 – 5 drops glycogen H.
ii. Add 2mL of Benedict’s reagent to each
test tube
iii. Mix well
iv. Boil the mixture in a water bath for 3
mins.

 d. ORCINOL TEST
i. Get 3 clean and and dry test tube

Test tube 1 – 1mL of 1% glucose
Test tube 2 – 1mL of 1% fructose
Test tube 3 – 1mL of 1% ribose

ii. Add 2mL of Bial’s reagent
v. Immerse the test tube at the
same time in a water bath of
boiling water
vi. Heat for 3 minutes
vii. Note color of solution or
formation of ppt.






























EXPERIMENT 6 3. Tertiary
CHEMISTRY OF PROTEINS - Refers to interactions of the protein
backbones causing folding and refolding
Proteins of the protein
- Complex, specialized molecules
4. Quaternary
- Builders of life - Refers to the interactions among
subunits of proteins
- Most abundant molecules in cells and
constitute about 50% Denaturation
- Proteins are made inactive or
- Consist of long chains of subunits called biologically non-functional
AMINO ACIDS covalently linked to each
other by PEPTIDE BONDS - a destructive process caused by
denaturing agents such as
For every peptide bond, heat
1 molecule H20 is released mechanical treatment
1 dipeptide bond is formed heavy metals
acids and bases
When MANY linkages are formed, organic solvents
Molecule formed is POLYPEPTIDE radiation

- Proteins are huge polypeptides of about - however, MILD DENATURATION is
50 amino acids and more sometimes reversible = RENATURATION

- Molecular weights of proteins reach up collagen – constituent of bone and cartilage
to millions

- Protein molecules are composed of
CARBON, HYDROGEN, OXYGEN AND
NITROGEN

- Proteins have VERY HIGH MOLECULAR
WEIGHTS

- Proteins have 4 LEVELS OF STRUCTURE

1. Primary
- Amino acid sequence which determined
protein properties

2. Secondary
- Conformation of the backbone which is
either ALPHA HELIX or BETA PLEATED
REACTIONS OF PROTEINS 3. COLOR REACTION TO VARIOUS TESTS
1. HYDROLYSIS
- Refers to the breaking of the peptide a. BIURET TEST
bonds that connect amino acids to - It uses a light blue solution which turns
compose protein purple when mixed with a solution
containing protein.
- Strong acid is used to hydrolyze
proteins - In this test, Cu 2+ and alkali are used to
treat compounds containing two or
- In body, faster because of hydrolyzing more peptide bonds that yield Cu2+
enzymes peptide complex in a base solution

2. DENATURATION - Negative = blue color; fewer than 2
- Many ways to precipitate proteins peptide bonds

- The resulting molecule differs from the b. HOPKINS-COLE REACTION
native protein as a result of - Also known as GLYOXYLIC ACID TEST
denaturation (or coagulation)
- Test for the presence of indole group
a. One ways is through application of HEAT
- In this process, peptide chains are - when strong acid is added to an amino
disorganized because there is a acid containing indole group like
cleavage of the H-bonds and other TRYTOPHAN, a VIOLET colored complex
linkages. will be formed

b. Another method is the USE OF c. Millon’s Test
ORGANIC SOLVENTS - This test is to recognize the presence of
- Such as acetone, methanol or ethanol hydroxyphenyl group in proteins like
those present in the amino acid,
- The addition of water-miscible organic TYROSINE
solvents decreases the solubility of
most globular proteins in water to such - The reaction between the protein and
extent that they precipitate out of mercury forms a WHITE PRECIPITATE
solution which turns RED upon heating

c. USE OF METAL IONS
- Such as zinc, lead, mercury, iron and
copper
- Act as denaturing agents
- Precipitation occurs because proteins
become cross-linked by heavy metals



d. NINHYDRIN TEST - ADDITION of an alkali such as NaOH will
- Very widely used reaction to detect the give an ORANGE COLOR SOLUTION
free amino group in amino acids
Casein
- in this test, PROTEINS MUST BE HEATED - Milk protein
first to digest or hydrolyzed and make - Can be isolated by acidification
the amino acids free - At this pH, protein is at its isoelectric
point that is no. + = no. –
- Ninhydrin reacts with the free ALPHA Result is least soluble in water
AMINO GROUP yielding to a DEEP BLUE
COLOR SOLUTION On other hand, if negative or
positive, increase in water solubility
- Proline and hydroxyproline DO NOT
GIVE positive instead, yellow color is Dissolving the precipitate W/ ALCOHOL
produced (negative) CONTAINING 95% ETHANOL
- to remove fat that is precipitated along
e. Sulfur Test with casein
- Few amino acids containing sulfur such
as methionine, cysteine and cystine PROCEDURE
(oxidized cysteine)

- The reaction between H2S gas and lead
acetate paper yields to lead (II) sulfide
producing a BROWN OR BLACK COLOR
on the lead acetate paper

f. XanthRoproteic Test
- Characteristic reaction of proteins
containing aromatic rings

- this reaction is based on the ability of
aromatic ring to undergo nitration
reaction by adding NO2 group to their
structure

- the test is positive for amino acids such
as
phenylalanine
tyrosine
tryptophan

- a LEMON YELLOW solution is obtained
upon heating