BIOCHEMISTEY LABORATORY ➢ The particles are suspended in a liquid medium

and placed in a centrifuge tube. The tube is then

THE CELL: EXPERIMENT 1A placed in a rotor and spun at a desire speed.
➢ Separation through sedimentation could be
Introduction:
done naturally with the earth gravity,
nevertheless, it would take ages. Centrifugation
is making that natural process faster.
➢ Rotation of the rotor about a central axis
generates a centrifugal force upon the particles
in the suspension.
Materials Ingredients Reagent
o Beaker o 7g o 75 ml of
o Blender chicken sucrose
o Scalpel liver solution
o Graduated (keep o Distilled
cylinder frozen Water
o Centrifuge before
machine use
o Test tube
brush
o Filter
paper
o Test tubes
o Test tube
rack
o Stirring
rod

Pre lab questions: Procedure:

1. What is differential centrifugation? A. HOMOGENIZATION OF LIVER

• To separate organelles. o Homogenization is the process by which
a sample is broken into identical parts so
that removing one portion of it does not
disrupt and still accurately reflects the
remaining sample’s molecular
composition.
1. Thaw the liver first
2. Wash the thawed liver with distilled water
then with 75 ml sucrose solution.
3. Place the liver in a filter paper to thoroughly
dry it.
4. Minced the liver in a watch glass using
scalper (video: using mortar and pestle) and
transfer in a beaker.
5. Add 35 ml of 75 ml sucrose solution and
homogenize using a blender for 5-10
Centrifugation- is used for separation of particles from a minutes.
solution according to their size, shape, density, viscosity 6. Transfer the homogenate in test tubes for
of medium and other speed. centrifugation.
Remember:
Sediment- solid
Supernatant- liquid
Homogenization- Cell fractionation- Cell
Homogenization.
B. DIFFERENTIAL CENTRIFUGATION
1. Centrifuge the homogenate at rate 2 in 5
minutes
2. Decant and label the residue as sediment I.
3. Centrifuge the supernatant at rate 5 in 15
minutes and decant. Label residue as sediment
II.
4. Centrifuge the supernatant at rate 8 for 20
minutes ad decant. Label the residue as
sediment III and supernatant III.
5. Seal the samples and keep in refrigerator until
next lab period.
Remember:
Decant-Decantation: In the laboratory, the process of
pouring away a liquid while leaving a solid (often a
precipitate) behind. Decanting a liquid from a solid using
a stirring rod.
QUALITATIVE DETERMINATION OF SUBCELLULAR between two liquids. Record your
COMPONENTS: EXPERIMENT 1B observation.
C. Biuret Test
Introduction:

D. Sudan Test
Objective:

❏ To perform the different qualitative tests in the

differentiated samples in 1A; To identify the
biomolecules, present in the samples; To be
acquainted on the different chemical tests that
may be encountered in biochemistry laboratory

DEFINITIONS:
❖ DISCHE TEST
▪ One major difference between DNA and RNA
is their sugar: DNA contains deoxyribose,
whereas RNA contains ribose.
▪ DNA can be identified chemically with the
Dische diphenylamine test.
▪ Acidic conditions convert deoxyribose to a
molecule that binds with diphenylamine to
form a blue complex (Libretexts, 2020)

(i-note nalang ninyo materials hehe from the video)

Procedures:

▪ identify if there’s a presence of deoxyribose in

A. Dische Test
the sample in which it contains because under
1. Add 3ml of diphenylamine solution to
acidic conditions nucleic acid tends to react with
each test tube containing 1ml of the
diphenylamine (Patterson and Mura, 2013)
sample and mix well
2. Heat in water bath for 10 minutes and ▪ The intensity of the blue color is proportional to
cool in an ice bath. A clear tube means the concentration of DNA
absence of DNA or RNA.
B. Molish Test ▪ Test will detect the deoxyribose of DNA & will
1. Add 3-5 drops of molish reagent to each not interact with the ribose in RNA (Libretexts,
test tube containing 1ml of the sample 2020)
and mix well. RESULT:
2. To test each test tube, add 1ml
concentrated sulfuric acid along the side ▪ Blue color indicates the presence of DNA.
of the walls of the inclined tube and do ▪ Negative result is clear.
not mix. Observed a colored ring in
 ❖ MOLISH TEST ▪ The principle of biuret test is conveniently used
Molisch’s test is a general test for all to detect the presence of proteins in biological
carbohydrates. A positive reaction is indicated by fluids
appearance of a purple red ring at the interface ▪ CuSO4 reacts with compounds containing two or
between the acid and test layers. more peptide bonds to give a violet colored
product (Nucum, 2005)
▪ Monosaccharides give a rapid positive test. ▪ due to complex formation of cupric ions with
Disaccharides and polysaccharides react slower.
unshared electron pairs of peptide nitrogen and
▪ The test solution is combined with a small O2 of water.
amount of Molisch's reagent (α-naphthol) then ▪ Positive biuret test: The formation of a pink-
heat with conc. H2SO4 violet color indicates the presence of a protein
▪ After mixing, a small amount of concentrated with two or more peptide bonds.
sulfuric acid is slowly added down the sides of ▪ If such a protein is not present, the blue color of
the sloping test-tube, without mixing, to form a the copper (II) sulfate will remain (negative
layer. result).

▪ All carbohydrates – monosaccharides,

disaccharides, and polysaccharides (except
trioses and tetroses)– should give a positive
reaction
▪ Concentrated H2SO4 (sulfuric acid) to form
furfural, classified as aldehyde and organic
compounds, and its derivatives.
▪ This product when reacted with sulphonated
alpha-naphtol turns into a purple color RESULT:
▪ Dehydrate pentose to form furfural & dehydrate ▪ Purple like color is a positive result of Biuret Test.
hexose to form 5-hydroxymethyl furfural
❖ SUDAN TEST
▪ Lipids include a variety of molecules that dissolve
in nonpolar solvents such as ether and acetone,
but not in polar solvents such as water.
▪ Tests for lipids, triglycerides and lipoproteins:
based on a lipid's ability to selectively absorb
pigments in fat-soluble dyes such as Sudan IV.
▪ Lipids less dense than water and insoluble, it will
form a layer or globules above the water and
appear as a red layer

RESULT:
▪ The formation of a purple ring is a positive
indicator for Molisch’s Test

❖ BIURET TEST
▪ Biuret test is a general test for compounds RESULT:
containing two or more peptide bond (CO-NH
▪ 2 layers; top layer is orange red
group).
▪ Since all proteins and peptides possessing at
least two peptide linkage ie. tripeptide it gives
positive biuret test.
 detoxifying agent (National Center for
Biotechnology Information, 2020).

o Egg Albumen Composition of Albumen

PROTEINS: EXPERIMENT 2A (Powrie 1973)
Introduction: ▪ Proteins Lipids Carbohydrates Ash Water 9.7-
10.6%0.03% 0.4-0.9% 0.5-0.6% 87.9-89.4%
▪ is the most abundant substance in the cell next MAJOR PROTEINS IN ALBUMEN (Powrie 1973)
to water, comprising 15% of its overall mass. ▪ Ovalbumin 54%
Proteins is composed of amino acids as its ▪ Conalbumin 13%
building block. It is linked together with peptide ▪ Ovomucoid 11%
bond with a positive charge nitrogen-containing ▪ Lysozyme 3.5%
group at one end and a negatively charged ▪ Globulins (G2, G3) 8.0% (?)
carboxyl group. ▪ Ovomucin 1.5%
▪ Some side chains are neutral, some are acidic, ▪ Other protein components include, flavoprotein
some are basic, and some are classified as polar (.8%), ovoglycoprotein (.5%), ovomacroglobulin
or nonpolar. (.5%), ovoinhibitor (.l%) and avidin (.05%).
Objectives:

❏ To perform qualitative test for different types of

proteins ❏ To identify proteins based on the different
tests performed ❏ Relate the test results to the chemical
structure of each protein or amino acid.
Samples:
o Aspartame - artificial sweetener,
commonly used as sugar substitute
▪ dipeptide, a carboxylic acid and a methyl ester
▪ alpha-carboxy group of L-aspartic acid with the
amino group of methyl L-phenylalaninate
(National Center for Biotechnology Information,
2020)

o Glutathione - tripeptide comprised of

three amino acids (cysteine, glutamic
acid, and glycine); antioxidant and
QUALITATIVE TEST: ❖ Hopkins-Cole Test
▪ Test for indole group, tryptophan (Nucum, 2005)
❖ Biuret Test ▪ Reagent: Hopkin’s Cole reagent and conc. H2
▪ General test for proteins, detecting peptide
SO4
bonds (Nucum, 2005) ▪ Positive Result: purple ring at the interface of
▪ Reagents – KOH, hydrated CuSO4, Potassium two liquids
sodium tartarate ▪ Dehydration of tryptophan, only amino acid with
▪ Positive result – Violet solution indole group
▪ blue color of a basic solution of Cu2+ turns to a ▪ reacts with the reagent glyoxylic acid in presence
violet color when a tripeptide or larger peptide is of H2 SO4
present.
▪ The NaOH is there to raise the pH of the solution
to alkaline levels; the crucial component is the
copper II ion (Cu2+) from the CuSO4.

❖ Sakaguchi Test
▪ Test for guanidine group, arginine
▪ Reagent: α-Naphthol and a drop of
sodium hypobromite
▪ Positive Result: red-colored complex solution; in
alkaline solution react with sodium hypobromite
❖ Ninhydrin Test
as oxidizing agent (Nucum, 2005)
▪ General test for proteins except proline (yellow)
(Hunt, n.d.)
▪ Test for the –NH2 group in free amino acid;
▪ Positive Result - a deep blue or purple color
solution
▪ Reagent – Ninhydrin degrades amino acids into
aldehydes, ammonia and CO2 (on pH range 4-8)
(Perret & Nayuni, 2014)
▪ Ninhydrin then condenses with ammonia and
hydrindantin to produce an intensely blue or
purple pigment (ruhemann‘s purple) ❖ Xanthoproteic Test
▪ Test for activated aromatic rings, Tyrosine and
tryptophan
▪ Reagents: Conc. Nitric Acid, ammonium
hydroxide
▪ Positive result: Yellow solution or precipitate
(Nucum, 2005)
▪ tyrosine and tryptophan contain activated
benzene rings and readily undergo nitration,
phenylalanine has benzene ring but not
REMEMBER:
activated
Q: WHAT IS DIFFERENCE BETWEEN BIURET TEST AND ▪ Nitric acid gives color when heated with proteins
NINHYDRIN TEST? containing tyrosine (yellow color) Tryptophan
(orange color) due to nitration ● adding 50%
-THEY ARE BOTH TEST FOR PROTEINS AND AMINO sodium hydroxide (strong base) will deepen the
ACIDS, HOWEVER IN BIURET TEST, IT NEEDS TO HAVE color to orange
TWO OR MORE PEPTIDE BOND FOR IT TO GIVE A
POSITIVE RESULT.
 ▪ Renaturation - limited denaturation changes
conditions can be reversed, in which the protein
is “refolded,”.
▪ For extensive denaturation changes, the process
is usually irreversible.
▪ Loss of water solubility is a frequent physical
consequence of protein denaturation.
▪ Coagulation precipitation out of biochemical
solution of denatured protein.
Protein denaturation involves loss of the protein’s three-
dimensional structure. Complete loss of such structure
produces an “unstructured” protein strand.

PROTEIN DENATURATION: EXPERIMENT 2B

What is Protein Denaturation?
▪ Protein denaturation is the partial or complete Household materials: Household Ingredients:
disorganization of a protein’s characteristic
three-dimensional shape. 1. Medicine cups 1. Egg white – 15ml
▪ Result of disruption of its secondary, tertiary, 2. Mixing bowl 2. Vinegar
and quaternary structural interactions. 3. Spoon and fork 3. 70% alcohol
▪ Because the biochemical function of a protein 4. Kitchen tong available at
depends on its three-dimensional shape, the 5. Egg separator home
result of denaturation is loss of biochemical 6. Syringe/Dropper 4. Distilled water
activity. 7. Cooking stove
▪ Protein denaturation does not affect the primary
PROCEDURE
structure of a protein.

A. Sample Preparation for Proteins

▪ Egg Albumin Solution- mix 5ml of egg white
in 45ml water
B. Effect of Mechanical agitation
1. Get 5 ml from the remaining egg white
Objectives: above.
2. Transfer the egg white into a bowl.
❏ To observe the effects of several denaturing reagents 3. Using a fork or whisk, beat the egg white
on a protein sample. ❏ To differentiate the effect of until change of color from transparent to
these denaturing reagents to the protein sample. ❏ To white.
provide practical applications of these denaturing
4. Record your observation.
agents.
C. Effect of Heat
▪ Some proteins lose all of their three-dimensional 1. Cook 5 ml of egg white in you pan at a very
structural characteristics upon denaturation, low heat for a minute.
most proteins maintain some 3D structure. 2. Remove the egg and place it in a plate.
 3. Compare the result with the original egg ▪ temperatures at which proteins denature may
white. vary, but most human proteins function
D. Effect of alcohol optimally at body temperature.
1. Place 2.0 ml of egg albumin solution in a
❖ Effect of Alcohol
medicine cup.
▪ Hydrogen bonding occurs between amide
2. Add 5.0 ml of 70% alcohol available at
groups in the secondary protein structure.
home. ▪ Hydrogen bonding between "side chains" occurs
3. Stir and leave for 5 minutes. in tertiary protein structure in a variety of amino
4. Compare the result with the original egg acid combinations.
white. ▪ Alcohol denatures proteins by disrupting the side
E. Effect of Acid chain intramolecular hydrogen bonding.
1. Place 2.0 ml of egg albumin solution in a ▪ New hydrogen bonds are formed instead
medicine cup. between the new alcohol molecule and the
2. Add 5.0 ml vinegar. protein side chains.
3. Mix thoroughly and leave for 5 minutes. ▪ 70% alcohol solution is used as a disinfectant on
the skin
4. Compare the result with the standard.
▪ 70% alcohol is able to penetrate the bacterial cell
wall and denature the proteins and enzymes
inside of the cell.
▪ 95% alcohol solution merely coagulates the
protein on the outside of the cell wall and
prevents any alcohol from entering the cell.

DEFINITIONS:
❖ Effect of Heat
▪ Heat disrupt hydrogen bonds and non-polar
hydrophobic interactions.
▪ heat increases the kinetic energy and causes the ❖ Effect of Heavy Metals
molecules to vibrate so rapidly and violently that ▪ Ions form strong bonds with the carboxylate
the bonds are disrupted. anions of the acidic amino acids or SH groups of
▪ proteins in eggs denature and coagulate during cysteine, disrupting ionic and disulfide linkages.
cooking. foods are cooked to denature the ▪ Heavy Metal Salts Disrupt Disulfide Bonds:
proteins to make it easier for enzymes to digest ▪ Heavy metals also disrupt disulfide bonds
them. because of their high affinity and attraction for
▪ Medical supplies and instruments are sterilized sulfur and which lead to the denaturation of
by heating to denature proteins in bacteria and proteins
thus destroy the bacteria. ▪ Heavy metals can disrupt bonds in the protein,
▪ High levels of thermal energy may disrupt the causing it to lose its structure.
hydrogen bonds that hold the protein together ▪ Salts of heavy metals such as mercury and lead
▪ As these bonds are broken, the protein will begin may be used to denature can interact with a
to unfold and lose its capacity to function as protein's functional side chain groups to form
intended complexes.
▪ Heavy metals also oxidize the protein's amino
acid side chains (Ophardt, 2003 & Stoker, 2017)
▪ Heavy metals (e.g. Hg2+, Pb2+, Cu2+) are high
molecular weight cations.
▪ (+) charge of cations counteracts the (-) charge
of the carboxylate group in proteins giving a
precipitate.

❖ Effect of Strong Acids

▪ Hydrogen bonding often involves these side
chains. Protonation of the amino acid residues
changes whether or not they participate in
hydrogen bonding, so a change in the pH can
denature a protein.
▪ All proteins have an optimal pH which is
dependent on the environment in which it
functions
▪ Salt bridges result from the neutralization of an
acid and amine on side chains; interaction is ionic
between (+) amino group and (-) acid group

❖ Effect of Alkaloidal Reagents

▪ Alkaloidal reagents (e.g. tannate & trichloro
acetate) are high molecular weight anions.
▪ These reagents combine with (+) amino groups
in proteins to disrupt ionic bonds.
▪ The negative charge of these anions counteracts
the (+) charge of the amino group in proteins
giving a precipitate.