BIOCHEMISTRY - LECTURE PRELIMINARY

EXPERIMENT 2A: PROTEINS ✓ Glutathione

- Tripeptide comprised of the three
Introduction
amino acids (cysteine, glutamic acid,
❖ Protein is the most abundant substance in and glycine); antioxidant and
the cell next to water, comprising 15 % of its detoxifying agent (National Center of
over-all mass. Biotechnology Information, 2020)
❖ Composed of amino acids as its building
block, linked together with peptide bonds
with a positive charged nitrogen-containing
group at one end and a negatively charged
carboxyl-group.
❖ Along the chain is a series of different side
chains from different amino acids.
❖ Some side chains are neutral, some are ✓ Egg Albumen
acidic, some are basic, and some are - Composition of Albumen (Powrie
classified as polar or nonpolar. 1973)
❖ The different tests in this experiment will
help you identify the different types of amino
present in a protein sample.

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
AMINO ACID CONTENT OF THE MAJOR
SAMPLES:
ALBUMENT PROTEINS
✓ Aspartame
- Artificial sweetener, commonly used as
sugar substitute
o peptide, a carboxylic acid and a methyl
ester
o alpha-carboxy group of L-aspartic acid
with the amino group of methyl L-
phenylalanine (National Center for
Biotechnology Informatic)
BIOCHEMISTRY - LECTURE PRELIMINARY

AMINO ACID CONTENT OF THE MAJOR

ALBUMEN PROTEINS cont.

✓ Hopkins-Cole Test
- Test for indole group, tryptophan (Nucum,
2005)
- Reagent: Hopkin’s Cole reagent and conc.
H2SO4
- Positive Result: purple ring at the interface
✓ Biuret Test
of two liquids
- General test for proteins, detecting dipeptide
- Dehydration of tryptophan, only amino acid
bonds (Nucum, 2005)
with indole group
- Reagents: – KOH, hydrated CuSO4,
- Reacts with reagent glyoxylic acid in
Potassium sodium tartarate
presence of H2SO4
- Positive result: Violet solution
- Blue color of basic solution of Cu2+ turns to
a violet color when a tripeptide or larger
peptide is present.
- The NaOH is there to raise the pH of the
solution to alkaline. ----

✓ Sakaguchi Test
- Test for guandine group, arginine
- Reagent: α- Napthol and a drop of sodium
hypobromite
- Positive Result: red-colored complex
solution; in alkaline solution react with
✓ Ninhydrin Test
sodium hypobromite as oxidizing agent.
- General test for proteins except proline
(Nucum, 2005)
(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)
BIOCHEMISTRY - LECTURE
✓ Xanthoproetic 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 activated.
- Nitric Acid gives color when heated with
proteins containing tyrosine (yellow color)
Tryptophan (orange color) due to nitration
- Adding ------- will deepen the color to
orange.
BIURET TEST
-test for proteins
✓ Reagents – KOH, hydrated CuSO4,
Potassium sodium tartarate or Cooper
Sulfate
✓ Positive result – Violet solution
PRELIMINARY
NINHYDRIN TEST
-used to detect the presence of amino acids
✓ Reagents- Ninhydrin (C9H6O4)
✓ Positive result – formation of red, blue
or purple color. We get a blue
coloration which we call Ruhemann's
purple
HOPKINS COLE
-used to detect the presence of tryptophan in
protein
✓ Reagents- Hopkins Cole reagent,
which consists of glyoxylic
acid.(Sulfuric Acid)
✓ Positive result – purple-colored ring at
the junction of two layers
SAKAGUCHI TEST
-used to detect the presence of arginine in
proteins
✓ Reagents- sodium hypobromite and 1-
naphthol (Sodium hydroxide and a-
naphthol)
✓ Positive result – red color
XANTHOPROTEIC TEST
-used to detect the presence of aromatic
amino acids (tyrosine and tryptophan)
✓ Reagents- 1 % tyrosine, 1 %
tryptophan, 1 % phenylalanine, 5 % egg
white (albumin), Nitric acid, 40 %
NaOH (Sodium Hydroxide)
✓ Positive result – dark yellow or
orange colored solution
BIOCHEMISTRY - LECTURE
PROTEIN DENATURATION (EXPERIMENT 2B)
What is Protein Denaturation?
✓ Protein denaturation is the partial or
complete disorganization of a protein’s
characteristic three-dimensional
shape
✓ Result of disruption of its secondary,
tertiary, and quaternary structural
interactions.
✓ Because the biochemical function of a
protein depends on its three-
dimensional shape, the result of
denaturation is loss of biochemical
activity
✓ Protein denaturation does note affect
the primary structure of a protein
✓ Some proteins lose all of their three-
dimensional structural characteristics
upon denaturation, most proteins
maintain some 3D structure
✓ 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 of precipitation out of
biochemical solution of denatured
protein
✓ Protein denaturation involves loss of
the protein’s three-dimensional
structure. Complete loss of such
structure procedures an
“unstructured” protein strand
PRELIMINARY
SELECTED PHYSICAL AND CHEMICAL
DENATURING AGENTS (DENATURANTS)
EFFECT OF HEAT
✓ Heat disrupt hydrogen bonds and non-
polar hydrophobic interaction
✓ Heat increases the kinetic energy and
causes the molecules to vibrate so
rapidly and violently that the bonds are
disrupted
✓ Proteins in eggs denature and
coagulate during cooking. Foods are
cooked to denature the proteins to
make it easier for enzymes to digest
them
✓ Medical supplies and instruments are
sterilized by heating to denature
proteins in bacteria and thus destroy
the bacteria (Ophardt, 2003; Stoker,
2017)
✓ High levels of thermal energy may
disrupt the hydrogen bonds that hold
the protein together
✓ As these bonds are broken, the protein
will begin to unfold and lose its
capacity to function as intended
✓ Temperature at which proteins
denature may vary, but most human
BIOCHEMISTRY - LECTURE PRELIMINARY

proteins function optimally at body attraction for sulfur and which lead to
temperature (37 degree centigrade) the denaturation of protein
✓ Heavy metals can disrupt bonds in the
EFFECT OF ALCOHOL
protein, causing it to lose its structure
✓ Hydrogen bonding occurs between ✓ Salts of heavy metals such as mercury
amide groups in the secondary protein and lead may be used to denaturation
structure interact with a protein’s functional side
✓ Hydrogen bonding between “side chain groups to form complexes.
chains” occurs in tertiary protein ✓ Heavy metals also oxidize the protein’s
structure in a variety of amino acid amino acid side chains
combinations ✓ Heavy metals (e.g., Hg2+, Pb2+, Cu2+)
✓ Alcohol denatures proteins by are high molecular weight cations
disrupting the side chain ✓ (+) charge of cations counteracts the (-
intramolecular hydrogen bonding ) charge of the carboxylate group in
✓ New hydrogen bonds are formed proteins giving a precipitate
instead between the new alcohol
molecule and the protein side chains
✓ 70% alcohol solution is used as a
disinfectant on the skin
✓ 70% of 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

EFFECT OF STRONG ACIDS (H+)

✓ Hydrogen bonding often involves these

side chains. PROTONATION of the
amino acid residue 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
EFFECT OF HEAVY METALS (Cu, Ag, Ba) which it functions
✓ Salt bridges result from the
✓ Ions form strong bonds with the
neutralization of an acid and amine on
carboxylate anions of the acidic amino
side chains; interaction is ionic
acids or -SH groups of cysteine,
between (+) amino group and (-) acid
disrupting ionic and disulfide linkages
group
✓ Heavy Metal Salts Disrupt Disulfide
Bonds:
✓ Heavy metals also disrupt disulfide
bonds because of their high affinity and
BIOCHEMISTRY - LECTURE PRELIMINARY

EFFECT OF ALKALOIDAL REAGENTS

✓ Alkaloidal reagents (e.g., tannate,

picric and 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. (“Denaturation”, 2019)