PROTEIN

GROUP 9
SAPALO
SIBUG
SIENNA
SORIANO
 CONTENT

1 Amino Acids & Proteins

2 Protein Denaturation

3 Color Reactions of Proteins & Amino

Acids
 PROTEINS AMINO ACIDS
- linear polymers of amino
acids

FUNCTIONS:

- Catalytic
- Contractile
- Structural
- Regulatory
- Protective
PRO TEIN STRUCTURE
AgNO
3

DENATURATION

Ethyl
Alcohol
PROTEIN
DENATURATION

Condition Result

Effect of heat 100° C Egg white

transparent at
first; after 5
minutes,
mixture is
cloudy;
precipitation
occurred (++)
 Reagent Result

Effect of alcohol 70% EtOH Clear at the

start; slightly
cloudy when
alcohol was
poured; slight
precipitation
occurred (+)

Clear at the
95% EtOH
start; turned
slightly cloudy
after alcohol
was poured; no
precipitation
occurred (-)
 Reagent Result

Effect of heavy metals Presence of

white
(A1) Silver 1% AgNO3
precipitate;
insoluble

(A2) Lead 10% Pb(Ac)2 Presence of

white
precipitate;
soluble
PROTEIN Aspartic
DENATURATION acid
AMINO ACIDS

Cysteine
Lysine

Glutamic
acid
Arginine
PROTEIN
DENATURATION
IN THE MEDICAL FIELD
ALCOHOL

HEAT 70%

HEAVY
METALS
COLOR REACTIONS OF
PROTEINS & AMINO ACIDS
Reagents Test for Results

Xanthoproteic Concentrated HNO3 Aromatic amino With HNO3: yellow to

Test acids clear solution

With 50% NaOH:

yellow to dark
orange solution
50% NaOH
XANTHOPROTEIC TEST
PRINCIPLE

- Xanthos (Greek) means yellow

- Aromatic groups can be derivatives of Benzene & can undergo

reaction e.g. NITRATION

● Nitration - adding Nitro Group (NO2) to a Benzene ring

- Tyrosine & Tryptophan are positive in this test

XANTHOPROTEIC TEST
PRINCIPLE
Equation

- Benzene ring undergoes nitration as it is heated with HNO3

- Appearance of Yellow Nitro Compounds = presence of aromatic

rings

- Alkaline condition (addition of NaOH) = orange colored due to

ionization of the aromatic group
ISOELECTRIC POINT

- the pH value at which concentration of anionic and

cationic groups are EQUAL (i.e. net charge = zero).

pH CHANGES & SOLUBILITY

- Solubility is dependent on molecule’s polarity as it is
affected by the solution’s

- If the pH of the protein solution = 0 net charge or in

ISOELECTRIC POINT (pI) = NON-POLAR = it is insoluble and
precipitates out of the solution.

- If pH is below or above the protein’s pI, (caused by adding

acid/base) = net positive charge or net negative charge =
BIURET TEST
Reagents Test for Results

Biuret Test NaOH Peptide With Egg White:

violet solution with
bonds blue precipitate

0.5% CuSO4

With Aspartame:
yellow solution with
orange precipitate
BIURET TEST
PRINCIPLE
- Cupric ions form violet-colored chelate complex with
unshared electron pairs of peptide nitrogen and oxygen of
water

- Violet color is due to the formation of a chelate complex

- The chelate complex absorbs light at 540 nm; hence, the

violet color

- The greater the concentration of peptide bonds = the

greater the color intensity.
BIURET TEST
ROLE OF REAGENTS
- CuSO4 provides the cupric ions which form the chelate
complex
BIURET TEST
ROLE OF REAGENTS
- NaOH raises the pH of the solution to an alkaline level
BIURET TEST & OTHER
PROTEINS

- 2 or more peptide bonds : required for the formation of the

chelate complex

- Proteins (multiple peptide bonds) yields POSITIVE RESULT

- Single amino acids (no peptide bond) yields NEGATIVE RESULT

- Dipeptide (containing 1 peptide bond) yields NEGATIVE RESULT

- Presence of free amino acids yields NEGATIVE RESULT

BIURET TEST
EGG WHITE VS. ASPARTAME
ASPARTAME EGG WHITE

Yellow Solution with Violet solution with

orange precipitate blue precipitate
BIURET TEST
EGG WHITE VS. ASPARTAME

ASPARTAME STRUCTURE

- Under alkaline conditions, aspartame’s peptide bonds become

hydrolyzed resulting to free amino acids

● Hydrolyze - chemical breakdown due to chemical reaction with

water

- Presence of free amino acids (not binded by peptide bonds) =

NEGATIVE RESULT
BIURET TEST
- Most amino acids account for a positive Biuret test result due to
the presence of multiple peptide bonds

BIURET TEST & PROTEIN HYDROLYSIS

Protein Hydrolysis
- occurs in the presence of the polypeptide chain, water, and enzyme

- water molecule binds to peptide chain to release an individual amino

acid from the polypeptide chain

- individual amino acid is called single amino acid

- Prevalent in digestion of essential amino acids from food

IMPORTANCE OF BIURET TEST IN PROTEIN
HYDROLYSIS

- Single amino acid produced by protein hydrolysis yields a negative

biuret test result

- Negative result is significant because it signifies that the proteins we

ingest in our body are being properly hydrolyzed/broken down so that
they can supply necessary nutrients in the bloodstream
COLOR REACTIONS OF
PROTEINS & AMINO ACIDS
Reagents Test for Results

Ferric Chloride Milk Phenolic – Without FeCl3

Test
containing solution: Light
FeCl3 solution amino brown
acids. With FeCl3
solution: Color
became lighter.
FERRIC CHLORIDE TEST

P R I N C I P L E & R O L E O F FeCl3

- Used to determine the presence of phenol through color test.

- The reaction of ferric chloride and phenol are both very dilute
and are both charge-transfer compound giving a very strong
visible spectral signal.
POSITIVE TEST

- Deep purple or green color

- Phenolic-amino acids
(NOT ALL phenols are positive)

- Addition of small amounts of

pyridine to FeCl3 and Phenol in
solvents benzene, toluene,
o=xylene, chloroform,
chlorobenzene, butyl bromide,
and ethylene dibromide
EQUATION

Phenol is reacted with Ferric Chloride and its

product is Ferric Phenoxide which is violet
colored.
COLOR REACTIONS OF
PROTEINS & AMINO ACIDS
Reagents Test for Results

Hopkins-Cole Hopkins – Cole Indole ring Positive:

Test
reagent of Purple ring in
between the
(Glyoxylic acid) tryptophan
sulfuric acid and
in proteins. egg white.

Concentrated
H2SO4
HOPKINS-COLE TEST
PRINCIPLE
- The formation of the purple ring is the result of the combination
of the indole group of tryptophan with glyoxylic acid in the
presence of H2SO4, forming two layers.

- The test is positive if the tryptophan is present.

COMPOSITION
- Hopkins - Cole reagent is composed of glyoxylic acid. When it is
added to a reagent, it gives color specifically in the shade of
blue.
EQUATION

Tryptophan reacts with glyoxylic acid in the

presence of sulfuric acid resulting to the violet ring.
POSITIVE RESULT
- Violet ring is visible.

- Tryptophan is
responsible for
positive result.

AMINO ACID
- Tryptophan
 Sakaguchi TestSakaguchi Test
NINHYDRIN TEST
Reagents Test for Results

NINHYDRIN Ninhydrin Amino groups With Eggwhite:

TEST except Proline Positive - Cloudy
and light violet
Hydroxyproline
With Aspartame:
Positive- Clear
violet
NINHYDRIN TEST
P Sakaguchi
R I N C I P L ETest
This test is used to detect a free amino group in all amino acids
except proline and hydroxyproline

EQUATION AND R O L E O F THE R E A G E N T

Ninhydrin, a strong oxidizing agent, reacts to the presence of
amino acids which will make the amino acid undergo oxidative
deamination and decarboxylation which frees an aldehyde, CO2
and ammonia, along with this, ninhydrin will undergo a reduction
to hydrindantin.
Ninhydrin then condensates with ammonia and hydrindantin
to produce an intensity blue or purple pigment, sometimes called
the Ruhemann’s Purple
Sakaguchi Test

Ninhydrin, a strong oxidizing agent, reacts to the presence of

amino acids which will make the amino acid undergo oxidative
deamination and decarboxylation which frees an aldehyde, CO2
and ammonia, along with this, ninhydrin will undergo a reduction
to hydrindantin.
Ninhydrin then condensates with ammonia and hydrindantin
to produce an intensity blue-purple pigment, sometimes called
the Ruhemann’s Purple
RESULTS
Sakaguchi Test Positive result

Negative result
APPLICATION
Sakaguchi Test
SAKUGUCHI TEST
Reagents Test for Results

SAKAGUCHI 0.2 % α naphthol Arginines Positive: Red or red

TEST (10- orange solution
hydroxynaphthalen
e)

Bleach (Sodium
hypochlorite)
Negative: No change
in color
Sakaguchi Test
PRINCIPLE

This test is used to detect amino acids that contain a

guanido group (Arginine)

Under alkaline solution, a guanidine compound like arginine

reacts with α naphthol which upon treatment of sodium
hypochlorite produces a red to red orange solution through
oxidation reaction.
LEAD ACETATE TEST
Reagents Test for Results

LEAD ACETATE 50% NaOH Cysteine Positive:Black

TEST (Sulfahydril or - Precipitate
Pb(CH3COO)2 SH group)
 Sakaguchi
LEAD ACETATETest
TEST
PRINCIPLE

Cysteine - SH + 50% NaOH Na2S

Na2S + Pb(CH3COO)2 PdS + CH3COONa

This test is used to detect the sulphide group in cystein or cystine.

 Sakaguchi
LEAD ACETATETest
TEST
PRINCIPLE

Cysteine - SH + 50% NaOH Na2S

Na2S + Pb(CH3COO)2 PbS + CH3COONa

When cystein containing proteins are boiled with strong alkali, the
sulfur present in the protein is liberated as Sodium sulfide. This reacts
with Lead Acetate to form a brown or black precipitate of Lead
Sulphide.