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A.

TITLE:
Learning Characteristic and Color Reaction from Protein
B. DATE OF EXPERIMENT:
November, 28th 2014
C. PURPOSE OF EXPERIMENT:
1. Differentiate the solubility properties of protein as reversible and irraversible
2. Differentiate the denaturation reaction of protein that caused by acid, salt, and
salt from hard metal, and also the heating based on observation
3. Understand the cause of precipitation in protein identify the protein occurence
based on the color reaction
4. Identify the protein occurence based on the color reaction

D. BASIC THEORY
Protein, highly complex substance that is present in all living organisms.
Proteins are of great nutritional value and are directly involved in the chemical
processes essential for life. The importance of proteins was recognized by the
chemists in the early 19th century who coined the name for these substances from
the Greek proteios, meaning “holding first place.” Proteins are species-specific;
that is, the proteins of one species differ from those of another species. They are
also organ-specific; for instance, within a single organism, muscle proteins differ
from those of the brain and liver. (Haurowitz:2014)
Proteins are organic compounds that contain the element nitrogen as well as
carbon, hydrogen, and oxygen. Proteins are the most diverse group of biologically
important substances and are often considered to be the central compound
necessary for life. In fact, the translation from the Greek root word means “first
place.” Skin and muscles are composed of proteins; antibodies and enzymes are
proteins; some hormones are proteins; and some proteins are involved with
digestion, respiration, reproduction, and even normal vision, just to mention a
few.
Amino Acids, There are obviously many types of proteins, but they are all
made from amino acids bonded together by the dehydration synthesis. By
continually adding amino acids, called peptides, two amino acids join together to
form dipeptides; as more peptides join together, they form polypeptides. Proteins
vary in length and complexity based on the number and type of amino acids that
compose the chain. There are about 20 different amino acids, each with a different
chemical structure and characteristics; for instance, some are polar, others are
nonpolar. The final protein structure is dependent upon the amino acids that
compose it. Protein function is directly related to the structure of that protein. A
protein's specific shape determines its function. If the three-dimensional structure
of the protein is altered because of a change in the structure of the amino acids,
the protein becomes denatured and does not perform its function as expected.
Protein Structure, The three-dimensional geometry of a protein molecule is
so important to its function that four levels of structure are used to describe a
protein. The first level, or primary structure, is the linear sequence of amino acids
that creates the peptide chain.

In the secondary structure, hydrogen bonding between different amino acids


creates a three-dimensional geometry like an alpha helix or pleated sheet. An
alpha helix is simply a spiral or coiled molecule, whereas a pleated sheet looks
like a ribbon with regular peaks and valleys as part of the fabric.

The tertiary structure describes the overall shape of the protein. Most tertiary
structures are either globular or fibrous.
Generally, nonstructural proteins such as enzymes are globular, which means
they look spherical. The enzyme amylase is a good example of a globular protein.
Structural proteins are typically long and thin, and hence the name, fibrous.
Quaternary structures describe the protein's appearance when a protein is
composed of two or more polypeptide chains. Often the polypeptide chains will
hydrogen bond with each other in unique patterns to create the desired protein
configuration.

Characteristic of Protein,
General Characteristics of Proteins are as follows:

 Proteins are organic substances, they are made up of nitrogen and also,
oxygen, carbon an d hydrogen.
 Proteins are the most important biomolecules, they are the
fundamental constituent of the cytoplasm of the cell.
 Proteins are the structural elements of body tissues.
 Proteins are made up of amino acids.
 Proteins gives heat and energy to the body and also aid in building and
repair.
 Amphoteric properties of proteins due to the presence of free carboxylic
and free amino groups at the end of protein it can react with acids and
bases.
Only small amounts of proteins are stored in the body as they can be used
up quickly on demand.
 Proteins are considered as the bricks, they make up bones, muscles, hair
and other parts of the body.
 Proteins like enzymes are functional elements that take part in metabolic
reactions.
 Antibodies, blood haemoglobin are also made of proteins.
 Proteins have a molecular weight of 5 to 300 kilo-daltons.

Properties of Proteins

a. Physical Properties of Proteins

 Proteins are colorless and tasteless.


 They are homogeneous and crystalline.
 Proteins vary in shape, they may be simple crystalloid structure to long
fibrilar structures.

b. Chemical Properties of Proteins

 Proteins when hydrolyzed by acidic agents, like conc.HCl yield amino


acids in the form of their hydrochlorides.
 Proteins when are hydrolyzed with alkaline agents leads to hydrolysis of
certain amino acids like arginie, cysteine, serine, etc., also the optical
activity of the amino acids is lost.
 Proteins with reaction with alcohols gives its corresponding esters. This
process is known as esterification.
 Amino acids reacts with amines to form amides.
 When free amino acids or proteins are said to react with mineral acids like
HCl, the acid salts are formed.
 When amino acids in alkaline medium reacts with many acid chlorides,
acylation reaction takes place.
 Sanger's reaction - Proteins react with FDNB reagent to produce yellow
colored derivative, DNB amino acid.
 Xanthoproteic test - On boiling proteins with conc. HNO3, yellow color
develops due to presence of benzene ring.
 Folin's test - This is a specific test for tyrosine amino acid, where blue
color develops with phosphomolybdotungstic acid in alkaline solution due
to presence of phenol group.

Denaturation of proteins involves the disruption and possible destruction of


both the secondary and tertiary structures. Since denaturation reactions are not
strong enough to break the peptide bonds, the primary structure (sequence of
amino acids) remains the same after a denaturation process. Denaturation disrupts
the normal alpha-helix and beta sheets in a protein and uncoils it into a random
shape.
Denaturation occurs because the bonding interactions responsible for the
secondary structure (hydrogen bonds to amides) and tertiary structure are
disrupted. In tertiary structure there are four types of bonding interactions between
"side chains" including: hydrogen bonding, salt bridges, disulfide bonds, and non-
polar hydrophobic interactions. which may be disrupted. Therefore, a variety of
reagents and conditions can cause denaturation.

The most common observation in the denaturation process is the precipitation


or coagulation of the protein.
a. Heat can be used to disrupt hydrogen bonds and non-polar hydrophobic
interactions. This occurs because heat increases the kinetic energy and causes
the molecules to vibrate so rapidly and violently that the bonds are disrupted.
The proteins in eggs denature and coagulate during cooking. Other 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.
b. In addition to heat, acids and bases can also make denatured proteins. As it
has been known that proteins can form a zwitter ion structure. Protein also
has the isoelectric point where the amount of positive charge and a negative
charge on the protein is the same. At that moment, denatured proteins can be
characterized by the forming clot and the solution becomes cloudy. At this
time the enthalpy of dissolution will be high, because the amount of heat
required to dissolve the amount of protein will increase. The mechanism is
the addition of acids and bases can disrupt the salt bridges found in proteins.
Positive and negative ions in the salt can be replaced with a pair of positive
and negative ions of an acid or base so that the salt bridge in a protein that is
one type of interaction on the protein, can be said to be chaotic and denatured
proteins.

The solubility of proteins in aqueous buffers depends on the distribution of


hydrophilic and hydrophobic amino acid residues on the protein’s surface.
Hydrophobic residues predominantly occur in the globular protein core, but some
exist in patches on the surface. Proteins that have high hydrophobic amino acid
content on the surface have low solubility in an aqueous solvent. Charged and
polar surface residues interact with ionic groups in the solvent and increase the
solubility of a protein. Knowledge of a protein's amino acid composition will aid
in determining an ideal precipitation solvent and methods.
Precipitation of Protein with Heavy Metal
Basic precipitation reaction by heavy metal is charge neutralization.
Precipitation can be occurred if protein is in negative isoelectric state. With
presence of positive charge in heavy metal, neutralization reaction will be
occurred from protein and then produce proteinat neutral salt which precipitates.
This protein precipitation will dissolve by adding alkali (NH3, NaOH, etc.). This
protein precipitation property is reversible. From this theory, protein often use as
metal poisonous medicine, as mercury, copper, etc.
Precipitation of Protein with Ammonium Sulphate
Precipitation is a method used to purify proteins by altering their solubility. It
is a specific case of a more general technique known as salting out. Ammonium
sulfate is commonly used as its solubility is so high that salt solutions with high
ionic strength are allowed. The solubility of proteins varies according to the ionic
strength of the solution, and hence according to the salt concentration. Two
distinct effects are observed: at low salt concentrations, the solubility of the
protein increases with increasing salt concentration (i.e. increasing ionic strength),
an effect termed salting in. As the salt concentration (ionic strength) is increased
further, the solubility of the protein begins to decrease. At sufficiently high ionic
strength, the protein will be almost completely precipitated from the solution
(salting out). Since proteins differ markedly in their solubilities at high ionic
strength, salting-out is a very useful procedure to assist in the purification of a
given protein. The commonly used salt is ammonium sulfate, as it is very water
soluble, forms two ions high in the Hofmeister series, and has no adverse effects
upon enzyme activity. It is generally used as a saturated aqueous solution which is
diluted to the required concentration, expressed as a percentage concentration of
the saturated solution (a 100% solution).
In this experiment crash and milk protein solution respectively - each added a
solution of ammonium sulphate produce a white precipitate. White precipitate was
formed as ammonium sulfate can cause dehidratasi water, so the solubility
decreases. Then the precipitate was dissolved on the addition of water. Means in
crash and milk proteins contain amino groups that have low solubility which
causes it to settle and the precipitate formed is reversible because it can be
dissolved again.
Precipitation of Protein with Concentrated Mineral Acid
Concentrated mineral acid treatment in protein can cause salt compound is
formed from acid reaction with protein amino group. Another effect, irreversible
denaturation can be occurred and form protein precipitate. But generally,
precipitation by adding strong mineral acid (except concentrated HNO3) has
reversible property.
General color reaction of protein

Biuret Test Add 5 drops of 1% CuSO4 to 4 ml of 5% NaOH. Divide into two


(procedure) portions. Add 2ml of distilled water to one and 2ml of protein solution
to the other.
Biuret Test In alkaline medium copper from CuSO4 will form a coordination
(principle) complex with the peptide bond. A minimum of 3 peptides are required
to answer this test. Biuret test is not performed on urine because of
presence of peptide like linkage.
Ninhydrin Test Boil 1ml of protein solution with 5 drops of ninhydrin reagent.
(procedure)
Ninhydrin Test This test is given by the free amino acids, small peptides and protein
(principle) will react to give purple color. Ninhydrin reacts with amino acids to
form hyrindantin and then it further forms Ruheuman's purple by
reacting with amonia and another ninhydrin. Imino acids give yellow
color owing to absence of alpha amino acids.
Xanthoproteic To 1ml of protein solution add 1ml of concentrated HNO3. Boil for 30
Test (procedure) seconds. Coo under tap water and add 2-3 ml of 40% NaOH.
Xanthoproteic This test is answered by aromatic amino acids like tyrosine and
Test (principle) tryptophan. Phenyl alanine gives a weak positive reaction. When a
preotein solution is heated with concentrated nitric acid, the benzene
ring under goes nitration to form yellow nitro derivatives. When treated
with NaOH the sodium salt formed is tense orange in color.
Millon's Test Add 2-3 drops of mercuric sulphate in 10% H2SO4 to 1ml of protein
(procedure) solution. Boil gently of flame for 30 seconds. Add 2-3 drops of 1%
sodium nitrate solution.
Millon's Test Specific to phenolic group of thyrosine. The hydroxy benzene group of
(principle) thyrosine reacts with Millon's reagent to form red colored complex.
Aldehyde Add 1 drop of 10% mercuric sulphate in 10%H2SO4 to 1 ml of protein
Reaction solution and 1 drop of formaline. Mix well. Add 2 ml of concentrated
(procedure) H2SO4 along sides of test tube.
Aldehyde The test indicates presence of indole group containing amino acid. The
Reaction oxidizing agents are HgSO4 and H2SO4 which oxidize indole nucleus
(principle) of tryptophan. This oxidized indole ring reacts with formaldehyde to
give the violet colored ring at the junction of two liquids.
Sakaguchi's Test To 3ml of protein solution and add 2 drops of Molisch's reagent. Then
(procedure) add 6 drops of alkaline hypobromite.
Sakaguchi's Test Test is answered by arganine. The guanidine group reacts with alpha
(principle) napthol and sodium hypobromite to form deep red color.
Alkali Labile To 3ml of protein solution in a boiling test tube. Add equal volume of
Reaction 40% NaOH. Boil on direct flame for at least two minutes. Then add 3-4
(procedure) drops of lead acetate.
Alkali Labile When a protein solution is boiled with an alkali, the sulfur of cysteine
Reaction and cystine splits to form sodium sulfide. This forms black lead sulfate
(principle) on acting with lead acetate.

This test is not answered by methionine because the sulphur is involved


in a thioether linkage. This is not split by boiling with strong alkali.
Pauly's Test To 2ml of protein solution, add diazotized sulphanilic acid followed by
(procedure) 1ml of 20% Na2CO3.
Pauly's Test Imidazole group reacts with diazotized aulphanilic acid to form highly
(principle) colored azocompouds. In alkaline medium this is red in color.
Diazotized sulphanilic acid is also called Van Den Berg's reaction.

Color reaction in this experiment


a. Biuret Reaction
The biuret test is a chemical test used for detecting the presence of
peptide bonds. In the presence of peptides, a copper(II) ion forms violet-
colored coordination complexes in an alkaline solution.[1] Several variants on
the test have been developed, such as the BCA test and the Modified Lowry
test. The biuret reaction can be used to assess the concentration of proteins
because peptide bonds occur with the same frequency per amino acid in the
peptide. The intensity of the color, and hence the absorption at 540 nm, is
directly proportional to the protein concentration, according to the Beer-
Lambert law. Despite its name, the reagent does not in fact contain biuret
((H2N-CO-)2NH). The test is so named because it also gives a positive
reaction to the peptide-like bonds in the biuret molecule.

b. Xanthoprotein Reaction
The xanthoproteic test is a method that can be used to determine the
amount of protein soluble in a solution, using concentrated nitric acid. The
test gives a positive result in those proteins with aminoacids carrying
aromatic groups, especially in the presence of tyrosine. If the test is positive
the proof is neutralized with an alkali, turning dark yellow. This chemical
reaction is a qualitative test, determining the presence or absence of proteins.
To quantify, it is used another reaction, such as the Biuret, and an analysis is
made by photometric spectrum.

c. Ninhydrin Reaction
Ninhydrin (2,2-Dihydroxyindane-1,3-dione) is a chemical used to detect
ammonia or primary and secondary amines. When reacting with these free
amines, a deep blue or purple color known as Ruhemann's purple is produced.
Ninhydrin is most commonly used to detect fingerprints, as the terminal
amines of lysine residues in peptides and proteins sloughed off in fingerprints
react with ninhydrin. It is a white solid which is soluble in ethanol and
acetone at room temperature. Ninhydrin can be considered as the hydrate of
indane-1,2,3-trione.

d. Millon Reaction
Millon’s test is given by any compound containing a phenolic
hydroxy group. Consequently, any protein containing tyrosine will give a
positive test of a pink to dark-red colour. The Millon reagent is a solution of
mercuric and mercurous ions in nitric and nitrous acids (caution: millon’s
reagent is highly toxic and highly corrosive). The red colour is probably due
to a mercury salt of nitrated tyrosine. Millon's test is not specific for proteins
(it actually detects phenolic compounds), and so must be confirmed by other
tests for proteins such as the biuret test and the ninhydrin reaction. The
reagent is made by dissolving metallic mercury in nitric acid and diluting
with water.
e. Hopkin-Cole Reaction
The Hopkins-Cole reaction, also known as the glyoxylic acid
reaction, is a chemical test used for detecting the presence of tryptophan in
proteins. A protein solution is mixed with Hopkins Cole reagent, which
consists of glyoxylic acid. Concentrated sulfuric acid is slowly added to form
two layers. A purple ring appears between the two layers if the test is positive
for tryptophan. Nitrites, chlorates, nitrates and excess chlorides prevent the
reaction from occurring. When the violet or purple ring appears after the two
layers within an indole nucleus meet, this confirms that concentrated sulfuric
acid was added to a mixture of some sort that contained glyoxylic acid and a
protein. However, there are some products that do not show the reaction, such
as gelatin and zein.

Hydrolysing proteins using hydrochloric acid

If you have already studied the hydrolysis of amides under acidic conditions,
you will find that this is basically the same reaction. That's not surprising because
what biologists and biochemists call a peptide link (in proteins, for example) is
what chemists call an amide link. With an amide like ethanamide, the carbon-
nitrogen bond in the amide group is broken and you get a carboxylic acid formed:

Now imagine doing the same thing with a simple dipeptide made of any two
amino acids.
Instead of ammonium ions, you get positive ions made from the -NH2 groups
reacting with hydrogen ions. You need the extra hydrogen ion in the equation
(compared with the amide equation) to react with the -NH2 group on the left-hand
end of the dipeptide - the one not involved in the peptide link. If you scale this up
to a polypeptide (a protein chain), each of the peptide links will be broken in
exactly the same way. That means that you will end up with a mixture of the
amino acids that made up the protein - although in the form of their positive ions
because of the presence of the hydrogen ions from the hydrochloric acid.

E. TOOLS AND MATERIALS


1. Tools
- Beaker glass 250 mL (1 pieces)
- Pipette (10 pieces)
- Test tube (10 pieces)
- Graduated cylinder 10 mL (2 pieces)
- Test tube rack (1 piece)
- Spirtus burner (1 piece)
- Tripod (1 piece)
- Gauze wire (1 piece)
2. Materials
- Milk protein - Ninhydrin solution 0,2%
- Crash protein - H2SO4
- CH3COOH 1 N
- Ammonium sulphate
- Formaldehyde
- Aquadest
- PbSO4
- Pb asetat
- NaNO2 solution 1%
- NaOH
- PP Indicator
- HNO3 concentrated
- HCl concentrated
- NH4OH
- CuSO4
- NaOH 40%
- ZnSO4
- FeSO4
- HgSO4
- Reaksi biuret
F. PROCEDURE
1. Denaturation Protein
a. Denaturation because of adding acetic acid

5 mL protein

 in the test tube


 Added 2 drops acetic acid 1N
 shake

Precipitate

 Heated the tube in the steam bath water for 5


minutes
 Observed the change of precipitate

Changed Precipitate

b. Denaturation because of heating

2-3 mL protein

 Heated the test tube for 1


minutes
Precipitate

 Cooled the solution

Test Tube I Test Tube II

 Added 1-2 drops  Heated the tube


ammonium sulfat
 Heated the tube

Precipited
Precipited
c. Denaturation because of adding formaldehyde

1-1.5 mL Formaldehide + 2 mL
Aquadest

 Added 2 drops protein solution

Precipitate

2. Characteristic of Amphoter Protein

3mL aquades
in test tube

 Added 1mL of HCl 1N


 Added kongo indicator

Blue solution

 Added 2-3mL protein solution


 Note the changing color

Changing
Colour

3mL NaOH solution

 Added 2 drops of pp indicator

result
2-3 ml protein solution in test
tube

 Added 2 drops of pp indicator


 Added NaOH solution

result

3. Protein Precipitation
a Protein Precipitated with Ammonium Sulphate

3-4mL protein solution


in test tube

 Added 3-4mL saturated ammonium


sulphate solution
 Shake the tube slowly

Turbid Solution

 Taken 1mL turbid protein solution into


test tube

 Added 2-3mL aquades


 Shake the solution

Precipitate dissolve
b Protein Precipitated with Mineral Acid

1mL HNO3 saturated 1-2mL HCl saturated

 Put into test tube added 1-15 mL


protein solution drop by drops from
partition tube
 Put the test tube vertically

White ring formed

 Shake
 Added CH3COOH

Get more precipitate The solution more


colourless
c. Protein Precipitated by Hard Metals

1mL protein solution 1mL protein solution

 Put into test tube


 Added drop y drop  Put into test tube
CuSO4  Added drop y drop
 Shake PbSO4
 Shake

precipitate precipitate

 Added CuSO4 until  Added PbSO4


dissolve until dissolve

Solution solution

4. Reaction of Protein Color


a Biuret Reaction

3mL protein solution + Added


1 mL 40% NaOH

 Put into test tube


 Added drop by drop 0.5% CuSO4
solution

Change color

b Ksanthoprotein Reaction

3mL protein solution

 Put into test tube


 Added 1mL concentrate
HNO3
 Heated

Yellow solution

 Cooled
 Added ammonia

Orange Solution
c Ninhydrin Reaction

1mL Protein
solution

 Added 10 drops of ninhydrin


 Heated the mixture for 10
minutes
Color
changing

d Millon Reaction
2mL Protein solution

 Put into test tube


 added 1 mL millon reagent
 Heated

Yellow precipitate

 Cooled
 Added a drop of 1%NaNO2 solution
 Heated

Precipitate change

e. Hopkin Cole Reaction


1mL Protein solution

 Put in the test tube


 Added 1 drop formaldehyde
 Added 1 drop HgSO4

Mixed solution

 Added 1mL H2SO4

Form 2 layers
5. Hydrolisis protein and suphur test

1 mL protein solution in
test tube

 Added 1mL 40% NaOH solution


 Heated the mixture for a minutes
 Added a drop of Pb-acetate

Pb-acetate
precipitate
G. TABLE RESULT OF EXPERIMENT

No Procedure Experiment result Hypothesis/reaction Conclusion

1. Denaturation Protein
a. Denaturation because of adding acetic acid Before :
Milk : white Protein in milk and white egg will Protein
5 mL protein Egg protein : denaturate because adding acid which sign denaturized
colorless, thick to form precipitate/ flake because add acid
CH3COOH: colorless
 in the test tube
 Added 2 drops acetic acid 1N
 shake After :
I egg protein
Added CH3COOH:
Precipitate colourless + white
precipitate+ heated :
 Heated the tube in the steam bath white solid
water for 5 minutes
 Observed the change of precipitate II milk protein

Added CH3COOH:
Changed Precipitate colourless + white
solution+ heated :
white solid

b. Denaturation because of heating


Protein in milk and

2-3 mL protein
Before : Protein in milk and whte egg will white egg
Milk : white color denaturate if heated denaturate after
Egg protein: heat
colorless
Ammonium sulfat
sulfat: colorless

After :
Milk :
 milk+heated: white
precipitate
coagulate,
 cooled: liquid
Tube I:
 Added ammonium
sulfate: white
solution
 heated: white
coagulate (++)
Tube II :
 heated: white
coagulate (+)
Egg protein :
 Egg protein+heated:
white precipitate
coagulate
 cooled: liquid
Tube I:
 Added ammonium
sulfate: colorless
 Heated : white
coagulate (++)

Tube II :
heated: white
coagulate (+)

Before : Protein in milk and white egg will Adding


Formaldehyde : denaturate if add formaldehyde formaldehyde
colorless caused protein
Aquades: colorless denaturation
c. Denaturation because of adding
formaldehyde After Tube I:
 formaldehyde+aqu
1-1.5 mL formaldehyde+2mL ades: colorless
aquadest  Milk :
 formaldehyde+aqu
in test tube ades+milk(2
 Put into test tube drops): white
 Added protein solution
precipited, turbid
Precipitat solution
e After Tube II:
 formaldehyde+aqu
ades: colorless
formaldehyde+aqua
des+egg solution (2
drops): white
precipited, turbid
solution
2 Characteristic of Amphoter Protein

Before : Milk protein has characteristic acid Protein as amfoter


3mL aquades Aquades: colorless Egg protein has characteristic Adding charteristic, it’s
in test tube HCl 1N: colorless base (increase pH up isoelectric point) mean can be acid or
Kongo : colorless caused the characteristic protein show base
 Added 1mL of HCl 1N Milk : white as acid. Protein has acid
 Added kongo indicator Egg protein : characteristic when
Blue solution colorless adding base.

After:
 Added 2-3mL protein solution
Aquadest + HCl :
 Note the changing color
colorless
Added kongo orange
Changing Colour paper in pH 2
Milk:
 Added milk protein:
White (aq)
 Paper : orange pH 3
Egg:
 Added Egg protein:
Colourless (aq)
 Paper : orange pH 3

Before :
3mL dilute NaOH solution in NaOH:colorless
test tube Pp indicator :
colorless
 Added 2 drops of pp indicator After:
Milk protein + NaOH
result
: white solution
2-3mL protein Added PP:pink
solution in test solution
tube Egg protein + NaOH
 Added a drop of PP indicator : white solution
 Added NaOH solution Added PP : pink
solution
Result

After:
 Milk protein +
NaOH : white
solution
 Added PP: pink
solution
 Egg protein +
NaOH :white
solution
 Added PP : pink
solution
3 Protein precipitation
a. Protein precipitate with Ammonium Before : No chemical reaction so the precipitate Precipitation at
Sulphate Milk protein : white easy to dissolved. protein in milk &
color white egg with
3-4mL protein solution
in test tube Egg : colorless ammonium
Ammonium sulphate is
 Added 3-4mL saturated sulphate: colorless reversible
ammonium sulphate solution Aquades : colorless precipitate.
 Shake the tube slowly
After :
Turbid Solution
 Egg+(NH4)2SO4
 Taken 1mL turbid protein saturated:turbid
solution into test tube solution + aquades
: colorless
 Added 2-3mL aquades
precipitate dissolve
 Shake the solution
Precipitate  Milk+(NH4)2SO4
dissolve saturated: turbid
white + aquades:
turbid white
solution(--)
b. Protein precipitate with mineral acid Before:  Forming salt substance from acid  Precipiate has
Milk : white reaction with amin group in protein irreversible
1mL HNO3 1-2mL HCl saturated
Egg protein : gotten precipitate  Precipiate has
saturated
colorless  Forming salt substance from acid reversible
HCl : colorless reaction with amin group in protein and
 Put into test tube added 1-15 HNO3: colorless gotten precipitate which easy to dissolve
mL protein solution drop in water
by drops from partition After:
tube milk
 Put the test tube vertically  HNO3+ milk: white
ring+ shake: yellow
turbid solution+
White ring formed HCl: show white
precipitate
 Shake Egg
 Added CH3COOH  Added HNO3: white
ring (++)+ shake:
yellow turbid
Get more The solution solution+ HCl+ egg
precipitate more colourless solution : white
precipitation+
shake: white turbid
solution
c. Protein Precipitate by Heavy Metals

1mL protein solution Protein will precipitate with any heavy


Before: Precipitate will
metals
foam in protein
 Put into test tube CuSO4: blue solution because positive
 Added drop y drop CuSO4 change of metal
 Shake After:
react with positive
Potein + CuSO4 : change foam
tosca solution(++) protein
precipitate
Egg protein + CuSO4 :
 Added CuSO4 until tosca solution(+)
dissolve

Solution
1mL protein
solution
 Put into test tube
 Added drop y drop PbSO4 Egg protein + PbSO4:
 Shake
blue solution +
shaked: precipitated:
precipitate Added PbSO4 : blue
solution
 Added PbSO4
until dissolve

solution
4 Reaction of Protein Color

a. Biuret Reaction
Tube I Biuret reaction is color reaction which is Protein in milk
3mL protein solution +  Egg peptida group (-CO-N) and protein positif and white egg
Added 1 mL 40% NaOH protein+NaOH: reaction with form complex substance contain peptida
colourless+ CuSO4 : with Cu 2+ and N foarm peptida bonding group which sign
purple turbid molecule. with purple
 Put into test tube Tube II colour
 Added drop by drop 0.5%
 Milk +NaOH: white
CuSO4 solution
solution+ CuSO4:
purple
Change color
b. Xanthoprotein Reaction
Before: Xanthoprotein reaction can be done
There is amino acid
3mL protein solution Concentrate HNO3: because nitrasi reaction at benzenaring
in the protein with
colorless from amino acid in protein. test called
the orange colour
NH3 : colorless positive if the colour become orange
 Added 1mL concentrate
HNO3 After :
 Heated the mixture
 Milk protein+ HNO3
+ heated : yellow
turbid, +
Yellow solution
NH3:orange turbid
Egg protein+ HNO3 +
 Cooled the solution heated: yellow
 Added ammonia until the turbid + NH3 :
color change into orange orange turbid

Orange
Solution
c. Ninhydrin Reaction

1mL Protein solution Before: Nynhydrin + protein become complex Milk and egg
Ninhydrin :colorless reaction become positif
 Put into test tube
 Added 10 dros ninhydrin result to
 Heated 10 minutes After : nynhydrin test
 Milk protein + because the color
nynhydrin + heated: dark purple from
dark purple turbid milk and egg.
Color
change  Egg protein +
nynhydrin + heated
: dark purple turbid
d. Millon Reaction Before: Foming red precipitate because the egg Protein in milk
Hg2SO4: colourless protein & milk protein contain of and egg contain
2mL Protein tirosin/tryptofan which is reacted with Hg amino acid tirosin
solution After: from millon reactant. and triptofan
Tube I
 Added 1 mL millon reagent
 Heated the mixture  Milk+egg + millon: 2
phase form
 Heated
Yellow precipitate
 Milk : orange
precipitate
 Cooled  Egg : orange
 Added a drop of 1%NaNO2 solution precipitate
 Heated the precipitate
 NaNO3adding +
heated :
Change
Milk : red
color
precipitation
Egg : red
precipitation
e. Hopkin Cole Reaction Before : The reaction is positive if form purple The result is
Milk: white solution ring because form condensation in zone positive because
1mL Protein Egg:colorless indole and triptofan with aldehide. there is purple in
solution solution middle of solution
Formaldehyde: / ring
 Put in the test tube olorless solution
 Added 1 drop formaldehyde Millon reagent :
 Added 1 drop HgSO4 colorless solution
H2SO4 : colorless
Mixed solution solution

 Added 1mL H2SO4 After :


 Egg +
Form 2 formaldehyde :
layers colorless (aq) +
HgSO4 + H2SO4 : 2
layers ( turbid
white & white
precipitate)
 Milk +
formaldehyde +
HgSO4 + H2SO4 :
(white & turbid ) +
white precipitate
Shaked:
Milk : yellow &
purple in
middle(+)
Egg : yellow and
purple in middle
5 Hydrolisis protein and sulphur test

Before : The reaction positive if form purple ring Protein from egg
Milk : white(++) because form condentation in zone indole and milk positive
1 mL protein solution in Egg solution: and triptofan with aldehide contain of Sulphur
test tube colorless Protein which contain of amino acid with , marked by the
NaOH 40%: colorless S atom will given black color if reacted formation of black
 Added 1mL 40% NaOH solution Pb-acetic: colorless with Pb-acetate formed PbS. precipitate (PbS)
 Heated the mixture for a minutes
 Added a drop of Pb-acetate After Tube I
 Milk+NaOH40%:
turbid white Pb(CH3COO)2 (aq) + 2S2- PbS (s) +
Pb-acetate solution 2CH3COO- (aq)
precipitate  Milk+NaOH
40%+heated : turbid
yellow
 Milk+NaOH
40%+heated+Pb-
acetic: black
precipitate

After Tube II
 Egg solution+NaOH
40%:colorless
 Egg solution+ NaOH
40%+heated:
colorless
Egg solution+NaOH
40%+heated+Pb-
acetic: black
precipitate
H. ANALYSIS

Analysis and Discussion :

1. Denaturation of Protein
a. Denaturation Because of Adding Acid
From the experiment that we did, can known that protein in egg
probably contain of albumin and protein in milk probably contain of casein
and triptofan did the denaturation which is cause the forming of precipitate
of protein with the adding of acetate acid, because protein did the structure
changing from functional structure of protein. Denaturation of protein will
be bigger happen by heating. This caused that protein did the separation of
non-covalent bonding because of the increasing of temperature. So the
precipitate which is produced is increase.
b. Denaturation Because of Heating
From the experiment that we did, it can be known that heating wil
denaturate the protein that signed with the white precipitate which is
caused of the sparating of non-covalent bonding and the decreasing of the
ability to bond the water in protein so that the protein was clot and form
precipitate. Tha adding of ammonium sulphate as a denaturation agent.
c. Denaturation Because of the Adding of Formaldehyde
In this experiment, the adding of formaldehyde will denaturate
protein because of the forming of derivate of dimethyl amino acid because
of the reaction between formaldehyde with amino group in protein. The
amount of the precipitate shows the quantity of protein which is
denaturated. The precipitate from egg is more than milk. From that three
experiment that we did about the denaturation of protein, known that
protein will denaturation or destroying with some did like the adding of
acid, heating, and the adding of organnic compound. The break of protein
can be identified with the precipitate produced.
2. The Amphoter Characteristic of Protein
To know that protein have amphoter characteristic, this experiment is
did in acid and base condition. This, the goal of the addition of acid (HCl) is
to know that protein can do as base and also acid showed by the changing
color into turbid white (for milk) and colorless (for egg) after the addition of
protein solution and also by the changing color into pink solution (for milk)
and clear pink (for egg) after the addition of PP indicator. This shows that
protein have amphoter characteristic i.e acid and base.
3. Protein Precipitation
a. Precipitation of Protein by Ammonium Sulphate

Egg

The egg solution after added by ammonium sulphate turbid and


shake slowly produced the turbid solution and there’s white precipitate.
Ammonium sulphate here as a denaturation agent that cause the broke of
protein structure and make that precipitate. This turbid solution took for 1 mL
then added by water for 2 mL. The precipitate which is form before, after
added by water, the precipotate is dissolve anymore. The dissolve of the
precipitate when added by water show the protein back into that fuctional
functional, because water will hydrolize salt ammonium sulphate so that the
ability to denaturate protein is disappear.

Milk

The milk solution after added by ammonium sulphate turbid and


shake slowly produced the turbid white solution. Ammonium sulphate here as
a denaturation agent that cause the broke of protein structure and make that
precipitate. This turbid white solution took for 1 mL then added by water for
2 mL. After added by water, the color becomes turbid white (--).
From this experiment can be known that if there’s inorganioc salts
precipitate at high concentration in protein solution, so the solubility of
protein will decrease so that will cause that protein precipitate. This caused by
the salt ions competition with protein molecules to bond the water. Because
the ability of salt to hydrate is bigger than protein molecule, so the molecules
of protein will be precipitate. Protein which is precipitated is not did rge
chemical change so that can be dissolved anymore by the addition of water.
This precipitation is reversible.
b. Precipitation of Protein by using Mineral Acid

Egg

1 mL of concentrate HNO3 and 1 mL of HCl concentrateentered into


two test tubes. Then, added that egg solution through the tube’s wall and form
white ring (+) in the top of solution. This solution then shake and form a
white precipitate. This precipitate is the product of reaction between acid with
amino group in protein. Then, added again that acids (HNO3). In addition of
HNO3, the precipitate which is form is decrease. While in addition of HCl,
the precipitate which is form before is decrease. This shows that precipitation
of egg protein with HCl is reversible and with HNO3 is irreversible.

Milk

1 mL of concentrate nitrit acid and 1 mL concentrate HCl entered


into two test tubes. Then, added that milk solution through the tube’s wall and
form white ring (++) in the top of solution. This solution then shake and form
a white precipitate. This precipitate is the product of reaction between acid
with amino group in protein. Then, added again that acids (HCl and HNO 3).
In addition of HNO3, the precipitate which is form is decrease. While in
addition of HCl, the precipitate which is form before is decrease. This shows
that precipitation of egg protein with HCl is reversible and with HNO3 is
irreversible.
The precipitation of protein which is reversible (precipitation with
HCl) not did the chemical changing, so the precipitate which is produced still
can dissolve anymore when adding by more reagent, this precipitation happen
if there’s a salt or inorganic mineral. Precipitation of protein which is
irreversible (precipitation with HNO3) did the chemical changing, the
structure become broken and the precipitate produced can’t be dissolved
anymore. This precipotation happen when adding by organic reagen.
c. Precipitation of Protein by using Hard Metal
1,5 mL of protein, i.e protein from egg and milk entered into test
tube, then added by some salts from hard metal (in different test tube). The
salt of hard metal like Cu, Zn, and Fe will form the precipitate of proteinat
metal. The bond which is form is so strong and will separating the peptide
bond, so the protein is denaturation. Together, -COOH group and -NH2 group
in protein can react with hard metal ion and form kelat compound. The
different color in precipitate which produced come from the color of hard
metal which is added. The addition with CuSO4 produce blue precipitate,
while with ZnSO4 produce white precipitate. And for FeSO4 produce orange
solution with precipitate After the addition of hard metal is continued, the
preciopitate which is formed before start to dissolve. This shows that the
precipitation reaction of protein with hard metal is reversible, because the
salts of this heavy metal are inorganic.

4. eaction of Protein Color


a. Biuret Reaction
In this biuret test did by adding 1 mL of NaOH 40% in 3 mL milk
solution, then added by some drops of CuSO4 0,5% solution. From
experiment result, we get the color of solution become purple. After added
excess CuSO4 the solution become purple turbid. The function of the
addition of NaOH is to make the suspension of milk solution in alkalis
condition. While the addition of CuSO4 is to make purple biuret. The purple
color in that solution shows the form of complex compound between Cu2+
and N from peptide molecule (n-CO-NH-), the reaction is as follow:

While in egg solution did the same do and produced the clear
purple solution. The purple color which is produced in egg protein is more
saturated than the milk protein. This show that the protein solution in egg
have more peptide bonding or longer than the peptide bonding in milk.

b. Xantoprotein Reaction

Egg

In this test reaction did by adding 1 mL of concentrate HNO3 in 3


mL egg solution, form a solution with white precipitate then after the
solution is heated changes into clear yellow turbid. The addition of HNO3
causing the nitration reaction at benzene ring from amino acid composing
protein producing benzene. While the yellow turbid caused by the forming
of some polinitrobenzene compound from amino acid protein. Then after
the addition of some drops of NH4OH form a orange turbid and there’s
precipitate at the bottom of the tube. This caused by the acidity of fenol
which is react with alkali.

Milk

At the addition of 1 mL saturated HNO3 into 3 mL of milk


solution, form a yellow solution. Then, after the solution is heated changes
into oranges yellow solution. The addition of HNO3 cause the nitration
reaction happen at benzene ring from amino acid composing protein
producing benzene. While the yellow precipitate cause the form of a
polinitrobenzene compound from amino acid protein. Then after the
addition of some drops of NH3 form a orange turbid. This caused by the
acidity characteristic of fenol react with alkali.

F
r
om this experiment can be identified that between egg protein and milk
give the positive reaction contain of amino acid with benzene core like
phenylalanine, tyrosine, albumin, tryptophan, etc which is signed by the
forming of yellow precipitate.

c. Ninhidrine Reaction

Egg

At this ninhidrin reaction test did with set the pH in egg protein
solution which is 8 before, becomes 6. Then, added 10 drops of ninhidrin
reagent solution 0,2%. After heated for 10 minutes, it form dark purple
turbid solution. The dark purple turbid solution produced shows the
positive ninhidrin test, because at amino acid there’s carboxyl group react
with NH3, with the process of decarboxilation produce an amina. Amino
group in amino acid can react with nitrite acid and release the nitrogen gas.
Amino acid, ammonia and primer amino group in protein if boiled with
protein solution at pH 6 and with the ninhidrin and also hidrindatin makes
the solution becomes purple.

Milk

At the addition of 10 drops of ninhidrin reagent solution 0,2% in


milk protein solution with pH 6, after heated for 10 minutes fom dark
purple turbid solution.The dark purple turbid solution produced shows the
positive ninhidrin test, because at amino acid there’s carboxyl group react
with NH3, with the process of decarboxilation produce an amina. Amino
group in amino acid can react with nitrite acid and release the nitrogen gas.
Amino acid, ammonia and primer amino group in protein if boiled with
protein solution at pH 6 and with the ninhidrin and also hidrindatin makes
the solution becomes purple.
At this experiment between milk protein and egg, the purple color
which is produced in milk is more saturated (++) because the amino acid
in milk protein is more than in egg protein. So make the color that we get
is different.

d. Millon Reaction

Egg

After added by millon reagent (HgSO4 give acid condition to make


Hg not precipice, hydrolyze tyrosine in protein) and heated producing
orange precipitate. After cooled and added by NaNO2 which has a function
to reduce Hg and heated anymore producing red precipitate.

Milk

After added by millon reagent (HgSO4 give acid condition to make


Hg not precipice, hydrolyze tyrosine in protein) and heated producing
orange precipitate. After cooled and added by NaNO2 which has a function
to reduce Hg and heated anymore producing red precipitate in the edges.

From this experiment can explained that the precipitate which is


produced comes from Hg bonding in hydroxifenil which is producing a
red complex. Where that red complex shows there’s hydroxifenil group
(tyrosine) in both of that protein. This experiment that we did prove
there’s tyrosine in protein.
e. Hopkin-Cole Reaction

Egg

After added 1 drop of formaldehyde the protein solution becomes


colorless. Then added 1 mL saturated H2SO4. There’s purple ring in the
middle at the sign between protein solution and reagent.

Milk

After added 1 drop of formaldehyde the protein solution becomes


white solution. Then added 1 mL saturated H2SO4, and become white
solution. There’s purple ring in the middle at the sign between protein
solution and reagent.

5. Protein Hydrolysis and Test of Sulphur

Egg

Egg protein after added NaOH and heated producing colorless


solution, then added 1 drop of Pb acetate there’s black precipitate. The
addition of NaOH will hydrolyze the peptide bonding and protein polimer.
This hydrolysis producing the monomer of amino acid. If in the protein
there’s amino acid contain of S atom like sistein or metionin so produce
the black color because S atom react with Pb acetate form PbS precipitate,
with reaction:

Pb2+ + S2-  PbS↓

Milk

Milk protein after added NaOH and heated producing turbid


yellow solution, then added 1 drop of Pb acetate there’s black turbid. The
addition of NaOH will hydrolyze the peptide bonding and protein polimer.
This hydrolysis producing the monomer of amino acid. If in the protein
there’s amino acid contain of S atom like sistein or metionin so produce
the black color because S atom react with Pb acetate form PbS precipitate,
with reaction:

Pb2+ + S2-  PbS↓

I. ANSWER THE QUESTION


1. Explain the function of each reagent in protein test! (CuSO4, HgCl2,
HNO3, Pb-acetate)

Answered:

 CuSO4 is used as reagent for heavy metal presence in protein.


The positive result is precipitation formed.
 HgCl2 is used as reagent for hydroxyl phenil (-OH) group in
protein.
 HNO3 is used as reagent for benzene ring presence from protein
amino acid salt. In the experiment, when concentrated HNO3 is
added the derivative of nitrobenzene produced.
 Pb-acetate is used as reagent cistein and metionin amino acid
presence. The positive result is formation of PbS precipitate
which has black color, because S atom reacted with acetic acid.

2. How do the effects of organic solvent (acetone, ethanol) to property of


protein denaturation?
Answered:
The effect of organic solvent to property of protein denaturation is causes
the denaturation protein can be occurred because protein or nucleic acid
will lose secondary and tertiary structure.

3. Sebutkan macam-macam ikatan yang menyebabkan polipeptida menjadi


stabil dalam bentuk heliks!
Answered:
 Disulfide bonding
This bond is formed between two residues which is related with two parts
of polypeptide chain through cistein residue

 Hydrogen bonding
This bond is formed between NH or OH group and C=O in peptide
bonding or COO in R group.
J. CONCLUSIONS

From the experiment that we did, can be conclude that:

1. Protein denaturation can happen because of the addition of acid or


base and heating.
2. Protein is amphoter, i.e can react with acid and base which is signed
with there’s a precipitate.
3. The precipitation of protein is reversible and irreversible.
4. Protein which is contain of amino acid with benzene core shows the
positive reaction in biuret test signed with the purple precipitate.
5. Xanthoprotein reaction shows the positive result signed with the
formation of yellow color because of nitrite reaction benzene ring
from amino acid composing protein forming a polinitrobenzene
compound.
6. Ninhidrin reaction used to know if there’s α-amino acid group.
Positive reaction if there’s purple color. Where α-amino acid group
react with nitrite acid and release nitrogen gas.
7. Millon reaction is positive if the protein contain of hidoksifenil group
(tyrosine amino acid) can react with mercuri nitrate solution
producing red solution or precipitate.
8. Hopkin-Cole reaction did to test the tryptophan amino acid in
protein.
9. The addition of NaOH will hydrolyze the peptide bonding and
protein polimer.

REFERENCES

Anonymous. 2014. Proteins. http://biology.tutorvista.com//proteins.html. Accessed on


December, 15th 2015
Anonymous. 2014. Color Reaction of Protein. http://quizlet.com/8801729/color-reactions-of-
proteins-flash-cards/. Accessed on December, 15th 2015
Dakin, H.D.. The Glycoxylic Acid reaction for Trytophan, Indol, and Skatol.
http://www.jbc.org/content/2/4/289.full.pdf. Accessed on December, 15th 2015
Fessenden, Ralp J. dan Joan S. Fessenden. 1999. Kimia Organik Jilid 2. Jakarta:
Erlangga.
Haurowitz, Felix. 2014. Protein. http://www.britannica.com/EBchecked
/topic/479680/protein. Accessed on 15 November 2014
Ophardt, Charles E..2003. Denaturation of Protein. http://elmhurst.edu/
~chm/vchembook/568denaturation.html. accessed on December, 15th 2015
ATTACHMENT

1. Protein Denaturation

a. Denaturation by Adding Acid

Put 5 ml egg protein into test Put 5 ml milk protein into Each tube was added with
tube 1 test tube 2 CH3COOH

Formed white precipitate in Then heated in steam bath Egg protein become white
egg protein solution for 5 minutes solid (coagulate)
Milk protein become solid
(coagulate)

b. Denaturation by heating

Put 3 ml egg protein (tube 1) Then heated in steam bath Egg protein (tube 1) divided
and 3 ml milk protein (tube for 1 minutes into 2 tubes
2) Become white solid
precipitate
Milk protein (tube 2) divided Tube 1 of each protein Then tube 1 and 2 of each
into two tubes solution added (NH4)2SO4 protein are heated

Egg protein that added Milk protein that added


(NH4)2SO4 (tube 1) coagulate (NH4)2SO4 (tube 1)
(++) coagulate (++)
Than tube 2 Than tube 2
c. Denaturation by adding Formaldehyde

Formaldehyde 1.5 ml + 2 ml Formaldehyde 1.5 ml + 2


aquadest + 2 ml milk protein ml aquadest + 2 ml egg
= white precipitate protein = white precipitate

2. The Characteristic Of Amphoteric Protein

3 ml aquadest Added 1 ml HCl Added kongo indicator +


egg protein
The kongo is in pH 3
In base condition

NaOH added PP indicator : 3 ml protein added NaOH Each tube was added with
pink solution and PP : milk (pink turbid) CH3COOH
Egg (pink clear solution)

3. Protein Precipitation
a. By adding ammonium Sulfate

Put 4 ml egg protein (tube Then each tube added 3- Then added aquades egg
1) and milk protein (tube 2) 4 ml (NH4)2SO4 saturated protein = colorless, milk
protein : white turbid
b. Adding by mineral acid

HNO3 + egg protein (yellow) HNO3 + milk protein Milk and egg protein then
and HCl + egg protein (yellow) and HCl + milk added NH3 become more
(white) protein (white) yellow turbid solution

c. By adding heavy metals

Milk and egg protein added Then heated in steam bath Egg protein become white
by CuSO4 (tosca solution) for 5 minutes solid (coagulate)
A. Protein Color Reaction

a. Biurette reaction

Put 5 ml milk and egg Both of tube added NaOH Both of tube added CuSO4,
protein solution into test the milk protein solution
tube become turbid purple and
egg protein become clear
purple

b. Xanthoprotein reaction

Putted 3 ml milk and egg Both of tube added HNO3 Then added NH3 become
protein solution into test and heated become yellow orange
tube turbid
c. Ninhydrin Reaction

Putted 3 ml milk and egg Both of tube added Then the tube heated in
protein solution into test ninhydrin steam bath
tube

The solution become turbid


dark purple to blue
d. Millon Reaction

Putted 2 ml milk and egg Both of tube added Hg2SO4 Then the tube heated in
protein solution into test steam bath
tube

Then added NaNO2 1% Heated again


e. Hopkin-Cole Reaction

Putted 2 ml milk and egg 1 ml protein egg solution Added 1 drop formaldehyde
protein solution into test
tube

Added Hg2SO4 formed two 1 ml milk protein +


layers formaldehyde + Hg2SO4
B. Hydrolysis Of Protein

1 ml protein solution Both of tube added NaOH Then added Pb acetate


40 % (milk + NaOH =
white solution) and (egg +
NaOH = colorless)

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