Analysis

1. Protein Denaturation
a. Denaturation cause by adding acetic acid (CH3COOH)
This experiment purpose is to investigate the denaturation of protein caused of the
acid addition.
Milk Protein
First 5 ml of milk protein entered into test tube than added 2 drops of
CH3COOH 1N, after shaken then there will be a white precipitate. After that is
conducted heating, the more the precipitate gathered. Based on the theory,
protein will undergone the greatest turbidity during which the pH reaches the
isoelectric pH where the proteins positive and negative charges are equal, at this
moment proteins undergo denaturation indicated by increased turbidity and the
occurence of clots or precipitate. So with the addition of acetic acid of milk
proteins undergo denaturation indicated by the formation of a precipitate.
Structural changes caused by the denaturation process is the change in the
configuration of -helical proteins become elongated. This is due to the
destruction of the hydrogen bonds and nonpolar bonds that occur in the structure
of the protein folding.

White Egg Protein

5 ml of white egg protein entered into test tube than added 2 drops of
CH3COOH 1N after shaked then there will be a white flake. After it is heated, the
more the precipitate formed, the solution was turbid (+).Based on the theory,
protein will undergone the greatest turbidity during which the pH reaches the
isoelectric pH where the proteins positive and negative charges are equal, at this
moment proteins undergo denaturation indicated by increased turbidity and the
occurrence of clots or precipitate. So with the addition of acetic acid denaturated
egg white proteins are characterized by the formation of precipitates or flake.
Structural changes caused by the denaturation process is the change in the
configuration of -helical proteins become elongated. This is due to the
destruction of the hydrogen bonds and nonpolar bonds that occur in the structure
of the protein folding.

The reaction in general

So both protein whether in milk solution or white egg solution is denatured when
added acid because of change in the configuration of -helical proteins become
elongated. The precipitate in milk is more than in egg.

b. Denaturation Cause by Heating

This experiment purpose is to investigate the denaturation of protein caused of
heating.

Milk Protein
Milk protein is heated to form a white precipitate. Then the heated milk
proteins are divided into two tubes. At first tube added ammonium sulfate
solution formed a white precipitate (++) and the tube 2 added nothing formed a
white precipitate (+). The amount of precipitate on test tube 1 is greater than in
tube 2.
White Egg Protein
White egg protein is heated then white precipitate formed. Then the solution
was divided into two tubes. At first tube added with acetic acid solution with a
white precipitate formed (+). In tube 2 is added ammonium sulfate ((NH4)2SO4)
solution and formed a white precipitate (-).
From these experiments it appears that protein added with acetic acid gives
more precipitates, because more of denatured proteins in the protein
supplemented with ammonium sulfate ((NH4)2SO4). Protein solubility in acidic
conditions is at the minimum point, so that the protein will precipitate.
The reaction occurs
O

NHCHC

NHCHC

H2O, H+
heat

H2NCHCO2H + H2NCHCO2H
R

So both protein whether in milk solution or white egg solution is denatured when
raised temperature because of change in the configuration of -helical proteins
become elongated. The precipitate in milk is more than in egg

c. Denaturation that is caused by adding formaldehyde

This experiment purpose is to investigate the denaturation of protein caused of the
acid mineral addition.
Milk Protein
1- 1.5 mL formaldehyde solution that is colorless and 2 mL aquades is entered
into a test tube. Then added milk protein that is white color, it will produce a
insoluble white precipitate in water and the white turbid solution. The addition of
formaldehyde will be occur denaturation of milk proteins, which occur is the
reaction of the amino groups on proteins to form aminodimetil acids that will be
insoluble in water.
White Egg Protein
1-1.5 mL formaldehyde solution that is colorless and 2 mL aquades is entered
into a test tube. Then add the white egg protein solution is colorless, it will
produce a slight white precipitate insoluble in water and the resulting solution
was turbid and colorless. Because of addition of formaldehyde will be occur
denaturation of milk proteins, which occur is the reaction of the amino groups on
proteins to form aminodimetil acids that will precipitate insoluble in water.
The reaction occurs
COO+

H 3N

O
H

+
R

H3C

C
H

H
C

CH3

NH2

So both protein whether in milk solution or white egg solution is denatured when
added formaldehyde because of change in the configuration of -helical proteins
become elongated. The precipitate in milk is more than in egg

2. Amphoteric properties
In this experiment there are two ways to prove the amphoteric properties of protein
that are acidic and basic condition.

Because protein is composed by amino acids they will have the similar properties.
Protein is electrolyte colloid that is amphoteric. Thus protein could serve as basic or
acid based on the amount of NH2 from amino and COOH from acid. In neutral
condition these compound forms two ion pole (zwitter ion). This condition called
isoelectric point. In isoelectric point the amount of positive and negative charge are
equal.
In acidic condition
Milk Protein
Taken 3 ml distilled water and added 1 drop HCl 1 N, because kongo indicator is
unavailable it replaces by litmus paper indicator. Both red and blue litmus paper is put
in test tube then added milk solution. The blue litmus change color into red, the red
litmus is still red. It means the addition of acid will give the acidic condition in tube.
In isoelectric point the amount of positive and negative charge are equal. Thus made
the protein serves as base in reaction.

White Egg
Taken 3 ml distilled water and added 1 drop HCl 1 N, because kongo indicator is
unavailable it replaces by litmus paper indicator. Both red and blue litmus paper is put
in test tube then added white egg solution. The blue litmus change color into red, the
red litmus is still red. It means the addition of acid will give the acidic condition in
tube. In isoelectric point the amount of positive and negative charge are equal. Thus
made the protein serves as base in reaction.
In this experiment proven that protein has an amphoteric properties, protein will
serves as base if the condition is acidic an it is on isoelectric point.

Basic Condition
Milk Protein
Taken 3 ml diluted NaOH added PP indicator until the colorless solution become
pinkish. Added drop by drop milk and the change is the color is pinkish.
White Egg Protein
Taken 3 ml diluted NaOH added PP indicator until the colorless solution become
pinkish. Added drop by drop white egg and the change is the color is pinkish.
In this experiment proven that protein has an amphoteric properties, protein will
serves as acid if the condition is basic an it is on its isoelectric point.

The reaction occurs

3. Protein Precipitate
a. Protein precipitation with ammonium sulphate
This experiment purpose is to investigate precipitate of protein caused of the
addition (NH4)2SO4.
Reaction occur

Milk Protein
After adding ammonium sulphate and shaken turbid solution is formed
because the addition of ammonium sulphate causes protein dehydration (water
loss). As a result of this dehydration process of the protein molecules has the
smallest solubility and will be easy to settle. The addition of (NH4)2SO4 is
irreversible. The precipitate can be easily dissolved again by the addition of water.
It means the addition of water is reversible.
White Egg Protein
After added 4 drops ammonium sulphate and shaked to form a white
precipitate. Precipitation occur because the addition of ammonium sulphate
causes dehydration (water loss) on protein. As a result of this dehydration process
of the protein molecules has the smallest solubility and will be easy to settle. The
precipitated protein does not undergo chemical change, so it can be easily
dissolved again by the addition of water.
From this experiment shows that this precipitation is reversible.

b. Protein precipitation with concentrated mineral acid

Milk Protein
In first step, when 1mL concentrated HNO3 is added with 2 mL milk drop by
drop from the edge of tube, there are two layers and ring that are formed. The
upper layer is white turbid and the lower layer is colorless, the ring is between

two layers. After it is added by HNO3 again (excess) and shaken, there are more
precipitate.
Next step, when 1 mL concentrated HCl is added with 2 mL milk drop by drop
from the edge of tube, there are two layers and ring that are formed. The upper
layer is white turbid and the lower layer is colorless, the ring is between two
layers. The result of layer and ring are almost same with HNO3 treatment. But,
after it is added by HCl again (excess) and shaken, there solution become
colorless.
The precipitation that is formed is caused by acid reaction with amino group in
protein. The addition of HNO3 produces more precipitate which is irreversible
that is shown by forming more precipitate when HNO3 is excess. But HCl is
reversible goes same as the other strong acid.

White Egg Protein

In first step, when 1mL concentrated HNO3 is added with 2 mL white egg
solution drop by drop from the edge of tube, there are two layers and ring that are
formed. The upper layer is whitish solution and the lower layer is colorless, the
ring is between two layers. After it is added by HNO3 again (excess) and shaken,
there are more precipitate.
Next step, when 1mL concentrated HCl is added with 2mL white egg solution
drop by drop from the edge of tube, there are two layers and ring that are formed.
The upper layer is whitish solution and the lower layer is colorless, the ring is
between two layers. The result of layer and ring are almost same with HNO3
treatment. But, after it is added by HCl again (excess) and shaken, there solution
become colorless.
The precipitation that is formed is caused by acid reaction with amino group in
protein. The addition of HCl dissolves the precipitate again. Thus, HCl addition
produce precipitation which is reversible that is shown by dissolved precipitation
when HCl is excess goes same as the other strong acid.

c. Protein precipitation with heavy metal

Milk Protein
The first heavy metal that is tested in milk is Cu. After 1mL milk is added
with drop by drop CuSO4, which is blue solution, the white solution of milk

becomes greenish blue turbid solution and there is precipitate formed. The second
metal that is tested is Zn. After milk solution is added by ZnSO4, which is
colorless, there is white precipitate formed. The last metal that is tested is Fe.
After milk is added with FeSO4, which is yellow solution, the solution changes
into white turbid solution and there is brownish precipitate.

White Egg Protein

The first heavy metal that is tested in white egg solution is Cu. After 1mL
white egg solution is added with drop by drop CuSO4, which is blue solution, the
color becomes greenish blue turbid solution and there is precipitate formed. The
second metal that is tested is Pb. After white egg solution is added by PbSO4,
which is colorless, there is white precipitate formed. The last metal that is tested
is Fe. After white egg solution is added with FeSO4, which is yellow solution, the
solution changes into yellow turbid solution and there is brownish yellow
precipitate.
From the experiment of milk and white egg solution and some metals, we can
see that precipitate reaction by heavy metal (such as Cu, Pb, Fe) is charge
neutralization. Precipitation is occurred when protein in negative isoelectric state.
Cation of heavy metal make neutralized reaction is occurred and proteinate
neutral salt will precipitate. COOH and NH2 group are reacted with heavy metal
ion which will be formed qhelat compound. The color of precipitation is the result
of heavy metal ions color which is added.

The precipitate that is produced in white egg is more than in milk. It means
that the protein in white egg is more than in milk, thus the protein in white egg
easily precipitate. And after the heavy metal is added (excess), the precipitate is
dissolved. It means that the protein precipitated reaction is reversible.
But when the experiment conducted the precipitate is more. So there is
discussion here. It could be error in observation or the addition of excess heavy
metal solution is not observed drop by drop. So the precipitate is more.

4. COLOR REACTION PROTEIN

a. Biuret is to identify protein in general, give positive result for peptides bond.
In the biuret test, when a few drops of a dilute solution of CuSO4 is added to a
strong alkali NaOH, peptides or proteins produced purple. It is a common test for
proteins and peptides that containing two or more peptide chains. Biuret is formed by
heating urea and has a structure similar to the structure of the peptides from the
protein. The reaction occur

This experiment purpose is to examine the properties of the proteins, the first step
in testing the protein using the biuret reaction is to prepare 3 mL solution of milk and
egg whites. Each inserted into a test tube and then added with 1 mL of 40% NaOH
solution then added to a solution of 0.5% CuSO4 drop by drop. After treatment the
milk solution becomes colored purple precipitate (++), while the egg solution into a
solution of purple colored solution(+++).
The function of the addition of NaOH is that to make the protein into basic
condition. While the addition of CuSO4 is used to produce a purple biuret. This is
because the complex compounds formed between Cu 2+ and N of molecules of peptide
bonds. The purple color resulting from this test showed positive milk and egg white
protein. From this experiment purple on egg white protein is more concentrated than
milk protein. This indicates that the peptide bond in the egg white protein more or
longer than the peptide bonds in milk proteins.

b. Xanthoprotein Test
In this experiment, first preparing a solution of 3 mL of milk and egg white solution.
Each put into a different test tube and then added to 1 mL of concentrated HNO3 and
heated colorless. After heating the milk solution contained yellow granules, then
cooled and added to a solution of ammonia produces orange granules (upper) and

turbid solution (lower). While the egg white solution contained yellow granules, then
cooled and added with ammonia produces orange granule. The yellow color after the
addition of HNO3 and heating showed that the positive result to solution of milk and
egg containing protein. The yellow color is due to the formation of a compound
polinitrobenzena of protein amino acids. When the addition of alkali (ammonia)
solution will change color due to the properties of acidity reacts with alkaline phenol.
The following equation:

From this experiment can be identified that the amino acids contained in both of
these proteins have amino acid groups nucleated benzene. The possibility of such an
amino acid is phenylalanine, tyrosine or tryptophan.

c. Ninhydrin reaction
This experiment was performed aimed at identifying proteins by using a color
reaction with ninhydrin. ninhydrin reaction was used to test for the presence of the
amino group, NH2 amino acids in a protein. The first step to do is adjust the pH of the
protein solution, which is milk and egg white until it has a pH of 7. After that, the
solution of 0.5 ml was taken and put into a test tube. Furthermore, in both the protein
solution was added 10 drops of 0.2% solution of ninhydrin. Then, the protein solution
was heated in a water bath for 10 minutes. Heating causes, milk and egg white
solution turns into a blue colored solution.
From the results of these experiments, it is known that milk and egg white when
tested with ninhydrin produces purple color indicates that the protein in milk and egg
white positive to ninhydrin test. From this it can be seen that the proteins in the milk
and the egg white contain amino group, NH2. This reaction occurs in the free amino
acid groups of amino acids with ninhydrin are written below:

d.

Millon reaction
This experiment purpose is identifying proteins by color reactions using reagents

millon. Protein containing tyrosine will give a positive result. The first step to do is
put 2 ml of milk protein and egg white into each test tube. Then add 1 ml of clear and
colorless reagent millon or reagent mercurysulfate. Subsequently, to a solution of
milk and egg white are heated. After that, add 1 drop of 1% solution of NaNO2 to a
solution of milk and egg white. The treatment solution of milk and egg white solution
became red granule.
From these results it can be seen that the milk and egg white proteins contain
amino acids that have a hydroxyphenyl group, or phenol. Thus, the possibility of milk
and egg white contains the amino acid tyrosine containing hydroxyphenyl group, or
phenol. The reaction occurs

Addition HgSO4 is to provide an condition that Hg does not precipitate acid,

hydrolyze proteins that are tyrosine, while the addition of NaNO2 serves to reduce Hg.
From these experiments can be explained that the binding of Hg occurred at
hydroxyphenyl that produces a red colored complex. Where the red colored complex
indicates hydroxyphenyl group (tyrosine) in both proteins.

e. Hopkins-Cole Reaction
This experiment purpose is identifying proteins by color reactions using
Hopkins-Cole reagent. Hopkins-Cole reaction was used to test for indole group of
amino acids in a protein. The first step to do is put 1ml solution of milk protein and
egg white into each test tube. next, add 1 drop of diluted formaldehyde in the form of
clear and colorless solution into each test tube. Next, add 1 drop of mercury sulphate.
Then, concentrated H2SO4 was added through the test tube wall is tilted up to form
two layers. Once added concentrated H2SO4 doesnt occur purple rings. The
formation of a purple ring, indicating the presence of proteins containing the amino
acid group-containing indole, such as tryptophan. The formation of this ring because

of the formation of condensation 2 indole nucleus of tryptophan with aldehydes. But

because the test result is negative thus, the possibility of milk and egg white proteins
containing tryptophan is not proven. Here could be because of the formation of
condensation 2 indole nucleus of tryptophan with aldehydes doesnt occur.

5. HYDROLYSIS OF PROTEIN AND SULPHUR TEST

Protein Hydrolysis and Test of Sulfur Presence
Milk Protein
In this experiment, 1mL of milk is added with 1mL NaOH 40%. The precipitation
is formed, and then it is heated for a minute. After heated, the solution becomes
yellowish turbid. And the last step is added by a drop of Pb-acetate. After addition of
Pb-acetate, the yellowish solution becomes blackish yellow turbid solutions, it means
that there is precipitate formed.
White egg Protein
In this experiment, 1mL of white egg solution is added with 1mL NaOH 40%. The
color which is white turbid changes into yellowish solution, and then it is heated for a
minute. After heated, the solution becomes yellow solution. And the last step is added
by a drop of Pb-acetate. After addition of Pb-acetate, the yellowish solution becomes
grayish black turbid solution, it means that there is precipitate formed.
From this experiment, the addition of alkaline in protein will make peptide bond
is hydrolyzed from protein polymer into amino acids monomers. Heating is done to
accelerate the hydrolysis reaction. The addition of Pb-acetate is used as an indicator
that the milk and egg whites are amino acid containing S atom such as cystine and
cysteine. Black precipitate was occurring showed PbS deposition due to one amino
acid from hydrolyzed proteins have S atoms, so that the addition of Pb in alkaline
salts will form a precipitate that is easily observed. Thus, amino groups are decreased.
Protein in milk and white egg, the protein contains S group which will be eliminated
into H2S compound. The proteins of milk and white egg may be cysteine or cystine.
Because there is black precipitation that is formed in milk and white egg solution after
Pb-acetate is added, the compound change from Pb into PbS precipitate:
Pb2+ + 4 OH- PbO22- + 2 H2O
S2- + 2 H2O + PbO22- PbS (s) + 4 OH

Protein in white egg is more than protein in milk because the precipitate that is
produced is more which is shown by differences of the color range of black
precipitate.

Conclusion
1. The protein in milk and white egg can be denatured causes the addition of acid that
is characterized by the formation of a precipitate. The precipitate occur in milk is
more than in egg.
2. The protein in milk and white egg can be denatured because of the heating, acid, and
salt. The precipitate occur in milk is more than in egg.
3. The protein in milk and white egg will be denatured because the addition of
chemical compounds, such as compounds formadehid characterized by the
formation of a precipitate. The precipitate occur in milk is more than in egg.
4. Protein has an amphoteric property, protein will serves as base if the condition is
acidic and it is on isoelectric point. Protein has an amphoteric property, protein will
serves as acid if the condition is basic and it is on isoelectric point.
5. The deposition of the protein in milk and white egg by ammonium sulphate will

give a precipitate which is reversible, when added to water.

6. Precipitation of proteins in milk and white egg with HNO3 concentrated will give a

precipitate which is irreversible when added with excess HNO3.

7. Precipitation of proteins in milk and white egg with HCl concentrated will produce a
dissolved precipitate which is indicated reversible when added with excess HCl.
8. Precipitation of proteins in milk and white egg with heavy metals will produce a
precipitate which is irreversible when added with excessive heavy metals, causes if
there is of proteins in isoelectric negative charge which is then neutralized by the
positive charge of metal ions.
9. Protein color reaction
a. Biuret Test
Protein in milk and egg containing peptide bond is marked by its form purple
solution. The purple color is shown by positive to biuret test.
b. Xantoprotein Test
Protein in milk and egg containing polynitro benzene compound is marked by its
form orange color. The orange color is shown by positive to xantoprotein test.
c. Ninhydrin test

Protein in milk and egg containing amino group, NH2. Milk and egg are shown
positive result to ninhydrin test with its formed blue solution.
d. Millons test
Protein in milk and egg containing tyrosin / triptophan that marked by formed the
red precipitate. Milk and egg are shown positive result to millons test with its
formed red precipitate.
e. Hopkin-cole
Protein in milk and egg containing indol group because show positive result in
Hopkin cole reaction.
10. Protein hydrolysis and sulfure test
Protein in milk and egg occur hydrolysis produce amino acid that have S atom (cysteine
and cystine) that marked by formed PbS precipitate (black) after addition Pb acetate.