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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.
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.
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
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.
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.
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.
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.
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.
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
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
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
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.