THE IDENTIFICATION OF AMINO ACID IN CHICKEN EGG, SPECIFIC SOLUTION, AND UNKNOWN SOLUTION Ni Wayan Krisna Dewi Chemistry Education Department Ganesha University of Education Abstract Basically, chicken egg is always founds in our daily life. Chicken eggs are foods we can eat directly or through the process beforehand. Egg is important food in this era, because many disease now is caused by protein deficiency. On the egg chicken, there are 12.8 % and egg chicken is cheaper than other animal protein. The experiment was conducted to determine the types of amino acids found in egg albumin. In the experiments have been conducted, the types of amino acids contained in the egg albumin are tyrosine, tryptophan, phenylalanine, glycine and other free amino acids. Keywords: The protein of egg chicken, testing protein. A. Introduction: Protein is the macromolecule that formed by amino acid that composed by nitrogen, carbon, oxygen and hydrogen, some of amino acid contains sulfur (methionine, cystine, and cysteine) that connected by peptide bond. On the living organism, protein has
Transcript
THE IDENTIFICATION OF AMINO ACID IN CHICKEN EGG, SPECIFIC SOLUTION,
AND UNKNOWN SOLUTION
Ni Wayan Krisna Dewi
Chemistry Education Department
Ganesha University of Education
Abstract
Basically, chicken egg is always founds in our daily life. Chicken eggs are foods we can
eat directly or through the process beforehand. Egg is important food in this era, because many
disease now is caused by protein deficiency. On the egg chicken, there are 12.8 % and egg
chicken is cheaper than other animal protein. The experiment was conducted to determine the
types of amino acids found in egg albumin. In the experiments have been conducted, the types of
amino acids contained in the egg albumin are tyrosine, tryptophan, phenylalanine, glycine and
other free amino acids.
Keywords: The protein of egg chicken, testing protein.
A. Introduction:
Protein is the macromolecule that formed by amino acid that composed by nitrogen,
carbon, oxygen and hydrogen, some of amino acid contains sulfur (methionine, cystine, and
cysteine) that connected by peptide bond. On the living organism, protein has function to
construct cell structure and some protein has function in physiological aspect (Bintang,2010).
Amino acids are organic molecules with low molecular period (between 100-200 Da) that
contain at least one carboxyl group (-COOH) and an amino group (-NH2). These amino acids are
essential components for protein biosynthesis. In the protein there are about 20 types of amino
standard amino acids. All items are a α-amino acids, except proline. Variations that occur
between amino acids is located in the R group or side chain. Based on its R groups will be able
to predict the properties of an amino acid. Instead, based on the properties identified will be
known to the R groups are contained in the amino acid or amino acid type will be known (Tika,
2007).
H2N C COOH
R
H
The structure of α-amino acids. R groups representing the side chain has a different
structure for each amino acid. Carboxyl and amino groups can be used to analyze a general
amino acid in the mixture. R groups used in analyzing the specific amino acid.
Based on its structure, amino acids are classified into seven groups, where the
classification is based on the chemical nature of its R group that will facilitate in given the
general properties of each amino acid. The seven groups were included in the table below:
Table 1: The classification of amino acid based on the structure
R-group properties Example of amino acid
Aliphatic Gly, Ala, Val, Leu, ILe
Aromatic Phe, Tyr, Trp, His
Hydro cyclic Ser, Thr, Try
Carboxylic Asp, Glu
Contain sulfur Cys, Met
Imines Pro, Hyp
Amino Lys, Arg
Amide Asn, Gln
Source: Murray.et.al, 1995: 29-30
Besides that, amino acids are also classified into two major groups of the amino acids
essential and nonessential amino acids.
Table 2: Classification of amino acid based on essential and nonessential
Essential amino acid Nonessential amino acid
Arginine Alanine
Histidine Asparagine
Isoleusine Aspartic acid
Leusine cystein
Lysine Glutamic acid
Metionin Glutamine
Phenylalanine Glisine
Treonin Proline
Tryptophan Serine
Valine Tyrosine
Qualitative test is can be done for knowing the protein that contain on protein solution.
To find out the types of amino acids in a protein that already found in foods such as egg albumin
can be identified with several test reactions such as reaction Millon test, Hopkins-Cole,
ninhydrin, PbS and Test Nitropruside.
1. Millon Test
Millon reagent prepared from mercury (Hg) is dissolved in nitric acid and diluted with
distilled water. The addition of these reagents on proteins that contain tyrosine will give a
positive test result and is characterized by the formation of red color. The red color is a salt
of mercury from the nitrazed tyrosine. However, this reaction is not specific because it also
gives a positive test (red form) in the presence of phenolic compounds (Anwar, 1996). This
is basically a positive reaction for phenols, due to the formation of mercury compounds with
hydroxyphenyl groups are colored.
2. Hopkins-Cole Reaction
In this test, the reagents used glyoxylate acid (CHO.COOH). Hopkins-Cole reagent
containing glyoxylate acid. This reagent is made of oxalic acid with magnesium powder in
water. The positive test results are indicated the formation of the purple ring at the boundary
between the solution of indole ring-containing compounds and reagents. Ring formation is
due to the formation of condensation of two indole nucleus of tryptophan with aldehydes.
The assay is specific for the amino acid tryptophan which contains only the tryptophan
indole ring. Of the test compound is an acid complex formed 2,3,4,5-tentrahidro-β-carbonyl-
4-carboxylate. Structural formula is:
3. Ninhydrin Test
This test is used to detect the presence of free amino groups of amino acids at which this
reaction reagents used were ninhydrin (triketohidrindene hydrate). When heated with
ninhydrin amino acid complex is formed at the blue or purple. The reaction is
The overall reaction of amino acids with ninhydrin are as follows:
a. Oxidative Decarboxylation of amino acids and result in a reduced ninhydrin, ammonia
and carbon dioxide.
b. Reduced ninhydrin reaction with ninhydrin molecule to another and with the release of
ammonia molecules form a complex in blue or purple.
H
H H
COOH
H
H
H
NHN
+
H
+OH
OHO
O
R
CNH2 COOH
O
OC
C
N
R
C COOH
H
CH
O
OC
CN
R
C H
HCH
O
OC
CN 2
OH
OHO
O
O
O
C
C
N
C
C
O
O
Complex color (violet or blue)
+H2O-RCHO
-CO2
H2O
For one amino acid can be determined quantitatively by observing the intensity of color
formed is proportional to the concentration of these amino acids. In this case, NH3 and CO2
released so kemingkinan can be measured quantitatively. Some examples of these amino
acids are aniline, vanilina, leucina, isoleusina, and metionina. Proline and hidroxypidine will
produce different complex color with other amino acids.
4. Nitropuside Test (cystein test)
This test is used to detect the amino acid Cysteine in which the reagents used are sodium
nitropruside in aqueous NH3. Positive test results indicated the formation of red color. Here
the reaction between the sulfhydryl group of Cysteine, peptide or protein with a reagent to
form a red colored complex.
For some proteins that give negative results to this test turns out to be positive after
having heated to coagulation and denaturation.
5. PbS Test
In this test reagent used is a solution of NaOH and PbS which the sulfur contained in the
amino acid Cysteine is released as sulfide ion in the presence of NaOH. In contrast, the
sulfide ion reacts with Pb2+ ions form a black precipitate of PbS.
B. Method
This experiment was conducted in Organic Chemistry Laboratory in Ganesha University of
Education on March, 1th 2012.
B.1 Material and Equipments
Materials
+
HH
H
N +
O
O
OH
OH
O
OO
OH
HO
N
O
O
Blue or violet complex color
No Materials Concentration Total
1 Egg albumin - 5 mL
2 Larutan Pb(CH3COO)2 - Sufficiently
3 Cystein - Sufficiently
4 Sistine - Sufficiently
5 Millon reagent - Sufficiently
6 Hopkins-Cole reagent - Sufficiently
7 Ninhidrin reagent - Sufficiently
8 Sodium Nitropruside reagent - Sufficiently
9 Ammonia reagent - Sufficiently
10 Concentrated sulfuric acid - Sufficiently
11 Aquades - Sufficiently
12 Phenilalanin - Sufficiently
13 Triptopan - Sufficiently
14 Glycine - Sufficiently
15 Tyrosine - Sufficiently
Equipments
No Equipments Size Total
1 Test tube - 1 rack
2 Drop pipette - 3 units
3 Funnel - 1 unit
4 Beaker glass 25 mL 2 units
5 Beaker glass 100 mL 3 units
6 Volumetric pipette - 1 unit
7 Graduated cylinder glass 10 mL 1 unit
8 Erlenmeyer 100 mL 1 unit
9 Spatula - 2 units
10 Stirring rod - 1 unit
11 Spiritus burner - 1 unit
12 Matches - 1 unit
13 Beaker glass 250 mL 1 unit
14 Erlenmeyer 250 mL 1 unit
15 Watch glass - 2 units
B.2 Procedure
a. Millon Test
o Into 3 ml of albumin solution was added 5 drops of Millon. Then the mixture was
heated in a water bath
o Into a standard solution of tyrosine, triptopan, glycine and phenylalanine was
added 5 drops of Millon. Then the mixture was heated in a boiling water batch
b. Hopskin-Cole Test
o Into 2 ml of albumin solution was added a solution of Hopkins-Cole as much as 2
ml. Then a solution of concentrated sulfuric acid solution was added drop wise
through the tube wall as much as 5 ml.
o Into 2 ml of albumin solution was added a solution of Hopkins-Cole as much as 2
ml. Then a solution of concentrated sulfuric acid solution was added drop wise
through the tube wall as much as 5 ml.
o Into a standard solution of tyrosine, triptopan, glycine and phenylalanine was
added a solution of Hopkins-Cole as much as 2 ml. Then a solution of
concentrated sulfuric acid solution was added drop wise through the tube wall as
much as 5 ml.
c. Ninhydrin test
o Into 3 ml of egg albumin solution was added 0.1% ninhydrin solution of 0.5 ml.
then the mixture was heated to boiling.
o Into 3 ml solution of tyrosine, triptopan, phenylalanine and glycine each was
added 0.1% ninhydrin solution of 0.5 ml. then the mixture was heated to boiling.
d. PbS test
o To the 5 ml protein solution was added 2 ml of NaOH solution. Then the Pb-
acetate solution was added 2 drops. After that, the solution was heated on a water
bath to boiling.
o To the 5 ml solution of tyrosine, triptopan, glycine and phenylalanine was added a
solution of NaOH to 2 ml.Then it was added Pb-acetate solution 2 drops. After
that, the solution was heated on a boiling water bath
e. Nitropuside Test
Some of the crystal grains of cysteine hydrochloride was dissolved in aquades as
much as 5 ml. then added a solution of 0.1% sodium nitropruside of 0.5 ml. This
mixture was added a solution of ammonia of 0.5 ml.
Beside the materials that used above, there are also unknown solution, such as solution A,
solution B, solution C, and solution D. These was tested in each test above.
C. Result and Discussion
In this experiment, carried out the test amino acids found in protein albumin (egg white).
Egg white or albumin previously separated from the egg yolk and then diluted with aquades in
the ratio 1: 5. Then the protein solution was tested with Millon test, Hopkins-Cole test, test
Ninhidfrin, PbS test, and test Nitroprusida with specific reagents. The results of experiments
conducted can be described as follows:
1. Millon Test
In the test Millon, Millon reagent is used which is made of mercury and dissolved
in nitric acid and a little nitrous acid. The use of this acid has the objective to make the
protein is not in basic conditions so that no precipitate is formed Hg (OH) 2. In addition,
the reaction is not advisable to use hydrochloric acid as it can cause the formation of
chlorine radicals that can disrupt the complex will be formed.
In this experiment the standard tests previously conducted tests of tyrosine amino
acid types, triptopan, phenylalanine and glycine. At the amino acid type thyrosin give a
positive tests with the formation of red complex. Thyrosin standard solution is white,
after adding 5 drops of Millon, the white color of the solution immediately thyrosin be
lost and the solution became clear. Subsequently the mixture was heated in a water bath
for several minutes. The results showed changes in color and heating to form a red
complex.
Hg+ -+ ( )HO CH2CH C
OH
O
NH2
HO CH2CH CO
NH2
OHg
+
White precipitate from protein e salt
The next test of the protein in egg albumin. Albumin solution has a white
colorafter the poured with 5 drops of Millon reagent mixture causes the formation of
white. Further chemical changes in the color changes to red when the mixture is heated.
This indicates the presence of amino acid type thyrosin on egg albumin. The red color
formed is derived from the nitrazed thyrosin made of mercury in nitric acid. The reaction
is as follows:
2. Hopkins-Cole Test
In the Hopkins-Cole tests used Hopkins-Cole reagent containing glyoxylate (HOOC-CHO).
This test will give positive tests for forming tryptofan with a purple ring on the tube wall. On
these tests, the standard solution tryptofan which colorless was added with Hopkins-Cole
solution of the brown lead is formed of two layers. At the top layer is light brown solution
and the bottom layer is green solution. However, when was added a solution of concentrated
sulfuric acid cause the tube to form a purple ring.
In the next tests, a solution of albumin was added Hopkins-Cole reagent. The result is
formed of two layers that do not dissolve each other. The solution at the top is colorless
solution and the bottom layer of light brown. Then, after adding a solution of concentrated
sulfuric acid slowly through the tube wall. This is done so that the rings formed are not
broken. On addition of concentrated sulfuric acid solution is formed of three layers where
the upper layer in the form of colorless solution, on the bottom layer of the solution is brown
and the middle layer to form a purple ring. Purple ring formed by condensation of an
aldehyde group of tryptofan by glyoxylate and sulfuric acid.
HgOO2NHO CH2CH COOH
CH2
+HNO3 CH2CH COOH
CH2
+
tirosin Nitrazed tyrosine
3. Ninhydrin Test
In this experiment performed tests using the ninhydrin reagent ninhydrin. This is done to
identify the presence of free amino acids such as glycine and bergugus phenilalanin. Into a
solution of tyrosine satndar, triptopan, phenylalanine and glycine, respectively was added
colorless solution of ninhydrin. The result is no change in color
The next test is tests for albumin in egg white solution. The addition of a solution of
ninhydrin in albumin cause the solution becomes blue after heating.
4. PbS Test
In this tests, use a solution of NaOH to remove the sulfide ion in the sulfide-containing
amino acid. Subsequently will be identified by the presence of sulfide formation of a black
precipitate of PbS by the addition of lead acetate. On this tests, the solution tirosisn,
triptopan, phenylalanine and glisisn was added a solution of NaOH and the solution of lead
acetate. Mixture does not form changes. After a heated black precipitate is formed which
indicate the presence of PbS. This means there is no sulfhydryl group at the fourth amino
acid. In the test of the protein solution, the addition of 2 mL of NaOH solution and 2 drops
of Pb (CH3COO) 2 does not give changing color. When heated, forming a black precipitate.
This shows the amino acid cysteine or methionine in egg albumin. The reaction is as
follows:
S2- + Pb2+ PbS(s)
5. Nitropuside Test
Nitropruside test is used to identify the presence of cysteine sulfhydryl. The reaction
between the sulfhydryl of cysteine, peptides, or proteins with sodium nitropruside in excess
of ammonia produces a red complex. In this experiment, crystal white, cysteine
hydrochloride was dissolved in aquades after forming colorless solution. Further addition of
sodium nitropruside cause the formation of two phases in which the upper layer of the
brown solution and at the bottom of the form of a colorless solution. This indicates that the
solution is not yet reacted. However, the addition of ammonia solution causes the formation
of complexes in maroon. The reaction is as follows:
[Fe(CN)5NC]2- + NH3 + RSH → NH4[Fe(CN)5NOSR]2-
Testing Unknown Sample
The sample that’s given is 4 samples. The marked of each sample are sample A, sample
B, sample C, and sample D. The result is as follow
SAMPLEACTION
Millon TestHopkins-
Cole Test
Ninhydrin
TestPbS Test
Nitropruside
test
A - - + _ -
B + + - _ -
C + - - - -
D - - - - -
1. Sample A
In Millon test, there gives negative respond, it also negative in Hopkins-Cole test, PbS
test, and Nitropuside Test. From the experiment, the sample A is phenylalanin.
2. Sample B
Sample B gives positives respond in two tests, in Millon test, and Hopkins test. So the
sample B is tryptophan.
3. Sample C
In the sample C, there are one positive respond only in Millon test. So, sample C is
tyrosine.
4. Sample D
Sample D gives negative respond in all tests. It show that the solution is not contain
amino acid. So, sample D is water.
D. Conclusion
Based on the experiment above, it can be conclude that:
1. The proteins that contain in egg albumin are cystein, tryptopan, tyrosine, and glycin.
2. On the experiment by using unknown sample, it can be concluded thatL
