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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 -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).

R H2N C H COOH

The structure of -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 Arginine Histidine Isoleusine Leusine Lysine Metionin Phenylalanine Treonin Tryptophan Valine Nonessential amino acid Alanine Asparagine Aspartic acid cystein Glutamic acid Glutamine Glisine Proline Serine 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.
O

HO

CH2CH NH2

+ Hg+ OH

HO

CH2CH

O O

Hg

NH2 White precipitate from protein e salt

HO tirosin

CH2CH CH2

COOH+ HNO 3

O2N

CH2CH CH2

COOH+ HgO

Nitrazed tyrosine

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--carbonyl-4-carboxylate. Structural formula is:

H COOH NH

N H

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
O R OH + NH2 C H O C CH C O
O C N C O

O COOH H2 O C N C O -CO2

R C H R C H COOH

OH O

N +H2O -RCHO

O OH C O

O C C O

+ OH O

CH C O

NH2

Complex color (violet or blue)

The overall reaction of amino acids with ninhydrin are as follows: a. b. Oxidative Decarboxylation of amino acids and result in a reduced ninhydrin, Reduced ninhydrin reaction with ninhydrin molecule to another and with the ammonia and carbon dioxide. release of ammonia molecules form a complex in blue or purple.

O OH + OH O H

H H N + H HO

O N

Blue or violet complex color

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 No 1 2 3 4 5 6 7 Materials Materials Egg albumin Larutan Pb(CH3COO)2 Cystein Sistine Millon reagent Hopkins-Cole reagent Ninhidrin reagent Concentration Total 5 mL Sufficiently Sufficiently Sufficiently Sufficiently Sufficiently Sufficiently

8 9 10 11 12 13 14 15 No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Sodium Nitropruside reagent Ammonia reagent Concentrated sulfuric acid Aquades Phenilalanin Triptopan Glycine Tyrosine Equipments Size 25 mL 100 mL 10 mL 100 mL 250 mL 250 mL -

Sufficiently Sufficiently Sufficiently Sufficiently Sufficiently Sufficiently Sufficiently Sufficiently Total 1 rack 3 units 1 unit 2 units 3 units 1 unit 1 unit 1 unit 2 units 1 unit 1 unit 1 unit 1 unit 1 unit 2 units

Equipments Test tube Drop pipette Funnel Beaker glass Beaker glass Volumetric pipette Graduated cylinder glass Erlenmeyer Spatula Stirring rod Spiritus burner Matches Beaker glass Erlenmeyer Watch glass 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. o Hopskin-Cole Test 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.

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. o o boiling. d. o PbS test To the 5 ml protein solution was added 2 ml of NaOH solution. Then the Ninhydrin test Into 3 ml of egg albumin solution was added 0.1% ninhydrin solution of 0.5 Into 3 ml solution of tyrosine, triptopan, phenylalanine and glycine each

ml. then the mixture was heated to boiling. was added 0.1% ninhydrin solution of 0.5 ml. then the mixture was heated to

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. 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:

HO

CH2CH NH2 HO tirosin CH2CH

+ Hg+ OH

HO

CH2CH

O O-

Hg

COOH+ HNO 3

NH2 White O2N precipitate from protein e salt+ HgO CH2CH COOH Nitrazed tyrosine CH2

CH2

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.

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]2Testing Unknown Sample The sample thats given is 4 samples. The marked of each sample are sample A, sample B, sample C, and sample D. The result is as follow SAMPLE Millon 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 + + Hopkins- Cole Test + ACTION Ninhydrin Test + Nitropruside test -

PbS Test _ _ -

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 Sample A is Phenilalanin Sample B is tryptophan Sample C is tyrosine References Bintang, Maria. 2010. Biokimia Teknik Penelitian. Jakarta: Erlangga. Morrison, Robert Thornton and Robert Neilson Boyd.1983.Organic Chemistry Fourth Edition.Singapore: Allyn and Bacon, Inc Murray Robert K.et al.1995.Biokimia Harper Edisisi ke-22., alih bahasa dr.Andry Hartono.Harpers Biochemistry 22nd edition. Jakarta: Penerbit Buku Kedokteran Tika, I Nyoman.2010.Buku Penuntun Praktikum Biokimia.Singaraja: Undiksha Voet, Donald et al.1999.Fundamentals of Biochemistry.USA: John Wiley & Sons, Inc Sample D is water