IDENTIFICATION OF PROTEIN IN DUCK EGG FROM THE PEPTIDE BOND THAT CONTAINING IN THE PROTEIN

Ni Putu Yani Novi Arysanti (0913031057)Chemistry Department of Education, Faculty of Mathematics and Natural Sciences, Ganesha University of Education, Singaraja

ABSTRACTProteins are complex organic compounds with high molecular weight composed of various types of amino acids linked by peptide bonds. Structures of protein can be grouped into four classes, namely the primary, secondary, tertiary, and quaternary structure. The presence of a protein in food ingredients such as eggs can be identified through a certain way by utilizing a unique bond in the protein. The purpose of this experiment is to identify the protein in duck eggs by utilizing the peptide bonds in proteins by using biuret test, precipitating by metal, the precipitating by salt, protein denaturation and the effect of physical changes in pH and chemical substances on the protein structure.The results obtained after testing the albumin solution of duck egg are, give a positive result on a test Biuret with the formation of the purple color as a complex of Cu2 + , precipitating by metal give a positive result by the formation of white precipitate due to reaction of heavy metals with proteins produce salt proteinat neutral, the precipitating by salt produce red precipitate after test by Millon test, indicating that the protein contains the amino acid tyrosine and produce purplish-blue filtrate through Biuret test. In the coagulation test produces a red precipitate when tested with Millon reagent, precipitation with alcohol to produce a white precipitate and the denaturation of protein formed a white precipitate on addition of HCl and acetate buffer pH 4.7 after heating.Keywords : albumin, protein, amino acids, peptide bond.

I. INTRODUCTIONThe human in their lifve always conduct activities. To conduct the activites are needed the amount of energy. Energy can be obtained from food that we often eat which is containing a wide range of nutrients that body needs. These nutrients can be carbohydrates, protein and fat. Protein is one of complex organic compounds that our bodies need for vital bodily functions, especially in the metabolic process. Proteins found in a wide range of foods, especially milk, cheese, meat, grains, peanuts and soybeans. Protein has a high molecular weight and it is a polymer of amino acid monomers that are linked to each other by peptide bonds.Polypeptide chain folded in such a way as to produce a specific shape (conformation) of protein. This conformation is a three-dimensional arrangement of atoms in the structure and determined by the sequence of amino acids. There are four levels of protein structure based on the type and amount of bond / chemistry interaction is the primary, secondary, tertiary and quaternary structure.Primary StructureThe primary structure of proteins (10) is a linear sequence of amino acids linked by peptide bonds. This sequence is determined by the sequence of nucleotide bases in a gene that encodes a protein. Other bonds involved in the formation of the primary structure is a disulfide bond formed by oxidation of two sulfhydryl groups (-SH) on cysteine residues. Secondary structureThe secondary structure of proteins (20) is defined as the folding of the regions in the polypeptide chain on a regular basis. Protein secondary structures include -helices and flat sheets. Most of the secondary structure of proteins form -helical structure in which the structure of these amino acids arranged in a helical conformation on a regular basis. The carbonyl oxygen of an amino acid hydrogen bond with the hydrogen atom of the amino group at the fourth amino acid formed in the -helical structure is in a position parallel to the helical axis. While the structure of the -sheet, hydrogen bonds are formed between different polypeptide chains or on different parts of the same polypeptide chain.Tertiary structureProtein tertiary structure (30) is an arrangement of three-dimensional folding of proteins that includes elements of secondary structure of proteins with varying composition. At this tertiary structure, secondary structure elements are packed into a certain shape. On the packaging it involved a variety of bonding and chemical interactions, such as disulfide bond between cysteine amino acids, hydrogen bonding, ionic bonding between the ionized groups, and hydrophilic and hydrophobic interactions are also likely coordinate covalent bonds as in metaloprotein. All bonding and interaction in addition to forming the tertiary structure also serves as a stabilizer.Quartener StructureThis structure occurs because of the association of two or more polypeptide subunits form a protein dimer, trimer, tetramer or greater. In the quaternary structure of the interaction between the protein tertiary structure to form an aggregate that has a particular biological function. Ties involved are usually non-covalent bonding and hydrophobic bonding mostly occurs in nonpolar regions.Identification of the protein can be done by utilizing a unique bond in the proteins, that is peptide bonds using biuret test and also observed the effect of physical changes caused by changes in temperature, pH and chemical substances on the protein structure.

Figure1. Protein structure. (a) Primary structure, (b) Secondary structure, (c) Tertiary structure, and (d) Quartener structureBiuret TestBiuret is a compound with two peptide bond which is can be formed at heating process two molecule of urea at temperature of 1800C.

(Tika, 2010)Cu2+ ions of biuret reagent under alkaline condition will reacts with the polypeptide or peptide bonds that make up proteins to form a complex compound of purple or violet color. The positive reaction is characterized by the formation of purple or violet color due to the complex compounds formed between Cu2+ and N of the molecules of peptide bonds. The number of amino acids bound to the peptide bond affects the color of the reaction. Dipeptide compounds provide the color blue, purple tripeptida, and tetrapeptida and peptide complexes to give a red color. In general, the positive color of biuret reaction is to form complex compounds in which an exception for the dipeptide and amino acids (except histidine, serine, and threonine) mamberikan not test positive on this test.

Reaction of Protein and Biuret (Tika, 2010)Prcipitating protein by metalGenerally, heavy metal salt containing Hg2+, Pb2+, Cd2+ and othe metal with high atomic weight. Reaction that is occurred between heavy metal salt will causing the formation of protein metal salt that insoluble. Positive ions that can precipitate the protein is ion Hg2+, Fe2+, Cu2+ dan Pb2+, while the negative ions that can precipitate the protein is a salicylate ion, trichloroacetic piktrat, tannates and sulfosalisilat. Basic reaction of precipitating by heavy metal is the neutralization of the charge. By the presence of positive charge of the heavy metals will be joined with the negative charge of the protein occurs neutralization protein and produced precipitating of protein. This protein will precipitate redissolve on the addition of alkali base (NaOH, KOH, etc.) where the properties of this precipitation are reversible.Precipitating by SaltIf there are inorganic salts at high concentrations of proteins in solution, the solubility of the protein is reduced, which causes precipitation of the protein. This happens because the salt ions compete with protein molecules to bind water. Because of the ability of hydrated salt ions is greater than the protein molecules, the protein molecules will precipitate.The precipitation is commonly done by using ammonium sulfate salt. The technique is based on the fact that the solubility of most proteins in salt solutions with high concentrations are very low. When the salt concentration increased, the protein will come out of solution and precipitate. This process is called salting out. Salt concentration is required in this condition varies from protein to protein. For example, 0.8 M ammonium sulfate can precipitate proteins from blood serum fibrinogen whereas 2.4 M ammonium sulfate required to precipitate the albumin.Coagulation Test by AcidProtein will experience the greatest turbidity when the pH reaches the isoelectric pH where the protein has a positive and negative charges are equal. At this moment experiencing coagulation proteins. Acid may disrupt the salt bridge with the ionic charge. A type of replacement reaction occurs when the positive and negative ions in the salt change partners with positive and negative ions from the acid. This reaction is one of them can occur in the digestive system, when stomach acid coagulate milk consumed.Precipitating protein by AlcoholBasic protein precipitation with alcohol is a competition between the protein bond formation water with alcohol water. Alcohol can precipitate the protein because of the alcohol functional group (-OH) is stronger bind water through hydrogen bond formation compared to the protein molecule so that the protein solubility in water decreases. In addition, the protein is also capable of destroying the hydrogen bonds between the amide group are contained in the secondary structure of proteins so that protein loss of water (hydrated) and finally settles.The objective of this experiment is to identify proteins by making use of peptide bonds in proteins by biuret test, precipitating by metal, precipitating by salt, protein denaturation and the effect of physical changes in pH and chemical substances on the protein structure.Benefits obtained after conducting this experiment is can identify proteins by making use of peptide bonds in proteins by biuret test, a metal deposition, the deposition of the salt, protein denaturation and the effect of physical changes in pH and chemical substances on the protein structure. In addition to open the knowledge of society about the importance of amino acids for a healthy body.

II. MATERIALS AND METHODThis experiment was done in Organic Chemistry Laboratory Chemistry Education Department, Ganesha University of Education Singaraja on 29 February 2012 at 08.00-12.00 WITA. In this experiment was conducted identification of proteins in duck eggs using a peptide bond through biuret test, the deposition of the metal, salt, and alcohol, coagulation and protein denaturation. The tools and materials used in this lab are as

Tabel 1. Equipment and MaterialNama alatJumlahNama bahanJumlah

Drop pipetteTest TubeStirring rodFunnelErlenmeyer 250 mLSpatulaBeaker 100 mLBeaker 250 mLHeaterWatch glassTest tube holderTest tube rack3 pieces12 pieces1 piece1 piece2 piece1 piece3 pieces2 pieces1 piece2 pieces2 pieces1 pieceEgg Albumine NaOH 0,1 N solutionNaOH 0,25 N solutionCuSO4 solutionHgCl2 solutionPb(CH3COO)2 solution Ammonium sulfate crystals Millon ReagentBuffer acetate pH 4,7HCl 0,1N solutionAquadesEthyl Alcohol 95%Acetic Acid solution 1MBiuret Reagent50 mL10 mL11 mL3 mL5 mL5 mL5 grams3 mL3 mL10 mL100 mL20 mL10 mL10 mL

ProcedurePembuatan Larutan ProteinThe albumine of duck egg was taken as musch as 10 mL, then added by 10 mL of aquades until the volume become 100 mL, then stirred. Biuret TestAmount 10 mL of NaOH 0,25 N was added into 3 mL of albumine solution and stirred. Then was added by CuSO4 0,01 N drop by drop nad stirred. If not appear color changing was added again CuSO4 until produce color change.Precipitating protein by metal Amount 5 5 drops of HgCl2 0,2 M solution was added into 3 mL of protein solution and take noted the changing that was occured. Amount 5 drops of Lead Acetate 0,2 M was added into 3 mL of protein solution larutan protein lalu perubahan yang terjadi dicatat.Precipitating of protein by SaltAmount 3 mL of protein solution was saturated by ammonium sulphate. Ammonium sulfate salt was added little by little and stir until evenly distributed. Little ammonium salt is added and stirred again, and continued until a little ammonium sulfate salt is left behind does not dissolve. If it is saturated, the mixture was filtered. Test solubility in water and sediment deposition test with Millon reagent which is about 5 drops of Millon reagent is added to the sediment that has been added to distilled water and then heated. To the filtrate was tested with the biuret reagent by as much as 1 ml of 2.5 N NaOH was added to 3 ml protein solution and stirred. Then added dropwise a solution of CuSO4 0,01 N and stirred. Coagulation TestTwo drops of acetic acid 1M solution was added into 5 mL of protein solution and put on the boiling water during 5 minutes. After 5 minutes, the precipitate that was formed taken by using stirring rod and put on another test tube. Test the solubility of the precipitate by Millon reagent. Amount 5 drops of Millon reagent was added into the precipitate that already added by aquades. Precipitating of protein by AlcoholPrepared three test tubes and filled with 5 mL of albumin solution respectively. Test tube I filled with 1 mL of 0.1 M HCl; test tube II with 1 mL of 0.1 M NaOH, and III tubes with 1 mL acetate buffer pH4, 7. Then each test tube was added by 6 mL of ethyl alcohol 95%. The changes that occurs was observed.Protein DenaturationPrepared three test tubes, each test tube was added by 9 mL albumin solution respectively. Then test tube I was added by 1 mL 0.1 N HCl; test tube II was added by 1 mL of 0.1 N NaOH, and test tube III added by 1 mL of acetate buffer pH 4.7. The third test tube was placed in boiling water for 15 minutes and then cooled at room temperature.

III. RESULT AND DISCUSSIONPreparation of Protein SolutionIn this experiment was used Duck Egg that will be taken the albumin and diluted by aquades with comparison 1:5 so that produce protein solution with white turbid color. This solution was used to identify the protein.

Figure 2. Albumin Solution

Biuret TestBiuret test is one test to identify the presence of protein in general. The result that was obtained after albumin solution added by NaOH solution and continuing by the addition of CuSO4 solution is produce violet solution. Its indicate that in the albumin was formed complex of Cu2+ ion from Biuret with N atom from peptide bond molecule.

Figure 3. Positive Biuret Test The addition of NaOH before the addition of Adapun CuSO4 solution has function to make basic condition because Cu2+ ion of Biuret Reagent only reacts with peptide bond composing of protein in acidic condition. The reaction is as follows. (a)

(b) (Tika, 2010)(a) Reaction of Biuret Formation, (b) Complexs structure of Cu2+ with ProteinNot all protein if tested by Biuret test will producing violet collor. Its depend on the type of peptide bond that presence in the protein solution of duck egg. The formation of violet complex indicates that peptide bond that containing in albumin of duck egg is tripeptide bond which is consist of 3 amino acid with two peptide bonds. The general formula of tripeptide bond is as figure below.

Precipitating by MetalIn this experiment, was used two kinds of solution that made of heavy metals like HgCl2 0,2 M and Pb(CH3COO)2 0,2 M. The albumin was put on two test tube. The result that was obtained after first albumin solution was added by 5 drops of HgCl2 0,2 M and second albumin solution added 5 drops of Pb(CH3COO)2 0,2 M is both of solution producing white precipitate.

(b)(a)Figure 4. (a) Reaction of albumin and HgCl2 and (b) Reaction of albumin and Pb(CH3COO)2This is caused by neutralization reaction between cation charge of heavy metal with anion of protein. The presence of CH3COO- and Cl- ion of Pb(CH3COO)2 and HgCl2 solution causing the solution become little bit acidic so that protein solution become base and become its anion. Because proteins are amphoteric which may be present as a neutral form at the isoelectric point and in the form of anion or cation.

Acidic ConditionIsoelectric pointBasic conditionBy the change of protein become its anion so will react with cation of metal ion to produce proteinat salt that unsoluble in water. This is causing the formation of the precipitate when added heavy metals. The reaction is as follows.

Proteinat salts are not soluble

Precipitating of protein by SaltIn this experiments used ammonium sulfate salt because it has a high solubility in water, not too affected by the change in temperature and not too damaging most of the protein. Ammonium sulfate salt in the form of yellowish white crystals in which the precipitating is done by adding ammonium sulfate salt into albumin solution until saturated in the meaning that until little salt that is not soluble in the solution of albumin.After the addition of salt is continuously followed by stirring the solution of albumin that was initially cloudy yellow color change to white milk. The addition of salt was stopped when there was still a bit of salt which is insoluble in water and the solution became milky white color. This change is due to the competition of salt ions with protein molecules that bind water in which the ability of hydrated salt ions is greater than the protein molecules. At the time of addition of salt, the salt concentration will also increase so that the protein will come out of solution and become precipitate. This process is called salting out.

Figure 5. Precipitate of protein Then the solution was filtered, the filtrate obtained is colorless and yellowish white precipitate. The filtrate obtained was tested by using the biuret test give a color change from colorless to blue-purple. This indicates that the filtrate still contains a protein that has not been settled.

(d)(c)(b)(a)

Figure 6. Filtrate after filtering process (a), Positive Biuret Test (b), Reaction between precipitate and aquades (c) and Positive Millon Test (d)Testing was continued by testing the solubility of precipitate in water and Millon test. The results obtained after the precipitate enterred into aquade, the precipitate little bit soluble. Then precipitate in aquades was added by 8 drops of Millon reagent and obtained results in the form of a white precipitate formed and after heating, a white precipitate which turns red. This indicates that the albumin in the solution containing the amino acid tyrosine in which the deposition is a complex salt of nitrated tyrosine.In the testing of the filtrate and precipitate both showed positive results that indicate the filtrate still contains protein. This means that the protein was precipitated has low solubility than the protein that not precipitate (still soluble) which is having a high solubility is still competing with ammonium sulfate salt. This shows that the addition of ammonium sulfate is not maximized.Coagulation TestOn this testing was done by adding 2 drops of 1 M acetic acid solution into the protein solution to produce a white precipitate. Precipitate formation due to the addition of acid to the protein solution causes H+ ions from the acid will be bound to groups which are have negatively charged so that the polarity changes of protein molecules.This polarity change of the protein causes a conformational change or damage to the structure of tertiary or quaternary structure of proteins that have protein coagulation. The precipitate which formed more increase when heated in a water bath because the temperature is not too extreme so that the precipitate that has formed due to multiply the number of modifications have been made to the structure of the secondary, tertiary and quaternary protein molecules.

Figure 7. Reaction result of albumin solution and acetic acid The precipitate obtained then tested with Millon reagent and test solubility in water. When testing solubility in water, precipitate does not dissolve. This is because the folds of the tertiary or quaternary structure of proteins so that the nonpolar will be outside the resulting insoluble precipitate. When added to the Millon reagent to form a white precipitate and the precipitate turned red after heating which showed positive results for protein that precipitated in the process of salting out. Red precipitate is a complex salt of ternitrasi tyrosine resulting in a that precipitated of proteins that contain amino acid tyrosine.

(b)(a)

Figure 8. Positive Millon test (a) and Reaction between precipitate and water (b)

Precipitating protein by AlcoholIn this experiment protein solution was given different treatment in each test tube which is already containing 5 mL of egg albumin. In the first test tube was added 1 ml of HCl solution resulting a turbid white solution. This indicates that the protein was partially precipitated due to the addition of acid causes the proteins to be under the isoelectric point so that reduced the solubility in water and become precipitate. After a turbid white solution was added by ethyl alcohol coagulation occurs. Competition is due to the formation of bonds between the protein-water with an alcohol-water. Functional group of the alcohol (-OH) is stronger bind water through hydrogen bond formation compared to the protein molecule so that the protein solubility in water decreases. In addition, the protein is also capable of destroying the hydrogen bonds between the amide group are contained in the secondary structure of proteins so that proteins lose water and eventually precipitate.In the second test tubes which had contained 5 mL of egg albumin was added 1 mL of 0.1 M NaOH and the results obtained is the solution became colorless. This is due to the addition of NaOH resulted in a change in pH above the isoelectric pH thus increasing their solubility in water. Having added a solution of the solution remains clear ethyl alcohol. This is due to increased solubility of protein molecules become less competitive in binding water than alcohol groups of the ethyl alcohol.In the third tube, after adding 1 mL of acetate buffer in a 3 mL solution of protein, then formed and slightly turbid colloidal solution. Once added etilalkohol 95%, then the white precipitate formed and more increase turbid solution. The addition of acetate buffer to achieve the protein isoelectric pH (4.7) where the pH of this protein will reach the highest turbidity.

(a)(b)(c)

Figure 9. Results Albumin Solution Reaction with HCl solution and etilalkohol (a), results Albumin reaction solution with NaOH solution and ethyl alcohol (b) and the reaction results with Albumin Buffer Solution and alcohol (c)

Protein DenaturationOn protein denaturation experiments are given different treatment in each test tube already containing 9 mL of egg albumin. In the first tube was added 1 mL of 0.1 M HCl solution and heated, the second tube was added 1 mL of 0.1 M NaOH, while the third tube was added 1 mL of acetate buffer pH 4.7 and heated. The results obtained are in tubes 1 and 3 give similar results which are turbid solution and white precipitate formed and after cooling a white precipitate. While in the tube 2 solution did not change (remains colorless) because the presence of the OH-base will lead to increased hydrogen bonding to their solubility in water increases.Changes in test tube 1 due to the addition of HCl causes damage the balance of zwitterions resulting protein solution was under the isoelectric point. Thus was occurred protein denaturation that is the changing natural properties of of proteins that occur caused by changes in pH and temperature when heating. Denaturation of the protein conformational changes resulting in -helix becomes elongated. This is due to layers of molecules in the hydrophobic part will come out while the hydrophilic part to be folded in so that the protein will coagulate and precipitate. Changes in the test tube 3 is caused by making the protein reaches the isoelectric pH (4.7) where the pH of this protein will reach the highest turbidity.Besides pH, denaturation also occurred caused by changes in temperature are not too extreme in the protein solution. 80-100oC warming could causes destruction of hydrogen bonding and hydrophobic interactions at nonpolar protein that decreases water binding capacity and protein was coagulated.

(b)(a)(c)

Figure 10. Tube 1 after addition of HCl and heating, the tube 2 after the addition of NaOH and heating, and tube 3 after addition of acetate buffer and heating

CONCLUSION Based on the experiments and discussion, it can be concluded that the albumin solution of duck egg give positive test for biuret test (purple form), producing precipitate causing by precipitating by metal, alcohol and salt, producing colloid on coagulation tests and can be experienced denaturation caused by the presence of acid and heating process.

REFFERENCESAnwar, Chairil. 1994. Pengantar Praktikum Kimia Organik. Yogyakarta: Direktorat Pendidikan Tinggi Redhana, I Wayan dan Siti Maryam. 2004. Buku Ajar Biokimia Jilid 1. Singaraja: IKIP Negeri SingarajaRedhana, I Wayan dan Siti Maryam. 2010. Penuntun Praktikum Biokimia. Singaraja: IKIP Negeri Singaraja1

Tika, I Nyoman. 2007. Penuntun Praktikum Biokimia. Singaraja: Universitas Pendidikan