QUALITATIVE ANALYSIS OF PROTEINS IN EGG ALBUMEN
Gusti Ngurah Putu Eka Putra (1013031020)
[email protected] Chemistry Education Department Faculty of Math and Natural Sciences, Ganesha University of Education ABSTRACT
There are two points as the aims of this experiment: (1) To identify protein by using peptide bond at protein through biuret test, sulfur test, precipitate by metal, precipitate by salt and protein denaturation. (2) To identify the influence of physical change such as pH and chemical substance to the protein structure. This is qualitative laboratory experiment in which the egg albumen used as the protein sample. The results were obtained that the protein identification can be conducted by biuret test, protein can be precipitated by heavy metal ions, salt and alcohol, protein capable of undergoing coagulation with the addition of acid, and proteins proteins can undergo denaturation by addition of acid and buffer acid and by heating process. The sample gave positive respond due to the biuret test which means, it contains tripeptide bonds, precipitated by metal, sat, alcohol and coagulation test and gave negative respond due to milon’s tests.
Key Words Protein’s identification, Egg albumen, Protein INTRODUCTION
Protein has four structures, namely the structure of primary, secondary, tertiary and quaternary. Here is an explanation of the four structures; Primary Structure The o primary structure (1 ) is a linear sequence of amino acids linked by peptide bonds (Redhana, 2004). The primary structure of a polymer chain that describes the basic arrangement of amino acids in the chain its peptide is regardless of the possibility of interactions between amino acids. In the primary structure contained amino acids sequences that make up proteins. Secondary Structure Ties contained in the secondary structure include bond contained in the primary structure (covalent) and the hydrogen bond between the carbonyl oxygen and amide hydrogen of the peptide bond. This hydrogen bond is formed by a regular pattern so as to form unique structures such as αα-helices and β-sheet β-sheet (Tika, 2007) Tertiary o Structure Protein tertiary structure (3 ) is a
three-dimensional arrangement of proteins that includes the folding of secondary structure elements. Secondary structure elements of the primary (α(α-helices and ββsheet), but the proportions and combinations vary widely (Redhana, 2004). In the secondary structure, secondary structure elements are packed in some form. On the packaging it involved a variety of bonding and chemical interactions such as disulfide bond between cysteine amino acids, hydrogen bonding, and ionic bonding between the ionized groups, hydrophilic and hydrophobic interactions and covalent bonding coordination. All of these interactions in addition to bond and form a tertiary structure also act as a stabilizer (Tika, 2007). Quaternary structure Protein quaternary structure occurs because the association of two or more polypeptide subunits forms a protein dimer, trimer, tetramer or larger (Redhana, 2004). There are
various ways you can do to make the testing of the protein which is often called by the reaction of the protein test. The reaction carried out with test biuret protein, by metal deposition, the deposition of the salt and alcohol, coagulation tests with acids, denaturation of proteins and sulfur in the protein test. Biuret test Biuret is a compound with two peptide bonds that are formed on heating 2+ two molecules of urea. The Cu ions under alkaline biuret reagent reacts with the polypeptide or peptide bonds that make up proteins form a complex compound of purple or violet. The positive reaction is characterized by the formation of purple color due to the complex compounds formed 2+ between Cu and N of the molecules of peptide bonds (Tika, 2010). The number of amino acids bound to the peptide bond also affects the color of this reaction. Compounds with dipeptide give blue, purple and tetrapeptide tripeptide peptide complexes as well as giving a red color. In general, the positive color of biuret reaction is to form complex compounds which are described as follows. Deposition by Metal Generally 2+ 2+ 2+ contain heavy metal salts of Hg , Pb P b , Cd and other metals with large atomic weight. The reaction that occurs between the heavy metal salts will result in the formation of protein-salt-insoluble protein (Ophart in Tika, 2010). Ions - positive ions that can precipitate 2+ 2+ 2+ 2+ the protein is Hg , Fe , Cu and Pb ,, while the negative ions that can precipitate the proteins were: ion of salicylic, trichloroacetic piktrat, Tannates and sulfosalisilic (Tika, 2010). Basic reagent is a heavy metal deposition by neutralizing the charge. It given the positive charge of the heavy metals will undergo neutralization and the resulting protein precipitate proteins. This protein will precipitate redissolve on the addition of an alkali base. The nature of this deposition is reversible (Achmad in Tika, 2010)
Precipitation by Salt If there is salt inorganic salts at high concentrations of proteins in solution, the solubility of the protein is reduced thus causing the precipitation of proteins. p roteins. This occurs because the ion - ion salts compete with the molecule - a protein to bind water molecules or hydrated. Because of the ability of ion - ion is greater than the hydrated salt than protein molecules; protein molecules will precipitate (Tika, 2010). Deposition is commonly done by using ammonium sulfate salt. The technique is based on the fact that the solubility of most proteins in a salt solution with high concentration is very low (Redhana in Tika, 2010). When the salt concentration increased, the protein will come out of solution and precipitate. This process is called salting out. Salt concentration required to salting out of this condition varies. Coagulation by Acid Test Protein will have developed the greatest turbidity when the pH reaches isoelectric that is where the protein has h as positive po sitive and an d negative charges are equal. At this moment protein is experiencing coagulation. Acid may disrupt the salt bridge with the ionic charge. A type of neutralization reaction occurs when the positive and negative ions from the salt change partners with positive and negative ions from the acid. This reaction is one of them can occur in the digestive system, stomach acid when consumed milk coagulating (Tika, 2010). Precipitation by Alcohol Basis of protein precipitation with alcohol is a competition between the formations of protein-water bonds with the alcohol-water. Alcohol can precipitate 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, alcohol can also damage the hydrogen bonds between the amide group are contained in the secondary structure of
proteins so that protein loss of water (hydrated) and finally settled (Tika, 2010). Protein Denaturation Protein denaturation can be interpreted as an alteration or modification of secondary structure, tertiary and quaternary protein molecule without breaking the bond - a covalent bond. Therefore, the denaturations can mean as a process of splitting of the hydrogen bonding, hydrophobic interactions, salt bonds and the opening of the folds of the protein molecule (Tika, 2010). Protein solubility is reduced denaturated. Layer of molecules in the hydrophobic part will come out while the hydrophilic are folded into that. Folding or reversal will occur when the protein near pH isoelectric and protein will clump and settle. Viscosity will increase as the molecules expand into an asymmetric, optical rotation angle will also increase the protein solution (Tika, 2010). Protein denaturation includes disruption and damage that may occur in secondary and tertiary structures of proteins. At the tertiary protein structure there are four types of interactions that form bonds to the side chain such as hydrogen bonding, salt bridges, disulfide bonds and hydrophobic interactions i nteractions are likely non-polar disorder. Denaturation commonly encountered is the process of precipitation and coagulation of proteins (Tika, 2010). Sulfur Test in Protein Sulfur in the protein can be oxidized to sulfate ion by 2+ oxidizing agents. Acidic sulfate ions in Ba ions react forming a white precipitate (Tika, 2010). MATERIALS AND METHODS
Equipment used on this experiment was: test tubes drop pipettes, beaker glass, graduated cylinder, spatula, glass rod, spritur burner, Erlenmeyer, porcelain basin and wood clamp. Whereas, the materials used were: protein solution (albumen), NaOH 0.1N and 0.25N, CuSO4 solution, HgCl2, Pb(CH3COO)2 solution, milon’s reagent,
Buffer acetate pH: 4.7, HCl 0.1N, ethyl alcohol 95%, CH3COOH 1M and (NH4)2SO4(s). The procedures of this experiment were as follows: - Preparation of Protein Solution The albumine of egg was taken as musch as 10 mL, then added by 10 mL of aquades until the volume become 100 mL, then stirred. - Biuret Test Amount 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.
-
Precipi cipitati tation on by metal
Amount 5 drops of HgCl2 0.2 M solutions was added into 3 mL of protein solution and take noted the changing that was occurred. Amount of 5 drops of Pb-Acetate 0.2 M was added into 3 mL of protein solution and the changing occur was noted. - Precipitation by Salt Amount 3 mL of protein solution was saturated by ammonium sulphate. Ammonium sulfate salt was added little by little and it was stirred until evenly distributed. Little ammonium salt is added and stirred again, and continued until a little ammonium sulfate salt is left behind or does not dissolve. If it is saturated, the mixture was filtered. The solubility in water of the precipitate was tested. At about 5 drops of Millon reagent is added to the precipitate 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 drop wise a solution of CuSO4 0.01 N and stirred. - Coagulation Test Two drops of acetic acid 1M solution was added into 5mL 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. recipitati pitation by Alco Al cohol hol - Preci Prepared three test tubes and filled with 5mL of albumin solution respectively. Test tube I filled with 1mL of 0.1 M HCl, test tube II with 1 mL of 0.1 M NaOH, and tube III with 1 mL acetate buffer pH4, 7. Then each test tube was added by 6 mL of ethyl alcohol 95%. The change that occurs was observed. Denaturati uration on - Protein Denat Prepared 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 three test tubes were placed in boiling water for 15 minutes and then cooled at room temperature.
In the test of metal precipitation the white precipitate which is soluble in water was produced. This means a positive respond of the egg albumen contain protein. In this experiment protein solution was added by HgCl2. When the addition of this solution became slightly turbid and a white precipitate formed. The same thing happens when the Pb(CH3COO)2 was added. The formation of white precipitate indicates that the protein 2+ 2+ solution was deposited by ion Hg and Pb were respectively derived from a solution of HgCl2 and Pb(CH3COO)2. In general protein can be precipitated 2+ 2+ 2+ by heavy heav y metal ions such as Hg , Pb , Cd + and Ag . This can occur due to charge neutralization reactions between heavy metal ions with the anion of the protein to produce salt-insoluble protein. Need to be that the protein is an electrolyte that is amphoteric colloid. In the neutral form is the form of bi polar compounds whose condition is known as the isoelectric point. The reaction is as follows:
RESULT AND DISCUSSION
In biuret test, the dark purple color of product indicates a positive reaction. In a positive test, a copper (II) ion was wa s reduced to copper (I), which forms a complex with the nitrogen and carbons of the peptide bonds in an alkaline solution. The reaction seen as follows:
O
C
C
RHC
CHR
Cu
2+
C
C
NH
HN
CHR
Figure 2. Reaction between heavy metal ion and protein
NH
HN
O
O
Insoluble of proteinat
RHC
Figure1. Complex of Cu-protein
O
The third test was protein precipitation by salt. In this experiment, the salt used was (NH4)2SO4. It started from the saturation of protein solution by addition of salt (NH4)2SO4. It gave positive respond by the formation of white precipitate. The formation of a white color of the protein solution can be explained due to the addition of salt at high concentrations and causing the salting out of the protein. At this state, the
ions of the salt ammonium ions compete with the protein to bind water. Because of the ability of salt ions to bind to water is greater than the protein, the protein will come out of solution and form a white precipitate. The white precipitate was soluble in water and gave negative respond due to milon’s tests. Which means that the the protein sample don’t contain tyrosine. However, it gave positive respond in biuret test by the formation of purple color of product. This indicates that in the filtrate still contained protein. The fourth test was coagulation test. The protein solution produced white precipitate as the addition of CH3COOH. The formation of white precipitate indicates that the protein is present in the sample. Acid may disrupt the salt bridge with the ionic charge neutralization reaction that coagulating the protein solution. In addition, it is also capable of undergoing coagulation protein when it reaches the isoelectric pH. The product was soluble in water but gave negative respond to milon’s tests. After warm up during the milon’s tests the white precipitate became yellow and produced colorless solution. This indicates that in the protein don’t contain tyrosine. The fifth test was precipitated by alcohol test. In this experiment protein st solution was added with acetic acid. The 1 sample was added by HCl ethyl alcohol 95%. nd The 2 sample was added by NaOH and rd ethyl alcohol 95%. The 3 sample was added by the acetate buffer pH 4.7 and ethyl st alcohol 95%. In the 1 sample gave nd congealing (penggumpalan) product, the 2 rd sample gave colorless product and the 3 sample gave white precipitate product. The white precipitate indicates that the addition of acid (HCl) and acetate buffer pH 4.7 within the protein solution causing the precipitation. In this reaction the alcohol and protein is
compete to each other in bonds with water. The addition of alcohol made precipitate because 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, alcohol can also damage the hydrogen bonds between the amide group are contained in the secondary structure of proteins so that protein loss of water (hydrated) and finally precipitate. The last test is protein denaturation. In this experiment there were 3 samples: the st nd 1 sample was added by HCL 0.1M, the 2 rd sample was added by NaOH 0.1M and the 3 sample was added by buffer acetate pH 4.7. The protein denaturation itself is process of change or modification to the structure of the secondary, tertiary and quaternary protein molecule without breaking the covalent st bonds. During the treatment, the 1 gave congealing (penggumpalan) that become white milky congealing (penggumpalan) nd after heated. The 2 gave colorless products that became yellowish with clear solution rd after heated. However, the 3 gave congealing (penggumpalan) that became white solid after heated. The white precipitate indicates that there had been occurs protein denaturation. Denaturation can occur because of temperature and pH. Heating at high o temperatures (above 80 C) cause damage to the structure of the protein and loss of protein activity. Then the white precipitate formed in the protein solution by addition of HCl and acetate buffer pH 4.7. The acetate buffer maintains the pH balance to damage the zwitter ion during the acidic conditions below the isoelectric point. Clearly, the observation result of entire test given by the following data:
Table 1. Observation results Biuret test (+) Violet solution
Metal ppt. (+) White solution and insoluble in water
Salt ppt. (+) White ppt insoluble in water
Coagulation (+) White ppt and insoluble in water
Alcohol ppt. 1 sample: Congealing (penggumpalan) s
Denaturation. s 1 sample: turbid congealing (penggumpala n)
nd
(-) Milon’s test: white suspension
(-) Milon’s test: white ppt yellow ppt + colorless solution
2 sample: colorless
nd
2 sample: colorless yellowish with clear solution rd
(+) Biuret test: blue solution
CONCLUSION Based on the result and discussion above, it can be concluded that the identification of protein can be conducted by biuret test which means, it contains tripeptide bonds, protein can be precipitated by heavy metal ions, salt and alcohol, protein capable of undergoing coagulation with the addition of acid, and proteins can undergo denaturation by addition of acid and buffer acid and by heating process. It is also means that the egg albumen as the protein resource is positive containing protein.
3 sample: white ppt rd
3 sample: congealing (penggumpala n) white solid.
ACKNOWLEDGMENTS The reporter wish to thank Dr. I Nyoman Tika, M.Si as the lecture who gave g ave the guidance in several cases during the experiment conducted, Mr. Lasya as the laboran who kept care of us beyond the experiment and the patience on us. The classmate of RKBI 2010 who kept motivates each other during the experiment. Thanks to all of them, in particular for their attention to detail.
REFFERENCES Muderawan, IW. (2010). Organic Chemistry II: Amino Acids, Peptieds and Singaraja: Ganesha Protein. Unsiversity of Education. Parning. 2005. Kimia 3B SMA Kelas XII. Jakarta : Penerbit Yudhistira Redhana, I Wayan dan Siti Maryam. 2004. Buku Ajar Biokimia Jilid 1. Singaraja: IKIP Negeri Singaraja. Tika, I Nyoman. 2010. Buku Penuntun Praktikum Biokimia. Biokimia. Singaraja: Universitas Pendidikan Ganesha.
