TYPE OF TEST XANTHOPROTEIC TEST

QUALITATIVE TEST FOR PROTEINS RESULTS OBSERVATION + - after adding conc. Nitric acid there was a white precipitate formed, then after heating the solution went from being white into yellow color -the amino acid tyrosine and tryptophan contain activated benzene ring which can undergo nitration that is responsible for the color change + -after adding conc sulfuric acid the solution became cloudy, then there was a violet color produced at the point of contact -the cause for this color change is that the glyoxylic acid reacts with this indole ring + - the color of the solution turned to light violet after the sodium hydroxide and copper sulphate were added -peptide linkage is responsible for Biuret test - simple amino acid will not give positive biuret test because this test is detecting the presence of peptide bonds + -the color of the solution turned to blue after it was added with .01% aqueos solution of Ninhydrin and was heated to boiling -presence of alpha amino group of protein results to positive result

HOPKINS_COLE TEST

BIURET TEST

NINHYDRIN TEST

SAKAGUCHI REACTION FOR ARGININE

+ - the color of solution turned to red upon the addition of sodium hydroxide, dilute alcoholic a-naphtol solution and sodium hypochlorite solution

Objectives To determine proteins present in a solution through the use of Inorganic acid, Glyoxylic acid, CuSO4, Triketohydrindene hydrate and Alcohol. To determine the extent of protein hydrolysis and its value. Principles For Xanthoproteic Test, proteins when treated with concentrated HNO3 turn yellow and change to orange when neutralize with NaOH. This is due to the nitration of the benzene ring (C6H5) present in the structure of such amino acids as tyrosine, tryptophan and phenylalanine. For Hopkins_cole test, when proteins mixed with glyoxylic acid is treated with concentrated H2S04 a violet ring is produced at the point of contact of the two solutions. This is due to the presence of indole nucleus of tryptophan component. The tryptophan condenses with the aldehyde to form the colored compound. For Biuret Test, alkaline solution of proteins treated with CuSO4 solution results in the production of a rose- pink to violet then purple color due to the presence of peptide linkage (- CONH2). All substances therefore containing this linkage respond to this test. Biuret having similar linkage give positive result. Dipeptides do not give the biuret test, but all other peptides do. This test serves as good index for determining the extent of protein hydrolysis. When ammonium sulphate is used for salting process, an excess alkali should be added. For Ninhydrin Test, when protein is boiled with ninhydrin a blue color is produced. This is due to the presence of alpha amino group in the molecule. All proteins peptides and amino acids except proline and hydroxyproline gives this typical reaction. Sakaguchi Test is a specific type of protein with the amino acid containing the guadinine group. In basic conditions, alpha naphtol and sodium hypobromite/chlorite react with the aforementioned compound to form red-orange complexes Analysis In this experiment, we use egg albumin to determine the presence of protein. Qualitative color reactions have been devised for the detection of protein. Due to the protein molecules and single pure protein compound, these tests are used for specific chemical groups of the component amino acids. Protein or polypeptides are organic compounds made of amino acids arranged in a linear chain. The amino acids in a polymer are joined together by the peptide bonds between the carboxyl and the amino groups of adjacent amino acid residue. There were several tests that we performed for the detection of proteins. From our table analysis, we can conclude that Tyrosine, Tryptophan and Phenylalanine are present in egg albumin. We detect this certain amino acids by performing Xanthoproetic Test. The amino acids that contain benzene ring like tyrosine and tryptophan undergo nitration in this test and give yellow color. The test gives a positive result in those proteins with amino acids carrying aromatic groups, especially in the presence of tyrosine. Due to the accumulation of oxides of nitrogen (HNO3) it tends to acquire an intense yellow and it is also due to ionization of phenolic group. Based on our result, the test was positive after being treated with HNO3 and turned intense yellow. Therefore the egg albumin tested contains tyrosine, tryptophan and phenylalanine. Another qualitative test for proteins is Glyoxylic acid reaction or Hopkins- Cole test. This test specify for tryptophan, the only amino acid containing an indole group. The indolering reacts with glyoxylic acid in the presence of a strong acid to form a violet cyclic product. When protein mixed with

glyoxylic acid is treated with concentrated H2SO4 a violet ring is produced at the point of contact of two solutions. This is due to the presence of an indole nucleus in the tryptophan component. The tryptophan condenses with the aldehyde to form the colored compound. In this experiment, the test was positive result when violet color appeared of solution with glyoxylic acid that was treated with H2SO4. Therefore, there is a strongly presence of tryptophan. Aside from the two tests, we also performed Biuret test. This test identifies the presence of protein in solution with a deep violet color. Alkaline solution of proteins treated with CuSO4 solution results in the production of a rose- pink to violet then purple color due to the presence of peptide linkage (- CONH2). As students, the importance of studying hydrolysis of proteins using Biuret test is that we can determine the concentration of protein not just for the highly presence of different kinds of amino acids but for qualitatively assessing the presence or amount or the functional activity of a target entity. The Biuret test is used to detect peptide bonds and so when amino acids are not joined together they cannot be detected. Only polypeptides or proteins can be detected, not single amino acids. Based on our result, there was a positive reaction because purple color appeared. Thus, the substance contains peptide linkage not single amino acids. For Ninhydrin test, the alpha amino group of free amino acids reacts with ninhydrin, which is a powerful oxidizing agent. It undergoes oxidative deamination liberating ammonia, carbon dioxide and corresponding aldehydes and reduced form of aldehyde. The ammonia form from alpha amino group reacts with ninhydrin and its reduced products (Hydridantin) to give a blue substance, di-ketohydrin. If protein present, the color of solution after boiling will change to bluish violet. In this experiment, a dark blue color appeared in our solution after being boiled with ninhydrin which indicates the positive result of alpha amino group. And lastly, we performed the Sakaguchi test for detection of arginine. Under alkaline conditions, alpha naphthol (1- hydroxyl naphthalene) reacts with a mono- substituted guanidine compound like arginine, which upon treatment with hypobromic or hypochloric produces a characteristic red color. Base on our result, there was a tiny red particles dominated in our solution. At first, there was a dark red ring in the middle with tiny dark particles in the bottom and dark red liquid in the upper part. After a few minutes, the solution turned into red. Therefore, it is a positive reaction for the presence of arginine. Conclusion Through this experiment, we can now detect the different kinds of amino acids using egg albumin. Thus, we detect tyrosine tryptophan and phenylalanine using xanthoproetic test which gives intense yellow to the solution if positive. For strong presence of tryptophan, we can use Hopkins-Cole test. Violet colored compounds form for positive reaction on this test. On the other hand, we can use Ninhydrin and Sakaguchi test for the presence of alpha amino group and arginine. And lastly, Biuret test the most puzzling test for protein, it identifies the presence of protein in solution with a deep violet color. Positive reaction for this test indicates the presence of peptide bonds.