2A Objectives: 1.

2.

To detect proteins by performing qualitative color tests. To determine which of the tests can detect proteins and which can detect specific amino acids in a protein molecule.

Theoretical Background: Proteins are organic compounds of high molecular weight made up of alpha-amino acids joined by means of peptide linkage. They are the most important of all biological substances being the fundamental constituents of the protoplasm of the cells. In this experiment, proteins can be detected through the use of different qualitative color tests. Proteins give various color reactions which is not specific for protein molecules, but for certain groups of the component amino acids. The first test is Xanthoproteic test. This reaction involves the nitration of benzene nucleus in alkaline solution. As a result, amino acids that contain aromatic nucleus undergo this reaction. Due to the nucleophilic content of an aromatic ring that gave the aromatic amino acids such as Tryptophan, Tyrosine and Phenylalanine, a property to be nitrated, they form yellow nitro derivative with HNO3. The second test is Glyoxylic Acid reactions or HopkinsCole test. It determines the presence of the amino acid tryptophan. When protein mixed with glyoxylic acid is treated with conc. H2SO4, a violet ring is produced at the point of contact of the two solutions. This is due to the presence of an indole nucleus in the tryptophan component. The tryptophan condenses with the aldehyde to form a violet ring. The intact protein gives a negative result because the tryptophan is not hydrolyzed enough for it to react with the glyoxylic acid. The hydrolyzed protein gives a positive result. The third test is Biuret test. It is used to detect the presence of peptide bonds. It relies on the reduction of copper (II) ions to copper (I), the latter form a complex with the nitrogen of the peptide bonds in an alkaline solution. An invigorating violet color can be observed primarily because of the peptide linkage. The fourth test is Ninhydrin test. It is a test for amino acids and proteins with a free -NH2 group. When such an -NH2 group reacts with ninhydrin, a purple-blue complex is formed. The last test is Sakaguchi reaction for arginine. The determination of this amino acid is neither to been based on the formation of urea of ammonia. It consists in the formation of an unstable red compound on treatment of a protein with sodium hydroxide, alpha-naphthol and sodium hypochlorite. Analysis: The egg albumin solution was used in the experiment to detect proteins. The first test was Xanthoproteic test. The albumin produced a yellow precipitate because it contained aromatic amino acid that reacted with HNO3. It may have contained Tryptophan, Phenylalanine or Tyrosine. The benzene ring became nitrated, resulting in a yellow precipitate. The second test was Hopkins-Cole test. The egg albumin gave a negative result. It is because of the intact protein in the albumin. The tryptophan was not hydrolyzed enough to react with the glyoxylic acid. In the Biuret test, the albumin gave a violet color appearance, thus, it contained proteins. The fourth test was the Ninhydrin test, there was a dark violet color appearance. This means that the albumin contained protein or amino acids with free -NH2. The last test was Sakaguchi reaction for arginine. Tremendous red coloration has been detected. It indicates that arginine is highly present. Conclusion: We conclude that proteins can be detected through the use of different qualitative color tests for proteins. There are general tests for proteins and specific tests for amino acids in a protein molecule. The general test is Biuret test. The specific tests are Xanthoproteic, Hopkins-Cole, Ninhydrin tests and Sakaguchi Reaction for Arginine. References: Biochemistry (12th Edition) by Espino-Cabatit Electronic Source: http://www.uwplatt.edu/~sundin/351/351h-pro.htm