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Ilar, Quenie Mariel

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BS-Chemistry

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ASCHEM3 Activity 8 Color Reactions of Proteins and Amino Acids Abstract: Amino acids are the building blocks of proteins. Each amino acid varies in the side chains they possess which can be used as a tool in qualitative analysis in proteins. The qualitative tests conducted include: Ninhydrin, Biuret, Xanthoproteic, Millon’s, Hopmkins-Cole, Bromine water, Pauly, Reduced sulfur, and Sakaguchi reactions. Albumin was confirmed to have the presence of tryptophan, tyrosine, histidine, cysteine while gelatin only contains tyrosine, histidine, and arginine based on the range of amino acids tested. Hair was also included in the Reduced Sulfur test to check the presence of cysteine.Thus, amino acid derivatives are essential in confirming the existence of certain amino acid in proteins through color reactions. Introduction Amino acids are critical to life since they have particularly important functions like being the building blocks of proteins and being the intermediates in metabolism. Amino acids possess an amine group, a carboxylic acid group and a varying side chain that differs between different amino acids. The side chain structure determines the class of the amino acid: nonpolar, neutral, acidic, or basic (Milio and Loffredo, 1995). Proteins (also known as 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 residues. Like other biological macromolecules such as polysaccharides and nucleic acids, proteins are essential parts of organisms and participate in virtually every process within cells (Nigam and Ayyagari, 2007). Certain functional groups in amino acids and proteins can react to produce characteristically colored products. The color intensity of the product formed by a particular group varies among proteins in proportion to the number of reacting functional, or free, groups present and their accessibility to the reagent (Milio and Loffredo, 1995). In this experiment, various color-producing reagents were used to qualitatively detect the presence of certain functional groups in amino acids and proteins. The principles of these qualitative tests are defined below. Ninhydrin reaction

In the pH range of 4-8, all α- amino acids react with ninhydrin (triketohydrindenehydrate, shown in Figure 1), a powerful oxidizing agent to give a purple colored product (diketohydrin) termed Rhuemann’s purple. All primary amines and ammonia react similarly but without the liberation of carbon dioxide. The imino acids proline and hydroxyproline also react with ninhydrin, but they give a

yellow colored complex instead of a purple one. Besides amino acids, other complex structures such as proteins also react positively when subjected to the ninhydrin reaction. Figure 1. Structure of Ninhydrin

Biuret reaction The biuret test for protein positively identifies the presence for proteins in solution with a deep-violet color. Biuret, H 2NCONHCONH2, reacts with copper (II) ions in basic solution to form a deep violet complex. The peptide linkages in proteins resemble those in biuret and also form deep violet complexes with basic copper (II) ion in solution. The general or biuret complex formed between the protein linkages and the copper (II) ion of the biuret test is shown in Figure 2.

Figure 2. Structure of protein-copper(II) complex

Xanthoproteic reaction Some amino acids contain aromatic groups that are derivatives of benzene. These aromatic groups can undergo reactions that are characteristics of benzene and benzene derivatives. One such reaction is the nitration of a benzene ring with nitric acid. The amino acids that have activated benzene ring can readily undergo nitration. This nitration reaction, in the presence of activated benzene ring, forms yellow product. Figure 3 below shows the mechanism for Xanthoproteic reaction.

Figure 3. Xanthoproteic test reaction mechanism

Millon’s reaction Millon’s test is specific to phenol containing structures (tyrosine is the only common phenolic amino acid, Figure 4). Millon’s reagent is concentrated HNO 3, in which mercury is dissolved. As a result of the reaction a red precipitate or a red solution is considered as positive test.

Figure 4. Structure of Tyrosine

Hopkins-Cole reaction This test is specific test for detecting tryptophan (Figure 5). The indole moiety of tryptophan reacts with glyoxilic acid in the presence of concentrated sulphuric acid to give a purple colored product.

Figure 5. Structure of Tryptophan

Bromine water test The addition of bromine water and n-amyl alcohol to a solution containing free tryptophan results in the formation of a pinkish lavender or violet color in the alcohol layer. In the presence of excess bromine water, the pink color disappears and it may be masked by the color of the reagent. Pauly reaction This test is specific for the detection of Tryptophan or Histidine (Figure 6). The reagent used for this test contains sulphanilic acid dissolved in hydrochloric acid. Sulphanilic acid upon diazotization in the presence of sodium nitrite and hydrochloric acid results in the formation a diazonium salt. The diazonium salt

formed couples with either tyrosine or histidine in alkaline medium to give a red coloured chromogen (azo dye).

Figure 6. Structure of Histidine

Reduced Sulfur Test Proteins containing sulfur (in cysteine and cystine) give a black deposit of lead sulfide (PbS) when heated with lead acetate in alkaline medium. The structures of cystine and cysteine are shown in Figures 7 and 8, respectively.

Figure 7. Structure of cystine of cysteine

Figure 8. Structure

Sakaguchi reaction The Sakaguchi reagent is used to test for a certain amino acid and proteins. The amino acid that is detected in this test is arginine (Figure 9). Since arginine has a guanidine group in its side chain, it gives a red color with α-naphthol in the presence of an oxidizing agent like bromine solution

Figure 9. Structure of arginine

Methodology The following test samples were subjected to different qualitative tests to detect the presence of certain functional groups in amino acids and proteins: Tryptophan, Glycine, Albumin, Gelatin, Hair, and distilled water as blank sample.

The reagents used in conducting the qualitative tests were: n-amyl alcohol, freshly prepared bromine water, 0.55 copper(II) sulfate, glyoxylic acid, 10% lead acetate, Millon’s reagent, α-naphthol, freshly prepared 0.2% Ninhydrin solution, concentrated HNO3, solid sodium acetate, Na2CO3, 10% and 20% NaOH, freshly prepared 0.5% NaNO2, sulfanillic acid, and concentrated H2SO4. A. Ninhydrin Reaction Each of the following samples was neutralized with NaOAc: Glycine, Albumin, Gelatin, and distilled water. To one mL of each sample, 2-3 drops of 0.2% freshly prepared Ninhydrin solution was added and was boiled in a water bath for more than 2 minutes. The heated mixture was allowed to cool and the resulting color was noted. B. Buret (Piotrowski’s) Reaction A mixture was prepared with 1mL of 10% NaOH, , 1-2 drops of 0.5% CuSO4 and 1 mL of each of the following samples: Glycine, Albumin, Gelatin, and distilled water. C. Xanthoproteic Reaction One mL of each of the following samples was added with 1 mL concentrated HNO3: Tryptophan, Albumin, distilled water. Each mixture was immersed in a boiling water bath for 5 minutes, cooled, and was made alkaline with 20% NaOH. Color change was noted. D. Millon’s Reaction Millon’s reagent was added to 1mL of each sample: Gelatin, albumin, Peptone, and distilled water. For 10 minutes, the mixture was heated in boiling water bath and was cooled afterwards. To the mixture, 0.5 mL of 0.5% NaNO2 was added and was gently warmed. The resulting color of the precipitate and/or the solution was observed. E. Hopkins-Cole Reaction In separate test tubes, 1 mL of Gelatin, Albumin, Tryptophan, and distilled water was placed. Glyoxylic acid reagent (0.5 mL) was added to each sample. One mL of concentrated H 2SO4 was layered in every test tube with different samples. Tryptophan was present if a violet ring appeared at the junction of the two fluids after a few seconds. F. Bromine Water Test Tryptophan, Gelatin, Albumin and distilled water were the samples obtained and 1 mL was poured in separate test tubes. Two drops of freshly prepared bromine water was mixed in ever test tubes. With 1 mL n-amyl alcohol was mixed and shaken until the color of the alcohol layer was observed. G. Pauly Reaction One mL of cold sulfanic acid with 1 mL of cold 0.5% NaNO 2 was mixed in separate test tubes. For 3 minutes, mixtures were cooled with ice and shaken constantly. In separate test tubes with the mixtures, 1 mL of Gelatin

and Albumin was added and alkaline with 10% Na 2CO3. Color formed was noted and blank test was performed for blank test. H. Reduced Sulfur Test In separate test tubes, 1 mL of Gelatin, Albumin, Hair and distilled water was poured and added with 1 mL of 20% NaOH and 2 drops of 10% Pb(OAc)2. With a marble, the mixtures were covered and boiled in a water bath for few minutes. The solution darkens if cysteine was present; the color was deepening into black if sufficient sulfur was present. I. Sakaguchi Reaction Two mL of 20% NaOH was mixed in separate test tubes with 1 mL samples of Gelatin, Albumin and distilled water and added with 2 drops of alpha-naphthol reagent. The solution was mixed and added 0.5 mL fresh bromine. The color formed was noted.

Results and Discussion Tables 1. Ninhydrin and Biuret Reactions Samples

TESTS NINHYDRIN

BIURET

1. Glycine

intense blue

blue/light blue solution

2. Albumin

purple solution

purple solution

3. Gelatin

purple solution

purple solution

colorless solution

blue/light blue solution

4. Distilled water

Ninhydrin test Amino acids contain a free amino group and a free carboxylic acid group that react together with ninhydrin to produce a colored product. When an amino group is attached to the alpha carbon on the amino acid’s carbon chain, the amino group’s nitrogen atom is part of a blue-purple product as shown in Equation 1 below. Proteins also contain free amino groups on the alpha-carbon and can react with ninhydrin to produce a blue-purple product. From the data on the table, Glycine is an amino acid therefore it yielded the Ruhemann’s purple. Since both albumin and gelatin are proteins, they also react with ninhydrin to form a purple product.

Equation 1. Mechanism for Ninhydrin test

Biuret test This test is used to differentiate between proteins and amino acids. The biuret reagent (copper sulfate in a strong base) reacts with peptide bonds in proteins to form a blue to violet complex known as the “Biuret complex” shown in Figure 2 back in the first page of this paper. Since two peptide bonds are at least required for the formation of this complex, only proteins respond positively to this test. The protein samples, Albumin and Gelatin, formed the purple-colored Biuret complex. Glycine did not respond positively to this test since it is an amino acid. Table 2. Xanthoproteic Reaction Samples 1. Tryptophan 2. Albumin 3. Distilled water

Observation blood red solution orange solution colorless solution

Aromatic amino acids respond to this test. In the presence of concentrated nitric acid, the aromatic phenyl ring is nitrated to give yellow colored nitroderivatives. At alkaline pH, the color changes to orange due to the ionization of the phenolic group. Tryptophan, an aromatic amino acid, gives a blood red solution because there is partial oxidation of the substance by nitric acid. Albumin was observed to produce an orange solution after the reaction since it is a protein which contains an aromatic amino acid in its chain. Table 3. Millon’s Reaction Samples 1. Gelatin

Observation deep red precipitate

2. Albumin

light pink precipitate

3. Distilled water

colorless solution

Millon's reagent gives positive results with proteins containing the phenolic amino acid “tyrosine”.In this test, the phenol group of tyrosine is first nitrated by nitric acid in the test solution. Then the nitrated tyrosine complexes mercury (I) and mercury (II) ions in the solution to form a red precipitate or a red solution, both positive results. Proteins that contain tyrosine will therefore yield a positive result. However, some proteins containing tyrosine initially forms a white precipitate that turns red when heated, while others form a red solution immediately (Milio and Loffredo, 1995). From the observed results in Table 3, both protein samples give a positive result thus confirming the presence of tyrosine. Table 4. Hopkins-Cole and Bromine Water test TESTS

Samples

Hopkins-Cole

Bromine Water

ring formed

pink

2. Gelatin

colorless, violet ring formed

turbid white

3. Albumin

violet ring formed at the junction of two fluids, light brown at top fluid

turbid white

colorless, no ring formed

very light yellow

1. Tryptophan

4. Distilled water

These tests are specific for tryptophan, the only amino acid containing an indole group. In the Hopkins-Cole reaction, the indole ring reacts with glyoxylic acid in the presence of a strong acid (H 2SO4in the experiment’s case) to form a violet cyclic product. The mechanism for this reaction is shown in Equation 2 below. The Hopkins-Cole test confirms the presence of tryptophan in Albumin. The protein solution is hydrolyzed by the concentrated H 2SO4 at the solution interface. Once the tryptophan is free, it reacts with the glyoxylic acid to form the violet product. However, the results for the two qualitative tests were questionable. In the case of Gelatin, according to Cole (2000), the protein lacks tryptophan which does not agree to the results obtained in Hopkins-Cole test. In addition, albumin contains tryptophan but showed negative results towards bromine water test.

Equation 2. Reaction Mechanism for Hopkins-Cole test

Table 5. Pauly Reaction Samples

Observation

1. Gelatin

intense yellow orange

2. Albumin

intense yellow orange

3. Distilled water

light brown

The basic principle in pauly's test is diazotization (Equation 3). Sulfanilic acid will be diazotized with the addition of sodium nitrite and sodium carbonate to form diazonium component. Diazonium component react with the imidazole ring of histidine and a phenol group of tyrosine to form dark red compound. Gelatin and albumin both yielded intense yellow orange products which is still considered as a positive result. The test confirms the presence of tyrosine, histidine, or both. According to Cole (2000), Gelatin contains less than 1% of histidine and less than 0.5% of tyrosine.

Equation 3. Diazotization of sulfanilic acid

Table 6. Reduced Sulfur Reaction Samples

Observation

1. Gelatin

colorless

2. Albumin

dark brown solution

3. Hair 4. Distilled water

hair dissolved; dark brown solution colorless

Sulphur containing amino acids, such as cysteine and cysteine, upon boiling with sodium hydroxide, yield sodium sulphide. This reaction is due to partial conversion of the organic sulphur to inorganic sulphide, which can be detected by precipitating it to lead sulphide, using lead acetate solution. The balance equation for this reaction is on Equation 4. Among the three protein samples: gelatin, albumin, and hair, only gelatin responded negatively to the test. According to Galewska et al. (2013), gelatin (denatured collagen) does not contain cysteine while albumin contains all the protein-building amino acids. Hair is composed of keratin which is a combination of

18 amino acids including cysteine. Cysteine, being rich in sulphur, plays an important role in the cohesion of the hair (Beveridge & Lucas,1944).

RSH +2 NaOH → ROH + Na 2 S + H 2 O 2−¿ +¿+ S ¿ Na2 S → Na¿ −¿ CH 3 COO¿ ¿ −¿ 2−¿ → PbS↓+2 CH 3 COO¿ ¿ Pb¿ Equation 4. Balance equation for Reduced Sulfur reaction

Table 7. Sakaguchi Reaction Samples

Observation

1. Gelatin

reddish color solution

2. Albumin

reddish color solution

3. Distilled water

deep green solution

This test is specific for arginine, as it is the only amino acid containing a guanidine group. This moiety reacts with α-naphthol and an oxidizing agent (bromine water in the experiment’s case) to give the red-colored complex. Gelatin and Albumin was confirmed to have the presence of arginine from the results obtained. Gelatin is said to have 8% of arginine (Cole, 2000). Conclusion Amino acids are building blocks of proteins. They possess an amine group, a carboxylic acid group and a varying side chain that differs between different amino acids. These side chains are essential in determining the presence of amino acids in proteins through qualitative test. The qualitative tests conducted in this experiment make use of various color-producing reagents which is dependent upon the side chains present in the test samples. Ninhydrin reaction was used to confirm the sample as an amino acid or protein. Biuret test was significant in distinguishing proteins from amino acids. Xanthoproteic reaction is for classifying amino acids with benzene derivative. Millon’s test was used to check the presence of tyrosine in the test samples. Both Hopkins-Cole and Bromine water test are important for tryptophan determination. Pauly reaction confirms the existence of tyrosine and histidine in the sample. Reduced Sulfur test makes use of the sulphur in determining

the presence of cysteine and cysteine. And Sakaguchi’s reaction is useful in arginine determination. Albumin was confirmed to have the presence of tryptophan, tyrosine, histidine, cysteine, and arginine. While for gelatin, only the existence of tyrosine, histidine, and arginine were observed. Hair was also included in the Reduced Sulfur test to check the presence of cysteine. References Beveridge J. M. R. and Lucas C. C. (1944). The analysis of hair keratin.Journal on Biochemistry. 38(1): 88– 95. Cole, CGB. Francis, F.J., editor. (2000). Gelatin.. Encyclopedia of Food Science and Technology, 2nd ed. 4 Vols. New York: John Wiley & Sons, pp. 1183-1188. Galewska Z., Gogiel T., Małkowski A., Romanowicz L., Sobolewski K., Wolańska M. (2013). Biochemistry Workbook. Medical University of Białystok. Milio, F. & Loffredo, W. (1995). Qualitative testing for amino acids and proteins. USA: Chemical Education Resources, Inc. Nigam, A & Ayyagari, A. (2007). Lab Manual in Biochemistry: Immunology and Biotechnology. New Delhi: Tata McGraw-Hill Education. ISBN: 9780070617674 vlab.amrita.edu,. (2011). Qualitative Analysis of Amino Acid. Retrieved 15 January 2016, from vlab.amrita.edu/?sub=3&brch=63&sim=1094&cnt=1