QUALITATIVE COLOR REACTIONS OF MYOGLOBIN Christine H. Umali, Jon Lester Uy, Chloe P. Valena, Jose Maria Q.

Veloso, Norjem R. Villanueva, Jaquelyn C. Wodi Group 9 2A Medical Technology BioChemistry Laboratory. ABSTRACT
The experiment aims to isolate the intact protein myoglobin from a beef sample and to analyze the chemical group responsible for color reaction and explain the principle involved in each test. Myoglobin is a protein that forms pigments responsible for making meat red. It is alsoa sensitive markerfor muscle injury, making it a potential marker for heart attack in patients with chest pain. The intact protein was isolated by cutting the beef sample into smaller pieces and treated with (NH4)2SO4 crystals and 70% buffer-diluted (NH4)2SO4 solution. Myoglobin was subjected for color reaction using the Biuret, Ninhydrin, Xanthoproteic, Millon, HopkinsCole, Sakaguchi, Nitroprusside, Pauly, Fohls and Amide test which determined the structure, functional group and presence of amide and aromatic side chains.

INTRODUCTION
Amino acids are molecules containing an amine group, a carboxylic acid group and a side chain that varies between different amino acids.

between side chains or R-groups of amino acid and carboxyl group. [6] Myoglobin is a protein isolated from beef samples. A myoglobin polypeptide is comprised of 8 separate right-handed -helices, designated A through H, that are connected by short non helical regions. Amino acid R-groups packed into the interior of the molecule are predominantly hydrophobic in character while those exposed on the surface of the molecule are generally hydrophilic, thus making the molecule relatively water-soluble.

Figure 1. General Structure of Amino Acid. Figure 1 shows the general structure of an amino acid with R being the functional group. Amino acids are amphoteric, behaving as amines in some reactions and as carboxylic acids in others. Amino acids are critical to life. They have particularly important functions like being the building blocks of proteins and being the intermediates in metabolism. Proteins, also known as peptides, are organic compounds made of amino acids arranged in a linear chain. Like other biological macromolecules, proteins are essential parts of organisms and participate in catalyzing biochemical reactions, structural and mechanical functions, cell signaling, and other processes within cells. [1] Proteins have a precise three-dimensional configuration. The primary structure refers to its amino acid sequence, which determines the protein shape. The secondary structure contains helix and pleated sheet structures, while the tertiary structure shows the further interaction of the backbone of a protein. The quaternary structure displays the interaction between subunits of a protein. He bonds that hold a protein together can either be hydrogen bonds, disulfide bonds, ionic and covalent bonds, and other bonds. These interactions take place

EXPERIMENTAL
A. Compounds used: Intact protein on Myoglobin B. Procedure: Ten sample test tubes each containing the intact protein was prepared by adding 5 drops of the intact protein to 1mL of distilled water. Another ten test tubes each containing the enzymatic hydrolisate was prepared by adding 5 drops of the enzymatic hydrolisate to 1 mL of distilled water. Biuret Test: 20 drops of 2.5 M NaOH was added to a test tube containing the intact protein and another 20 drops were added to the test tube containing the enzymatic hydrolysate. Then to each test tube, 2-3 drops of 0.1 M CuSO solution were added. Both test tubes were shaken and the color was noted. Ninhydrin Test: In each test tube, 6-10 drops of 0.1% ninhydrin solution were added into the intact protein and enzymatic hydrolysate. Both test tubes were then heated in a boiling water bath. Xanthoproteic Test: Ten drops of concentrated HNO3 solution

was slowly added to the diluted samples and were mixed. Then, 10 drops of concentrated NaOH was added and the color was noted. Millons Test: To each of the diluted samples, 5 drops of Millons reagent were added while noting the change in color. Hopkins-Cole Test: To the diluted samples, 20 drops of Hopkins-Cole reagent was slowly added and mixed well. The test tubes were then inclined the test tube and 20 drops of concentrated H2SO4 was added along the side. The change in color was then noted. Sakaguchi Test: To each of the diluted samples, 10 drops of 10% NaOH and 10 drops of 0.02% naphthol solution was added and the test tubes were then left untouched for 3 minutes. [3] Nitroprusside Test: 0.5 mL of 3M NaOH was added to the diluted samples. Then, 0.25 mL of 2% of nitroprusside solution was added. Fohls test: Five drops of 30% NaOH and 2 drops of 5% (CH3COO)2 Pb was added to the diluted samples. Both test tubes were then places in a boiling water bath and the change in color was observed. Test for Amides: To each of the diluted samples, 1 mL of 20% NaOH were added and both test tubes were placed in a boiling water bath. While in the water bath, a red litmus paper was held at the opening of each test tube to test for the evolution of gas. Pauly Test: First, the diazo reagent was prepared by mixing 3-5 drops of 1% sulfanilic acid with 3 drops of 5% NaNO2 solution. Then, 5 drops of the sample and 3-5 drops of 10% Na2CO3 were added to the diazo reagent. A red coloration was noted.

amino acids have a variety of chemically reactive groups that are used to characterize both free amino acid and proteins, the following tests were conducted to detect the presence of certain amino acids in Myoglobin. Table 1 shows the different results of the tests done on Myoglobin. Table 1. Results of Color Reaction of Intact protein and Enzymatic Hydrolysate Color Reaction Biuret Test Ninhydrin Test Intact Protein Purple Solution Blue- Violet Solution with brown ppt. Yellow solution Colorless solution to milky white ppt. Dark flesh to a colorless solution No change Yellow solution with a small amount of ppt. Light yellow solution with dark brown sediments Red to Blue Litmus Paper Red Solution Enzymatic Hydrolysate Green Solution Light Brown solution

Xanthoproteic Test Millons Test

Yellow layer on top of clear solution Flesh turbid solution

Hopkins-Cole Test Sakaguchi test Nitroprusside Test

Light yellow solution No change Yellow solution

Fohls Test

No Change

Test for Amide Pauly Test

Red to Blue Litmus paper Red Orange Solution

RESULTS AND DISCUSSION

In this experiment, we isolated the protein myoglobin and the intact protein was used in the enzymatic hydrolysis experiment to produce the enzymatic hydrolysate of Myoglobin. The intact protein and the enzymatic hydrolisate then underwent different reactions to test for the presence of specific amino acids. Since

1. Biuret test The Biuret test is a test done for the presence of peptide bonds. It is a positive test for proteins but not for amino acids. The evidence for a peptide bond is the formation of a violet-pink solution. The formation of a violet-pink solution is due to when the cupric ion, in a basic solution is added to any polymer such as proteins, which contains multiple amide bonds. The intact protein sample showed positive for peptide bonds, while the enzymatic hydrolysate shows negative result for peptide bonds. 2. Ninhydrin test- The Ninhydrin test is a test for the presence of amino acid. The

positive result of amino acid is detected by the yielding of a purple solution.

Figure 2. Two Ninhydrin molecules Figure 2 shows the removal of the amino (NH2) group from an amino acid to form a blue violet complex. The intact protein yielded a positive result, while the enzymatic hydrolysate showed negative results. 3. Xanthoproteic Test - Some amino acids contain aromatic groups that are derivatives of benzene such as tyrosine and tryptophan. 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. The intact protein yielded a positive result for the presence of aromatic groups. The enzymatic hydrolysate on the other hand, showed some presence of aromatic groups. 4. Millons Test- Millons test is specific for the detection of tyrosine. Tyrosine is the only amino acid that contains a phenol group on which a hydroxyl group is attached. A positive result will yield a red precipitate. The myoglobin sample and the enzymatic hydrolysate both showed a negative result. 5. Hopkins-Cole Test Hopkins-Cole test is specific for the detection of the presence of tryptophan. The color produced is due to the formation of a compound from the glyoxylic acid in the reagent and the tryptophan in the protein. A similar color is produced when sulfuric acid is added to a protein solution in the presence of a trace of formaldehyde. The reaction is used as a test for formaldehyde in milk. The formation of a purple solution indicates a positive result. The myoglobin sample and the enzymatic hydrolysate both yielded a negative result. 6. Sakaguchi Test- The only amino acid, which contains a guanidine group, is arginine. Arginine gives a red color with naphthol, in the presence of an oxidizing agent like Bromine solution. The intact protein and enzymatic hydrolysate both

showed negative result for the presence of arginine. [3] 7. Nitroprusside Test- Nitroprusside Test is specific for the detection of cystein, the only amino acid containing sulfahydryl group. This group reacts with nitroprusside in the presence of excess ammonia. Positive results show a red complex. However, the intact protein and enzymatic hydrolysate both showed negative results, yielding a yellow solution. [2] 8. Fohls Test- Fohls test is performed for the determination of S- containing amino acids. The soutions were heated for the formation of sulfide. If the test yielded a positive result, the solution shows a red solution or a further reaction of NA2S will lead to a dark brown precipitate. Figure 3 shows Fohls reaction. [2]

Figure 3. Fohls Reaction The intact protein shows a positive result, while the enzymatic hydrolysate yielded a negative result. 9. Test for Amides This test is done for the presence of Asparagine and Glutamine. When the red limus paper turned blue, it indicates a basic component of the myoglobin and enzymatic hydrolysate. 10. Pauly test The sulfanilic acid gets diazotized in the [presence of sodium nitrite and sodium carbonate. Positive result will show yellow to dark orange solution and residues. The residues are histidine and / or tyrosine residues. Both the intact protein and enzymatic hydrolysate yielded a positive result. [4]

REFERENCES
[1] http://chemistry.about.com/od/lectureno teslab1/a/Amino-Acids.htm [2] http://faculty.ksu.edu.sa/75115/BCH%2 0221Lectures/Enzyme%20lectures%20fo r%20Cat%201.pdf [3] http://www.jbc.org/content/86/1/217.full .pdf [4] http://www.ncbi.nlm.nih.gov/pmc/article s/PMC1258626/pdf/biochemj012010077.pdf [5] http://www.slideshare.net/guest807eec1 /chap-11

[6] http://themedicalbiochemistrypage.org/h emoglobin-myoglobin.html