ISOLATION AND CHARACTERIZATION OF GLUTEN FROM WHEAT FLOUR

Dianne I. Pascua, Francesca F. Perez, Romulo S. Perez, Lia S. Priela, Zeth DR. Raquedan Group 8 2C Medical Technology Biochemistry Laboratory

ABSTRACT
Gluten was extracted from flour by kneading the flour, agglomerating the gluten into an elastic network, a dough, and then washing out the starch. The presence of starch was tested by adding iodine solution to the washings wherein a negative coloration (yellow) is needed. Gluten was isolated and separated into test tubes to prepare an intact protein solution and then subjected to qualitative color reactions. Biuret test, Ninhydrin test, Xanthoproteic test, Millons test, Hopkins-Cole test, Sakaguchi test, Nitropusside test, Fohls test, test for Amides, and Pauly test were performed . Biuret test and Ninhydrin Test yielded a blue-violet color, Xanthoproteic test resulted to a dark-red color, Millons test gave a clear solution, Hopkins-Cole test produced pink residue, Sakaguchi test contributed to the pale-yellow solution of the sample, Nitropusside test yielded canary yellow, Fohls test gave a black residue, test for amides turned red litmus paper into blue and Pauly test resulted to a yellow solution.

INTRODUCTION
Proteins, meaning first, are a class of organic compounds which are present in and vital to every living cell. In the form of skin, hair, callus, cartilage, muscles, tendons and ligaments, proteins hold together, protect, and provide structure to the body of a multi-celled organism. In the form of enzymes, hormones, antibodies, and globulins, they catalyze, regulate, and protect the body chemistry. In the form of hemoglobin, myoglobin and various lipoproteins, they affect the transport of oxygen and other substances within an organism. [1] Gluten is a protein composite found in foods processed from wheat and related grain species, including barley and rye. Gluten gives elasticity to dough, helping it rise and keeps its shape and often gives the final product a chewy texture. Gluten is used in cosmetics, hair products, and other dermatological preparations. [2] Isolation of proteins is the process of separating a type of protein from a complex mixture. The considerations in the isolation of an intact protein from its source is to determine the three-dimensional (3D) structure of the protein (fibrous or globular); interactions that keep the native conformation of the protein functional (electrostatic, covalent, hydrophobic, H-bonding, and van der Waals); acid-base property; and solubility of protein in different solvents. [3] Qualitative color reactions are used to determine the presence of certain amino acids and proteins in an intact protein solution. The biuret test is used to detect the presence of peptide bonds while the Ninhydrin reaction is a typical test for an -amino acid. The Xanthoproteic test detects side chains of aromatic amino acids while the Millons and Hopkins-Cole tests determine tyrosine and tryptophan residues, respectively. The

Nitropusside test is used to find out if sulfurcontaining amino acids are present; test for amides is used to detect R-groups of asparagines and glutamine. [4]

EXPERIMENTAL
A. Compounds tested (or samples used) Wheat flour, iodine solution, intact protein solution, distilled H2O, 6 M NaOH, 3 M NaOH, 2.5 M NaOH, 20% NaOH, 10% NaOH, Conc. NaOH solution, 5% NaNO2, 10% Na2CO3, 2% Nitropusside solution, 0.02% Naphthol solution, 1% Sulfanilic acid, 0.1% Ninhydrin solution, Conc. HNO3, Conc. H2SO4, Hopkins-Cole reagent, Millons reagent, 2% NaOBr, 0.1% M CuSO 4, 5% (CH3COO)2 Pb, gluten. B. Procedure Isolation of Gluten In isolating gluten, 1 cup of wheat flour and water were used to make thick dough. The dough was wrapped in cheesecloth and placed under running water until all starch is removed. The washings were tested by adding I2 solution until a negative result (yellow) is obtained. Gluten was isolated from wheat flour.

Ten drops of 10% NaOH and 10 drops of 0.02% naphthol solution were added to the sample, mixed and stood for 3 minutes. Another 3 drops of 2% NaOBr was added, mixed and color was noted. 7. Nitropusside Test In the sample, 0.5 mL of 3 M NaOH and 0.25 mL of 2% nitropusside solution were placed. The color of the solution was noted. 8. Fohls Test The sample was treated with 5 drops of 30% NaOH and 2 drops 5% (Ch3COO)2 Pb. The test tube was placed in a boiling water bath. The appearance was noted. 9. Test for Amides For this test, 1 mL of 20% NaOH was placed in the sample and placed in a boiling water bath. A red litmus paper was moistened in the mouth of the tube. The result was noted. 10. Pauly Test Diazo reagent was prepared by mixing 3-5 drops of 1% sulfanilic acid with 3 drops 5% NaNO2 solution. Five drops of sample and 3-5 drops of 10% Na2CO3 were added to the Diazo reagent. The color appearance was noted.

RESULTS AND DISCUSSION

Qualitative Color Reactions of Gluten 1. Biuret Test The intact protein solution was treated with 20 drops of 2.5 M NaOH and mixed. Another 2-3 drops of 0.1 M CuSO4 solution was added. The test tube was shaken and color of the solution was noted. 2. Ninhydrin Test In Ninhydrin test, 6-10 drops of 0.1% ninhydrin solution was placed to the diluted samples. The tbe is heated in a boiling water bath. Appearance of blue-violet coloration was noted. 3. Xanthoproteic Test The sample was treated slowly with 10 drops of conc. HNO3, mixed and color of the solution was noted. Another 10 drops of conc. NaOH was added slowly, mixed and color was noted again. 4. Millons Test In the diluted sample, 5 drops of Millons reagent was added and color was noted. 5. Hopkins-Cole Test The Hopkins-Cole reagent was added slowly to the sample and mixed. The test tube was inclined and 20 drops of conc. H2SO4 was slowly placed. The mixture should not be shaken. The color of the interface was noted. 6. Sakaguchi Test

Figure 1. Qualitative Color Reactions in Gluten

determination of sulfur-containing amino acids and based on the results the intact protein have sulfur-containing amino acids. Test for amides is conducted to detect R-groups of asparagine and glutamine and based on the results it exhibited a positive result. And lastly, Pauly test is a specific test for the detection of histidine, and based on the results the sample do not contain histidine.

REFERENCES
[1] Reush, William. April 5, 2013. "Proteins, Peptides & Amino Acids." Chemistry. Retrieved December 14, 2013, from https://www2.chemistry.msu.edu/faculty/reusch/ VirtTxtJmL/proteins.htm [2] Wikipedia. http://en.wikipedia.org/wiki/Gluten (accessed December 14, 2013). Figure 2. Ninhydrin test, Fohls Test, and Test for Amides in boiling water bath Table 1. Qualitative Color Reactions
Color Reaction Biuret Ninhydrin Xanthoproteic Millons Hopkins-Cole Sakaguchi Nitroprusside Fohls Test for Amide Pauly Intact Protein Blue-violet (-) Blue-violet (+) Dark-red (+) Clear solution (-) Clear violet ring (+) Pale yellow (-) Canary yellow (-) Black residue (+) Red litmus paper to blue (+) Yellow (-)

[3] Crisostomo, A., Daya, M., de Guia, R., Farrow, F., Gabona, M., Liu, I., Pea, G., Pea, L., Santiago, L., Santiago, M., Sarile, A., Torres, P., Vargas, A., & Ysrael, M. (2010). Laboratory Manual in General Biochemistry. Quezon City: C & E Publishing, Inc, pp. 17 [4] Crisostomo, A., Daya, M., de Guia, R., Farrow, F., Gabona, M., Liu, I., Pea, G., Pea, L., Santiago, L., Santiago, M., Sarile, A., Torres, P., Vargas, A., & Ysrael, M. (2010). Laboratory Manual in General Biochemistry. Quezon City: C & E Publishing, Inc, pp. 22

The Biuret test is used to detect the presence of peptide bonds and based on the results the intact protein sample yielded a blue-violet color and it indicates a negative test for peptide bonds. The Ninhydrin test is a test for an -amino acid and based on the table the sample gave a positive result and it means it has an -amino acid. Xanthoproteic test detects side chains of aromatic acids and the results showed a positive reaction. Millons test determines tyrosine residues and based on the data, the sample doesnt have tyrosine. Hopkins-Cole test is used to determine tryptophan residues and based on the table, it showed a positive result. Sakaguchi test is a test for the guanidino group of arginine and based on the results gluten showed a negative reaction. Nitropusside test is used to find out if sulfur-containing amino acids are present and according to the results, the sample yielded a negative result but it should be a positive result. Fohls test is also used for