Isolation, Hydrolysis, and Color Reactions of Intact and Hydrolyzed Protein Title: Isolation, Basic Hydrolysis, and Qualitative

Color Reactions of Gluten from Wheat Flour Abstract Introduction Results and Discussion Conclusion References

Color Reaction Intact Protein Biuret Violet solution Ninhydrin Blue violet solution Xanthoproteic Light yellow solution Clear, colorless Millons solution Violet coloration of Hopkins-Cole intact protein Clear, colorless Sakaguchi solution Nitroprusside Red to Yellow solution Light yellow solution Test for Amide Red --> Blue litmus paper :

Basic Hydrolysate Brown solution Yellow Orange solution Orange solution Brown precipitate *Light yellow solution w/ violet interface Light yellow solution Yellow solution Light Yellow Red --> Blue litmus paper Objectives

The aims of this experiment were to isolate gluten form wheat flour by difference in solubility; to analyze chemical groups responsible for color reactions and explain the principle behind each test; and to perform basic hydrolysis on the isolated gluten and enumerate the advantages and disadvantages of basic hydrolysis. Materials and Methods The reagents and materials used in the isolation of gluten were one cup wheat flour, cheese cloth and I2 solution. Enough water was added to one cup of wheat flour to produce thick dough. The dough was wrapped in a cheesecloth, and was placed under the running water until all starch is removed. To test the presence of starch in the dough, I2 solution was added until the negative results was obtained. The insoluble material left was the white solid elastic crude gluten. Isolated gluten, 4 M NaOH, 1 M HCl, red and blue litmus paper, hard glass test tubes, and 250-mL beaker were used in order to produce the basic hydrolysate. The basic hydrolysis was performed by adding 10 mL of 4 M NaOH to 0.5 g isolated gluten in a hard glass test tube that, and was labeled. The tube was covered with stopper and

was subjected to autoclaving, 15 psi for 15 hours. After the procedure, a clear yellow solution with urine-like odor was added with 10 mL distilled water and was transferred into 250-mL beaker. The mixture was neutralized with 1 M HCl. Red and blue litmus papers were used to check if the mixture was already neutralized. For quantitative color reactions, intact and hydrolyzed gluten, 3 M NaOH, 2.5 M NaOH, 20% NaOH, 10% NaOH, concentrated NaOH dolution, 2% Nitroprusside solution, 0.02% Naphthol solution, 0.1% Ninhydrin solution, concentrated HNO3, concentrated H2So4, Hopkins-Cole reagent, Millon's reagent, red and blue litmus papers, 2% NaOBr, 0.1 M CuSO4, droppers, beaker, test tubes and hot plate were used. The source of gluten used in the isolation was wheat flour. Enough water was added to one cup of wheat flour to produce thick dough. The different tests for the presence of amino acids are Biuret, Ninhydrin, Xanthoproteic, Millons, HopkinsCole, Sakaguchi, Nitroprusside, and test for amide were used in order to determine which would give the positive and negative results for intact and basic hydrolysate. For each test, eight separate test tubes were prepared for intact protein solution and 0.5 mL basic hydrolysate. Intact protein solution was prepared by adding 1 mL distilled water into the test tubes containing 0.5 g of gluten.

In Biuret test, each test tube containing the sample of intact and basic hydrolyzed protein was added with 20 drops of 2.5 M NaOH. After mixing well, 2-3 drops of 0.1 M CuSO4 solution was added. The test tubes were shaken until visible results were obtained. For Ninhydrin test, the samples were treated with 6-10 drops of 0.1% ninhydrin solution and were placed in a water bath to check which will give blueviolet coloration. For Xantoproteic test, 10 drops of Concentrated HNO3 was added slowly and was mixed. The color of the solution was noted; then another 10 drops of concentrated NaOH was placed in the solution. Color of the solution was once again noted. Another set of samples for Millons test was used. Five drops Millons reagent was added, and the change in color was again recorded. Another test was Hopkins-Cole test, wherein 20 drops of Hopkins-Cole reagent was added to the diluted samples and was mixed well. The test tubes were inclined and added slowly along the side with 20 drops concentrated H2SO4. It is important that the solution must not be disturbed in order to view the presence of the colored interface of the solution and be able record. Sakaguchi test was performed by placing 10 drops of 10% NaOH and 10 drops of 0.02% naphthol solution. After the solution was mixed, it was allowed to stand for three minutes. Three drops of 2% NaOBr was added and mixed. The visible results were again recorded. Another set of diluted samples were treated with 0.5 mL of 3 M NaOH, then 0.25 mL 2% nitroprusside solution was added. The formation of a red solution is a visible positive result for Nitroprusside test. The last qualitative test for intact protein and basic hydrolysate was the test for amide. The diluted samples were treated with 1 mL of 20% NaOH to 10 drops of the sample, and were placed in boiling water bath. The test for evolution of gas was performed by placing a moistened red litmus paper over the mouth tube. Results

were tabulated in order to confirm which gave the visible positive results, which could determine the amino acids present in gluten. Results and Discussion Solubility is the principle involved in the isolation of gluten from the wheat flour. The insoluble property of gluten made it possible to be isolated, on the other hand starch is soluble in water. Further addition of I2 solution, aid in the complete isolation of gluten from wheat flour, because I2 solution facilitate in the formation of the iodo-starch complex, which can be seen by the blue coloration of starch still present in the washings, so further addition of I2 solution, starch can be easily removed, and white rubbery crude gluten will be obtained. The qualitative color reaction tests are used to detect the presence of free amino acids and proteins, through the reactions for side chains, a-amino, and acarboxyl groups.