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Isolation and Characterization of Proteins (Qualitative Colored Reactions and Paper Chromatography) Jenny Lynn A. Karunungan, Daniel Adam Levy, Josell Mary Engelee Lim, Alphonse Leandro Lomotan, Ma. Eloisa Mateos Group 6 2B Medical Technology Biochemistry Laboratory ABSTRACT
In the experiment,Qualitative Color Reactions was performed in order to analyze the chemical goups responsibe for the color reaction of the protein Gluten. Different tests such as Biuret, Ninhydrin, Xanthhoproteicm Millon’s, HopkinsCole, Sakaguchi, Nitroprusside, Fohl’s, Test for Amides, and Pauly yielded different colored solutions as results. The different results was due to the difference in the side chains present in Gluten. Paper Chromatography was also conducted to be able to separate and determine the amino acid constituents of the acidic, basic, and enzymatic hydrolysate of Gluten, a protein found in wheat flour, based on the polarities of Proline, Alanine, Cysteine, Tyrosine, and Glycine. A 1.5 cm margin had been drawn across the longer bottom edge of the chromatograph paper. Eight equidistant points were plotted along the line where the given amino acids and protein hydrolysate samples had been applied. The TLC plate was then introduced in the solvent system consisting of 1-Butanol,acetic acid and water with a ratio of 4: 1: 5. Then, Rf values for the standards were computed and it was determined that there were 3 amino acids present in the acid hydrolysate of gluten and they are : Alanine (Rf value = 0.19), Tyrosine (Rf value = 0.33) and an amino acid with a Rf value of 0.43 (unknown since it did not match with any of the used standards of amino acids). For the Alkaline Hydrolysate of gluten, Cystein (Rf value = 0.11) and Tyrosine (Rf value = 0.34) was identified. And lastly, for enzymatic hydrolysate of gluten, Tyrosine (Rf value = 0.31) and Glycine (Rf value = 0.13) was identified.
INTRODUCTION Amino Acids are molecules that have an amino group and a carboxyl group attached to its carbon atom. It’s -carbon atom is also attached to hydrogen and a side chain that varies between different amino acids. There are 21 Amino Acids that are usually found in proteins. Proteins, also known as peptides, are organic compounds composed of amino acids arranged in a linear chain. They are essential to life and participate also in catalyzing biochemical reactions, structural and mechanical functions, and other cell processes. Proteins are also threedimensional. Their structure is complex thus they are defined in 4 terms: primary, secondary, tertiary, and quaternary structure. The primary structure defined the sequence of amino acids present in the protein, the secondary structure defines the arrangement in space of the atoms in the peptide backbone, the tertiary structure defines the 3-D arrangement of all the atoms in the protein, and the quaternary structure defines the arrangement of subunits with respect to one another in a protein. Gluten is a water insoluble protein composite found in foods processed from wheat and related grain species, including barley and rye. It is the one that gives elasticity to dough, helping it rise and keep its shape and often gives the final product a chewy texture. Gluten is composed of two major protein
components, glutenin and gliadin, interact in an aqueous system to produce this viscoelastic property. Glutenin, the higher molecular weight protein fraction, contributes elasticity. Gliadin, of lower molecular weight, provides extensibility. It is describe that gluten has a polar group level of about 10%, with a resultant net positive charge which is a low level of polarity of the total amino acid structure compared to normal Polar grouping levels of 30-45% for most food proteins, that cause the viscoelastic behavior of hydrated wheat gluten that persists even in the presence of excess water. The result of this reduced polarity is that excess water is repelled and the wheat gluten molecules associate closely together and resist dispersion.  This experiment wanted to also qualitatively analyze the amino acid components of Gluten, and so, seeral tests were made to analyze these. The tests that were involved in the experiment were Biuret tests, Ninhydrin test, Xanthoproteic test, Millon’s test, Hopkins-Cole test, Sakaguchi test, Nitroprusside test, Fohl’s test, and the test for amide. Paper Chromatography was also used to qualitatively separate and identify the amino acids present in the 3 hydrolysates of Gluten. Paper Chromatography is a common technique used to separate amino acids since it is quick and requires less materials. It contains a mobile and a stationary phase. The principle of separation in
paper chromatography is based on their polarity. Components of the sample will separate readily according to how strongly they adsorb on the stationary phase versus how readily they dissolve in the mobile phase. Identification of the separated components of unknown substance subjected in paper chromatography is determined by comparing each Rf values (relative to the front values) of the separated components with the standard component of known identity. The objectives of this experiment is to primarily qualitatively and quantitatively analyze and identify the amino acids present in the protein Gluten by use of Paper Chromatography and Qualitative colored analysis.
EXPERIMENTAL A. Compounds tested (or Samples used) The samples used in this experiment are Intact protein Gluten and Enzymatic Hydrolysate Gluten. The intact protein Gluten is light brown in color and has a chewy texture.
B.Procedure 1.Qualitative Color Reactions
Ten sample test tubes of intact protein gluten were prepared by adding pinch amount of intact protein to 3 ml distilled water; and another ten sample test tubes containing enzymatic hydrolysate gluten were prepared by adding 5 drops of the hydrolysate to 3 ml distilled water. One out of ten sample test tubes of intact protein and hydrolysate are subjected to different test: Biuret Test: 20 drops of 2.5 M NaOH were added to each sample test tubes of intact protein and enzymatic hydrolysate and then 2-3 drops of 0.1 M CuSO4 solution were added. Both test tubes were shaken and the color was noted. Ninhydrin Test: 6-10 drops of 0.1% ninhydrin solution were placed to each sample test tubes of intact protein and enzymatic hydrolysate. Sample test tubes were then heated in a boiling water bath and appearance of blue violet coloration was to be noted. Xanthoproteic Test: In each test tube of intact protein and enzymatic hydrolysate, 10 drops of conc. HNO3 were slowly added, then mixed well and the color was noted. Another 10 drops of conc. NaOH were added slowly and the color was noted also. Million’s Test: Five drops of Million’s reagent were added to the diluted samples of intact protein and enzymatic hydrolysate and the color was noted. Hopkins-Cole Test: In each diluted samples, 20 drops of Hopkins-Cole reagent were added and were mixed well. Then each diluted samples
are inclined and slowly added along the side with 20 drops conc. H2SO4. Do not shake the mixture and note the color at the interface. Sakaguchi Test: Ten drops of 10% NaOH and 10 drops of 0.02% naphthol solution were added to each diluted samples of intact protein and enzymatic hydrolysate, mixed well and let stand for 3 minutes. Then 3 drops of 2% NaOBr were added and the color produced was noted. Nitroprusside Test: In each diluted samples, 0.5 ml of 3 M NaOH and 0.25 ml 2% nitroprusside solution were added then formation of red solution was noted. Fohl’s Test: 5 drops of 30% NaOH and 2 drops of 5% (CH3COO)2Pb were added to one of each diluted samples of intact protein and enzymatic hydrolysate then placed in a boiling water bath and noted the appearance of dark(black or brown) sediment. Test for Amides: 1 ml of 20% NaOH was added to each diluted samples of intact protein and enzymatic hydrolysate, then was placed in a boiling water bath and test for evolution of gas while heated was noted by placing a moistened litmus paper. The color of the litmus paper was noted. Pauly Test: The diluted samples of intact protein and enzymatic hydrolysate and 3 drops of 10 % Na2CO3 were added to the diazo reagent (contains 3-5 drops of 1% sulfanilic acid and 3 drops of 5% NaNO solution). The appearance of red coloration was noted.
On a paper chromatogram, an origin was drawn with a pencil line 1.5-cm from the bottom of the longer edge of the chromatogram. 13 equidistant points were marked on the line for spotting of the amino acid standards and three hydrolysate samples.The standards were applied five times,and the samples ten times using capillary tubes. The sample was allowed to dry between applications. The chromatogram was placed inside the 1000 mL beaker which was used as a chamber. The level of the solvent was below the origin.The beaker was covered with a Petri dish. After approximately 30 minutes the chromatogram was removed and the solvent front was marked with a pencil. The chromatogram was air dried, and then 1% ninhydrin reagent was dropped on the whole surface of the chromatogram. The chromatogram was once again dried, revealing the amino acid spots. The densest areas of the spots were then encircled, and the Rf values were then computed.
RESULTS AND DISCUSSION QUALITATIVE COLOR REACTIONS
The experiment analyzed the chemical groups responsibe for color reactions and with the principle of each tests, qualitative analysis was done. 1. Biuret Test The Biuret test detects peptide bonds - and turns violet (positive result) in the presence of proteins. The Biuret reagent is made of sodium hydroxide and copper sulfate. Gluten rendered a positive result . 2. Ninhydrin Test Amines, or alpha-amino acids react with ninhydrin to yield a colored result. Gluten, like alpha-amino acids reacted to the test and yielded a light violet coloration - suggesting a positive result . 3. Xanthoproteic Test A xanthoproteic reaction is the reaction of tyrosine-containing proteins with nitric acid turning the product yellow. The xanthoproteic test is carried out by adding concentrated nitric acid to the substrate being tested. If proteins are present containing amino acids with aromatic rings - the mixture turns yellow (like in the case of Gluten) . 4. Millon's Test This test detects the presence of soluble proteins. The color produced is given by derivatives of benzene in which a hydrogen has been replaced by a hydroxyl group. The reaction serves as a test for the presence of tyrosine, trypthopane, and phenylalanine. A positive result would yield a reddish-brown color . 5. Hopkins-Cole Test This test determines the presence of the amino acid tryptophan - which creates a violet ring where two layers meet . 6. Sakaguchi Test This test is used (in this case) to detect arginine, and reacts with the guanidine group of the protein to give a red color. Gluten tested negative for arginine [1, 2]. 7. Nitroprusside This test is specific for cysteine, the only amino acid containing a sulfhydryl group (-SH). The gluten tested negative for cysteine . 8. Fohl's Test This test determines if the protein has sulfur containing amino acids, like cysteine, cystine, and methionine. A positive result would bring about a red decolorization. 9. Test for Amides The changing of the litmus paper's color from red to blue indicates a presence of a basic component/basic amino acid in gluten. 10. Pauly's Test The imidazole group reacts with diazotized aulphanilic acid to form highly colored
azocompounds. In alkaline medium this is red in color . Color Reaction Biuret
Light violet coloration (+) Colorless (-) Yellow to orange coloration (+) White or slightly turbid solution Colorless (-)
Cloudy solution Yellow solution (-)
Ninhydrin Xanthoproteic Millon’s
Enzymatic Hydrolysate Colorless (-) Colorless (-) Colorless (-) Colorless (-) Colorless or no layer formed (-) Colorless (-) Yellow Solution (-) Colorless (-)
Brown-black sediment (+) Test for Light pink Colorless (-) Amide coloration,negative in blue ltmus paper (+) Pauly Dark yellow or Yellow orange coloration solution (-) (-) Table 1. Qualitative Color Reaction Results
PAPER CHROMATOGRAPHY Paper Chromatography is the technique used to qualitatively analyze the acidic, basic and enzymatic hydrolysates of Gluten, a protein found in wheat flour. This technique is facilitated by the principle of polarity toward the two phases, the stationary and the mobile phase. Amino Acid Standard
The experiment utilized a 12x15 chromatograph paper plotted with 5 different amino acid standards (Alanine, Glycine, Cysteine, Proline and, Tyrosine) and 3 hydrolysates of Gluten (acid, basic and, enzymatic) along the line of its origin located 1.5 cm above its baseline. The chromatograph paper was placed inside a pre-equilibrated chamber consisting of 1-
Butanol,acetic acid and water with a ratio of 4: 1: 5. A higher Rf value would mean that the substance being identified is less polar or nonpolar while a lower Rf value would mean otherwise. Using the table above, it was made clear that Cysteine and Glycine had a lesser affinity to the mobile phase since it has a lesser Rf value and thus proving that it is a polar uncharged amino acid. On the other hand, Alanine and Proline had a high Rf value, it had greater affinity to the mobile phase and thus proving that it is a non-polar hydrophobic amino acid. As stated earlier, the solvent front was composed of 3 types of molecules and all of them had their own purpose. Water served as the stationary phase; 1-Butanol, a non-polar molecule carried the non-polar amino acids up to the mobile phase and; Acetic acid, a polar molecule that carried the polar amino acids up to the mobile phase. Since there was a 4:1 ratio of 1-Butanol and Acetic acid it could be said that non-polar amino acids are favored than the polar ones thus, non-polar amino acids like Alanine and Proline had a higher Rf value than the polar amino acids Cysteine and Glycine. These interpretations were made possible because of 1% Ninhydrin reagent. Since the 20 standard amino acids were all colorless, the chromatograph paper was sprayed evenly with Ninhydrin reagent to make the spots appear blue, yellow or purple. Lastly, by comparing the Rf values of the standards with the Rf values present in the hydrolysates, it was determined that Alanine, Tyrosine and an unknown amino acid with a Rf value of 0.43 was present in the acidic hydrolysate of gluten. For the basic hydrolysate, Cysteine and Tyrosine was present while for the enzymatic hydrolysate, Tyrosine and Glycine was present.
Figure 1. Paper Chromatogram Result REFERENCES  Alexander, D. Sakaguchi Test Amino Acid Proteins.
http://www.reference.com/motif/science/sakaguc hi-test-amino-acid-proteins 1/12/14  Bogazici University. Experiment 2 Qualitative Analysis of Amino Acids and Proteins. http://www.chem.boun.edu.tr/webpages/courses /Chem415/Chem%20415%20Experiment%202.p df 1/12/14  Hunt, I. Ninhydrin Test. http://www.chem.ucalgary.ca/courses/351/Carey 5th/Ch27/ch27-3-3.html 1/12/14  Merriam Webster Dictionary. Xanthoproteic Reaction. http://www.merriamwebster.com/dictionary/xanthoproteic%20reactio n 1/12/14  Norris, J. Experimental Organic Chemistry Proteins. http://www.books-aboutcalifornia.com/Pages/Experimental_Organic_Che mistry/Ex_Organic_Chem_Chap_26.html 1/12/14  Ohio University Department of Biological Sciences. Biuret Test. http://www.biosci.ohiou.edu/introbioslab/Bios170 /170_2/biuret.htm 1/12/14  Sairam, N. Color Reactions of Proteins. http://quizlet.com/8801729/color-reactions-ofproteins-flash-cards/ 1/12/14  (Author Unknown) Protein's Test. http://fulltimes.wordpress.com/protein/ 1/12/14 Wikipedia.Gluten. http://en.wikipedia.org/wiki/Gluten 12/30/2013  Excerpt from Wheat Gluten: A Natural Protein for the Future - Today by the International Wheat Gluten Association http://www.mpbio.com/detailed_info.php?family _key=02101815&country=168 12/29/2013