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J.C. Garbosa, A.T. Garcia, R.S. Garcia, R.C. Gomez, D.A. Gonong Group # 4, 2CMT, Faculty of Pharmacy, UST
Proteins are probably the most important class of biochemical molecules, although of course lipids and carbohydrates are also essential for life. Casein is a protein that is found in milk which is used independently in many foods as a binding agent. The general objective of this experiment is to isolate the protein Casein from skimmed milk and to analyze the chemical groups responsoble for color reactions and also to explain the principles involved in each test. Biuret Test, Ninhydrin Test, Xanthoproteic Test, Millon s Test, Hopkins-Cole Test, Sakaguchi Test, Nitroprusside Test, Fohl s Test and the Test for Amides were all done to a certain portion of the isolated Casein. Basic hydrolysis was done to the other portion of Casein. In addition to this, the different tests performed on the first portion of Casein were also done to the Casein that underwent basic hydrolysis. Various results were obtained and some differences were noted between the color reactions of the isolated Casein and the color reactions of the basic hydrolysates from the isolated Casein.
I. Introduction Amino acids have a variety of chemically reactive groups. The reactions for side chains, aamino, and a-carboxyl groups can be used to characterize both free amino acids and proteins. Biuret Test is used for detecting the presence of peptide bonds. It relies on the reduction of copper(II) ions to copper(I), the latter form a complex with the nitrogens of the peptide bonds in an alkaline solution. A violet color indicates the presence of proteins. It is possible to use the biuret reaction to determine the concentration of proteins because (for most proteins) peptide bonds occur with approximately the same frequency per gram of material. The intensity of the color, and hence the absorption at 540 nm, is directly proportional to the protein concentration, according to Lambert-Beer's law. Ninhydrin is a chemical substance mostly used in biochemical laboratories as a reagent for amino acids, which are small molecules that form proteins, as well as in forensics to detect finger prints and faint blood stains, Ninhydrin react with amino acids, producing a colored solution. This protocol is used to detect the presence of amino acids in certain substances by using a solution of alcohol, ninhydrim and water.
Xanthoproteic Test is a test for the detection of proteins in which concentrated nitric acid reacts with the proteins to form a yellow color that is intensified to orange-yellow by the addition of alkali called also xanthoproteic reaction Millon's test is given by any compound containing a phenolic hydroxy group. The Millon reagent is a solution of mercuric and mercurous ions in nitric and nitrous acids. Nitroprusside test is a test wherein sodium cyanide is added first to urine and let stand for approximately 10 minutes. The Sakaguchi Test is used to test for a certain amino acid and proteins. The tube was stoppered with cotton and was then submitted to the instructor for autoclaving (15 psi for 5 hours). Bacteria need proteins as their source for essential amino acids. The sample to be tested is treated with alpha-naphthol and sodium hypochlorite. The hopkins-cole test determines the presence of amino acid tryptophan. The Tryptophan creates the violet ring where the two layers meet. In this time disulfide bonds will be broken by the released cyanide. A black or brown color indicates the presence of the S-containing amino acids. any protein containing tyrosine will give a positive test of a pink to dark-red colour. A positive result yields a reddish wine color when arginine is present. The test will turn a red/purple color if the test is positive indicating that there were significant amounts of amino acid in the urine (aminoaciduria). Fohl s reaction is used for determination of S-containing amino acids. The amino acid that is detected in this test is arginine. Materials and Methods For the Alkaline Hydrolysis of Casein. Consequently. It was aslo labeled afterwards. Proteins are large molecules that cannot be brought directly into the bacterial cell. The . They need to be degraded into their component parts. Hydrolysis is a chemical reaction during which one or more water molecules are split into hydrogenand hydroxide ions which may go on to participate in further reactions. The destruction of disulfide bonds liberates cysteine from cystine and homocysteine from homocystine.5 grams of the isolated protein in a hard glass test tube. Some chemists no longer will use the hopkins-cole tst test becasuse it is not completely understood in terms of chemistry. 10 mL of 4M NaOH was added to 0. The tryptophan that the hopkins-col e test determines can be defined as an indole nucleus. Next sodium nitroprusside is added to the solution and it reacts with the newly freed sulfhydryl groups. II.
1M CuSO4.02% Naphthol solution was added to the samples. The test tube was shaken and the color of the solution was noted. After this.5mL of 3M NaOH was added to 0. It was then mixed well. 20 drops of 2. After this. 0. The appearance of a dark(brown or black) sediment was noted. The mixture was not shaken and the color of the interface was noted.appearance of the mixtured after autoclaving was then noted. Ten drops of concentrated HNO3 was slowly added to the diluted samples. This neutralized mixture was then used for the characterization tests. The test tube was then placed in a water bath. Twenty drops of Hopkins-Cole reagent was slowly added for the Hoplins-Cole test.5mL of the sample. After this. The mixture was neutralized using 1M HCl. 20 drops of concentrated H2SO4 was slowly added. while . For the Millon s test. 3 drops of 2% NaOBr was added and the color produced was noted. For each of the color reaction test. As for the Sakaguchi test. It was mixed and let stand for 3 minutes. Five drops of 30% NaOH and 2 drops of 5% (CH3COO)2Pb was added to the sample for the Fohl s test. Ten mL of distilled water was added. .25mL of 2% nitroprusside solution was added and the formation of a red solution was noted. The tube was the heated in a boiling water bath and the appearance of a blue-violet coloration was noted.5M NaOH was added to the samples and it was mixed well. The mixture was then transferred into a 250-mL beaker.1% Ninhydrin solution was added into the diluted samples.5g of the isolated pure Casein protein was mixed with 1mL of distilled water. While the test tube was inclined. mixed and the color of the solutionw as noted. The solution was then mixed and was noted of the color. 0.5mL of the hydrolysate was used. . In the Biuret test. As for the Ninhydrin test. 2-3 drops of 0. 5 drops of the reagent was added to the diluted samples and the change in color was noted. For the Nitroprusside test. 6-10 drops of 0. 10 drops of 10% NaOH and 10 drops of 0. 10 drops of NaOH was slowly added. After 3 minutes.
it was negative for the isolated casein and it was positive for the hydrolysate. brown solution white basic solution(red->blue litmus paper) Basic Hydrolysate light blue solution violet ring dark yellow solution pale yellow solution clear yellow solution pale brown solution yellow solution pale yellow solution white basic solution(red->blue litmus paper) The Biuret test for the intact protein was positive indicating the presence of a peptide bond. III. The tube was then placed in a water bath. since a positive result should yield a purple complex.For the test for Amides.1 shows the results obtained from the various color reactions Color Reaction Biuret Ninhydrin Xanthoproteic Millon s Hopkins-Cole Sakaguchi Nitroprusside Fohl s Test for Amides Intact protein (Casein) purple/ violet solution pale blue-violet solution pale yellow solution white solution white ring colorless solution yellow solution black precipitate.1 isolated casein Table 3. the test was positive though the result was not as intense as of the isolated protein s results. 1 mL of 20% NaOH was added to 10 drops of the sample. Results and Discussions Figure 3. A test for the evolution of gas during heating was done by placing a moistened red litmus paper over the mouth of the tube. The result was then noted. . As for the Ninhydrin test. This indicated the presence of either ammonia or primary or secondary amines. As for the hydrolysate.
Millon s test. For Millon s test. But for the hydrolysate.html http://www. both had positive results indicating the presence of an aromatic ring but based on the intensity of the yellow color.reference. For the Sakaguchi test. Since for both the samples.ehow. Sakaguchi test. Since both the hydrolysate and the casein had no violet color. References http://www. a wine red color indicates the presence of Arginine.com/ma/enwiki/en/ http://www. it turned to yellow solutions. since it had a pale brown color. the test for both of them was negative. it was positive for the Casein indicating that it has an S-containing amino acid. Since in the casein.com/what-is-casein.elmhurst.As for Xanthoproteic test. Ninhydrin test. it was negetive. Hopkins-Cole test only reacts with proteins containing Tryptophan. it is fine to say that it was positive for this test.ca/~chem2o6/labmanual/expt11/2o6exp11.worldlingo.com http://www.cerlabs. The Nitroprusside test only reacts with Cysteine to yield a red complex.com/experiments/10875404480. the test was negative to either. For both the 2 samples.mcmaster.com/motif/Science/ http://www. while it had the same result in Xanthoproteic test. Fohl s test and the test for Amides. IV.pdf . It turned out to be negative for the hydrolysate.wisegeek. it was negative for both the samples. As for the Fohl s test. since a brown precipitate or a brick red color is the indicator to confirm the presence of either Tryptophan or Tyrosine. Hopkins-Cole test.html http://www.chemistry. the casein and the basic hydrolysate had different reactions for Biuret test. Its indicator is the violet cyclic product. Nitroprusside test. it was observed that the hydrolysate has more aromatic rings in it.htm http://www. it was positive in the test for Amides because of the initially red litmus paper which was placed over the mouth of the tube turned to blue. only a colorless solution was obtained.edu/~chm/vchembook/565proteins. As for the conclusion.
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