BioOrganic Chemistry Experiment 8 Characterization of Proteins and Amino Acids

============= BIOORGANIC CHEMISTRY ============= BioOrganic Chemistry Laboratory – CH205 (2015-2016) Experiment 8

Characterization of Proteins and Amino Acids through Color Reaction Mary Bernardine G. Bagalay, Julie Ann Kim F. Berdonado, Jeanne Isabelle B. Bilasano, and Assumpta Minette C. Burgos*  Department of Speech Language Pathology, College of Rehabilitation Sciences University of Santo Tomas, Espana Street, Manila 1008 Date Submitted: April 5, 2016 Abstract: Proteins are carbohydrates responsible for growth and cellular maintenance in the body. It is made up of amino acids linked  by peptide bonds. Identifying the amino acid content of a protein can be feasible through different test specific specific for an amino acid. After conducting Biuret Test, Ninhydrin Test, Hopkins-Cole Test, Lead Acetate Test, Sakaguchi Test and Xanthoproteic Test, the amino acid in albumin and gelatin were determined. Albumin is made up of tryptophan, cysteine, methionine, arginine, arginine, tyrosine, and p henylalanine. henylalanine. On the other hand, ge latin is made up of arginine, tyrosine, tryptosan, and  phenylalanine

 Keywords: proteins, proteins, amino acids, acids, Biuret Test, Test, Ninhydrin Ninhydrin Test, Hopkins-Cole Hopkins-Cole Test, Lead Acetate Test, Test, Sakaguchi Test, Test, and  Xanthoproteic Test. Test.

Introduction

Biochemistry is the branch of science that tackles the chemical processes involved in living organisms. It has three factors namely carbohydrates, lipids, and proteins. Carbohydrates are sources of energy of one’s body. These are sugars, starches, celluloses and gums that occur in living tissues and food. Lipids is for energy reserve, signalling and serves as structural components of cell membranes. Some examples of which are fats, are fats, waxes,  waxes, sterols,  sterols, fat-soluble  fat-soluble vitamins  vitamins (such as vitamins A, D, E, and K), monoglycerides, diglycerides, triglycerides,  triglycerides,  and phospholipids. On the other hand, proteins are the  body’s source of energy for growth and cellular maintenance. Proteins  are the second largest component of cell, next to water. It consists of amino acid linked together by peptide bond. A peptide bond is formed when the carboxyl group of one molecule reacts with the amino group of the other molecule. This is a dehydration synthesis reaction, also known as a condensation reaction (Whitford, 2013). Amino acids   are the building blocks of proteins. A typical  protein has amino ‘folded’ into a 3-dimensional shape. Every protein has a unique shape and structure which determines its function. If the shape ‘unfolds,’ the protein will no longer function and will be destroyed by the cell. This unfolding of the protein is called denaturation.

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There are about 300 amino acids in nature, but only 20 are used to make proteins in the body. Essential amino acids are required in diet and which humans are incapable of forming requisite. Among the list of essential amino acids are histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. While, nonessential amino acids are not required in the diet namely alanine, asparagine, aspartic acid, and glutamic acid. When amino acids are tested on, it reacts due to its amphoteric nature and the R-group or side chain. An amphoteric means it is capable of reacting to both acids and bases. The side chain or functional groups present in amino acid determines the intensity of the product color. The reaction of a sample under different tests can be a key point in determining the components of it. The group’s objective was objective was to identify structural features common to amino acids by conducting specific chemical tests to characterize the different amino acids. Thereafter, the group had to identify the amino acids present in albumin, casein and gelatin using the principles of each chemical test as basis.

Methodology

The apparatus and materials used in this experiment depended on the test conducted by the group and the group’s number. Six chemical tests were conducted. These were  were   Biuret test, Ninhydrin test, Hopkins-Cole test, Lead Acetate test, Sakaguchi test, and Xanthoproteic test. Originally, there was supposed to be another test, the Millon’s test however , Millon’s test involves mercury which can be hazardous for the students. Hence, the professors decided to skip this test. Moreover, time constraints led to the distribution of samples to each group. Odd groups were assigned to use albumin, while the even groups on gelatin. In addition to the protein samples, each were given amino acid samples. Here is the assigned sample to each group arranged in progressing group number: arginine, cysteine (protein), glycine, histidine, methionine, phenylalanine, proline, serine, tryptophan, and tyrosine. Group 10 and 11 were assigned to tyrosine. Overall, 10 samples were involved in this experiment. Before the group started, 10 drops of gelatin and another 10 drops cysteine were collected in different test tubes. The tests were divided into general gene ral tests for proteins and amino acids and an d specific tests for amino acids. The general tests for proteins were Biuret an d Ninhydrin. In Biuret Test. 13 x 100 test tubes, 10% sodium hydroxide (NaOH), and 0.01% copper (I) sulphate (CuSO4) was used this test. Then, 10 drops of 10% NaOH and 2 drops 1% CuSO4 were added to each of the test tubes. The test was mixed and the color observed was noted. The other test, Ninhydrin test, required 13x100 test tubes, 0.1% Ninhydrin in ethanol and a boiling water bath. Beaker halfway filled water was placed on a hot plate for boiling. After adding 10 drops of 0.1% ethanolic ninhydrin to the samples, the test tubes were heating in a boiling bath for two minutes. The test tubes were taken out and color observed was noted. note d. The specific tests for amino acids were Hopkins-Cole Test, Lead Acetate Test, Sakaguchi Test and Xanthoproteic Test. 13 x 100 test tubes, Hopkins-Cole reagent and concentrated H2SO4 were needed for Hopkins-Cole Test.  20 drops of Hopkins-Cole reagent were added to the prepared test tubes filled with gelatin and cysteine and then mixed. The test tube was inclined at a 45º. Then, 20 drops of concentrated H2SO4 was carefully added into the solution. The color of the interphase was observed. It is imperative to note that after adding concentrated H2SO4, shaking of the test tubes must not be made as it may disrupt the process. *Assumpta Minette C. Burgos

 Next, Lead Acetate Test  involves 13x100 test tubes, 40% NaOH (or concentrated NaOH), 1% Pb(Ac)2 solution, and a boiling water bath. 10 drops of 40% NaOH (or concentreated NaOH) was added to the prepared test tubes filled with gelatin and cysteine. After 5 minutes of heating in a boiling water  bath, 5 drops of 1% Pb(Ac)2. Some yielded with a precipitate while some did not. Nevertheless, the color of the precipitate and/or the solution was observed and noted. 13x100 test tubes, 10% NaOH, 0.02% ethanolic α-naphthol, α-naphthol, and 2% NaOBr solution aided the group for the Sakaguchi Test . 10 drops of 10% NaOH and 10 drops of 0.02% ethanolic α-naphthol α -naphthol was added to the solution. Then, it was mixed. After 3 minutes, 5 drops of 2% NaOBr solution followed. The color of the solution was observed and noted. The last test was Xanthoproteic Test. This test required 13x100 test tubes, concentrated HNO3, 40% NaOH (or concentrated NaOH), a red litmus paper, and boiling water bath. 6 pieces of red litmus  paper was provided by b y the laboratory technician. technicia n. Next, 10 drops of concentrated HNO3 were added. The group then heated the test tube. There was no required number of minutes for heating the test tube. But, it is interesting to note that heating even for just 3 minutes would instigate a reaction with the nitric acid. The group then gradually added 40%NaOH (or concentrated NaOH) dropwise until the solution turns  basic. They were able to establish that the solution was indeed basic after the red litmus paper turned into blue after a drop of the solution. Both cysteine and gelatin turned basic after 20 drops of 40%NaOH (or concentrated NaOH). The color of the solution was then observed and noted. Observation and results of the different groups were then collected after the experiment.

Results and Discussion TABLE 1. Results of Biuret Test SAMPLE OBSERVATION 1 Purplish tinted clear solution Albumin 2 Clear light purple solution Gelatin 3 Light blue solution Arginine 4 Purple solution Cysteine (pale brown solution) 5 Colorless Glycine 6 Clear purple solution Histidine 7 Colorless solution Methionine 8 Phenylalanine Light blue solution 9 Clear light blue solution Proline 10 Serine Light purple 11 Tryptophan Clear solution with bluish tinge 12 Tyrosine Clear light blue solution

The Biuret Test  is a chemical test for detecting the presence of peptide bonds. If peptide bonds were present, copper (II) ion forms violet-coordination complexes in alkaline solution (Mohanty & Basu, 2006). The sole reagent, biuret reagent, in the Biuret Test for proteins contains hydrated copper *Assumpta Minette C. Burgos

sulphate, potassium hydroxide solution, and potassium sodium tartrate. Hydrated copper sulphate  provides Cu (II) ions which forms the chelate complex. Chelate involves the formation or presence of two or more separate coordinate bonds between a polydentate a polydentate (multiple bonded) ligand and a single central atom. The Cu (II) also gives off the characteristic blue color of the reagent. Potassium hydroxide solution does not directly participate with the reaction but is significant as it provides the a lkaline medium. Potassium sodium tartrate, on the other hand, stabilizes the chelate complex. As an alternative reagent to the Biuret Test, sodium hydroxide, c opper sulphate solutions and Fehling’s Solutions A and B may be used to produce comparable results. Interestingly, biuret reagent is not named after the scientist who formulated Biuret Test but by  peptide bonds in biuret delivers a positive result for the test. However, the protein must have at least three peptide bonds. A positive result yields a blue- to  – violet-colored violet-colored solution. The intensity of the color depends on the number of peptide bonds in the sample. Hence, the intensity of color is directly  proportional to the protein concentration in accord to the Beer-Lambert Law (David, 2001).

Albumin

Gelatin

Arginine

Cysteine

Glycine

Histidine

Methionine

Phenylalanine

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Proline

Serine Tryptophan FIGURE 1. Results of Biuret Test

Tyrosine

The result of the Biuret Test is expressed in Table 1 and can be observed in Figure 1. In the experiment, all of the sample except glycine and methionine are expected to deliver a blue to purple solution. This is because glycine, the simplest amino acid, does not have an amide link to which peptide  bonds can bind whereas, methionine is a free amino acid and does not contain a peptide bond. As observed in Figure 1, albumin and gelatin had the most saturated purple hue. It can be inferred that these two have the most peptide bonds and are proteins. On the contrary, cysteine exhibited a pale brown solution. Although Biuret test repeated twice by the group, it still exhibited the same pale brown color and this may be due to systematic error. TABLE 2. Results of Ninhydrin Test SAMPLE OBSERVATION 1 Albumin Indigo colored solution 2 Gelatin Clear blue violet solution 3 Arginine Colorless solution 4 Cysteine Pink solution (yellow solution) 5 Glycine Dark violet solution 6 Histidine Dark brown solution 7 Methionine Transparent dark purple solution 8 Phenylalanine Dark violet solution 9 Proline Dark red solution 10 Serine Light orange solution 11 Tryptophan Clear solution turned to apricot colored solution 12 Tyrosine Golden yellow solution

The second test made by the group was the Ninhydrin Test.  Ninhydrin Test is for detecting the existence of amino acids. Ninhydrin is an oxidating agent that leads to deamination of alpha-amino acids and proteins that contain free amino acids. It degrades amino acids into aldehydes, ammonia, and carbon dioxide. The net result is a reduced form of hydrindantin. When ninhydrin condenses with ammonia, hydrindantin produce a bluish- purple  purple pigment known as the Rhuemann’s purple. The Rhuemann’s *Assumpta Minette C. Burgos

 purple is used to detect fingerprints due to the terminal amines or lysine residues. In addition to that, ninhydrin also reacts with primary amines but the formation of carbon dioxide is quite problematic for amino acids.

Albumin

Gelatin

Arginine

Cysteine

Glycine

Histidine

Methionine

Phenylalanine

Proline

Serine

Tryptophan FIGURE 2. Results of Ninhydrin Test

Tyrosine

Result of the Ninhyrdin Test is expressed in Table 2 and can be observed in Figure 2. The expected positive visible result is a deep blue or purple solution with the exception of proline. Proline’s *Assumpta Minette C. Burgos

reaction is more yellow due to substitution of the alpha amino group that ninhydrin reacts with carbon rings. Cysteine was expected to have a pinkish solution as result but exhbitied a clear yellow solution. Again, the test was repeated by the group and yielded with the same result. Contamination of the test tube could be considered as the cause of error. TABLE 3. Results of Hopkins-Cole Test SAMPLE OBSERVATION 1 Clear solution with purplish interphase Albumin 2 Slightly yellow Gelatin 3 Colorless interphase Arginine 4 Colorless solution Cysteine 5 Colorless Glycine 6 Colorless Histidine 7 Colorless solution Methionine 8 Phenylalanine Colorless solution 9 Clear colorless Proline 10 Serine Colorless 11 Tryptophan Colorless solution with grayish interphase 12 Tyrosine Colorless interphase

The third test is the Hopkin’s Cole Test. This test is used to determine the presence of amino acid tryptophan. Tryptophan is the only amino acid containing an indole group. The structure of an indole can be visualized in Figure 3. Hopkins-Cole reagent contains glyoxylic acid (Mg powder, oxalic acid, and acetic acid) and concentrated H2SO4. The protein solution is hydrolysed by H2SO4 whereas reaction of glyoxylic acid with a free tryptophan will yield a violet product. Figure 3. Indole

Typtophan determines an indole nucleus and causes the formation of a violet ring in the junction where the two layers meet- this implies a positive result. Some products do not show the reaction, such as gelatin and zein. Hence, tryptophan in this experiment should exhibit a violet ring in the junction where two layers meets.

Albumin

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Gelatin

Arginine

Cysteine

Glycine

Proline

Histidine

Methionine Methion ine

Serine Tryptophan FIGURE 4. Results of Hopkins-Cole Test

Phenylalanine

Tyrosine

Result of the Hopkins-Cole Test is expressed in Table 3 and can be observed in Figure 4. Albumin and tryptophan have distinct results from the rest of the samples who exhibited a colorless solution. Tryptophan should have a violet interphase instead had a grayish interphase. Shaking of the test tube or any action made on it could have disrupted the interphase formation. It is very critical that the test tube should not be mixed or shook before observation as this interphase dispersion is fast. Human error can be considered for this.

1 2 3 4 5 6 7 8 9

TABLE 4: RESULTS OF LEAD ACETATE TEST SAMPLE OBSERVATION Clear solution turned to clear brown solution Albumin Colorless solution Gelatin Colorless solution Arginine Murky solution Cysteine Colorless solution Glycine Light yellow solution without precipitate Histidine Colorless without precipitate Methionine Phenylalanine Colorless solution Clear colorless solution Proline

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10 Serine 11 Tryptophan 12 Tyrosine

Clear light grayish tinted solution Colorless solution Colorless solution

Lead Acetate Test  is used determining the presence of sulphur from cysteine and methionine. Its reagent includes sodium hydroxide (NaOH) and lead (II) acetate. When NaOH is boiled, it converts into S in the amino acid to NaS. Then, NaS then precipitates as balck PbS with the addition of lead (II) acetate (Kulkarni, Rathod, Thonte, & Ghiware, 2008). To determine a positive result, solution should be color black or a presence of a precipitate should be observed.

Albumin

Gelatin

Arginine

Cysteine

Glycine

Histidine

Methionine

Phenylalanine

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Proline

Serine

Tryptophan

Tyrosine

FIGURE 5: Results of Lead Acetate Test

The result of this test is presented in Table 4 and can be observed in Figure 5. Although albumin exhibited a brown solution and not black, it can still be considered as a s a positive result. TABLE 5: RESULTS OF SAKAGUCHI TEST SAMPLE OBSERVATION 1 Clear grayish solution turned to red solution Gelatin 2 Clear red orange solution Albumin 3 Dark red solution Arginine 4 Saturated yellow solution Cysteine 5 Pale brown solution Glycine 6 Clear brown solution Histidine 7 Clear gold solution Methionine 8 Phenylalanine Cloudy light orange solution 9 Golden yellow solution Proline 10 Serine Light yellow solution 11 Tryptophan Clear grayish turned to clear brownish solution 12 Tyrosine Clear orange solution

The fifth test is Sakaguchi Test.  Contrary to the Biuret Test, Sakaguchi Test acquired is named after the scientist who first described it, Schoyo Sakaguchi. Sakaguchi reagent has 1-naphtanol and sodium hypobromite. Under alkaline condition, αα- naphthol (1-hydroxy naphthalene) reacts with a guanidine compound like arginine, which upon treatment with hypobromite or hypochlorite, produces a characteristic red color through oxidation reaction. Another distinct characteristic of Sakaguchi is that it is specific for arginine. Hence, samples with a positive result in Sakaguchi test is an arginine-containing sample.

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Albumin

Gelatin

Arginine

Cysteine

Glycine

Histidine

Methionine

Phenylalanine

Proline

Serine Tryptophan FIGURE 6. Results of Sakaguchi Test

Tyrosine

The results of the Sakaguchi Test is seen seen in Table 5 and observed observed in Figure 6. Positive test results are in an orange- to red-colored solution. In this test, albumin and gelatin generated a positive result for this test.

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1 2 3 4 5 6 7 8 9 10 11 12

TABLE 6. Results of Xanthoproteic Test SAMPLE OBSERVATION Cloudy white solution turned to clear solution Albumin after water bath then clear yellow solution After 20 drops NaOH clear neon yellow at 15 Gelatin drops (basic) Colorless solution Arginine Yellow solution Cysteine Colorless solution Glycine Colorless solution Histidine Colorless solution Methionine Phenylalanine Colorless solution Clear colorless solution Proline Colorless solution Serine Yellowish solution turned to red solution after 20 Tryptophan drops of NaOH Rusty orange solution Tyrosine

The last test involved in this experiment is the Xanthoproteic Test. It is used to determine the  presence of an activated benzene ring. Only amino acids that have activated benzene ring can undergo nitration(Mohanty & Basu, 2006). Nitric acid gives color when heated with proteins containing tyrosine and tryptophan. The re results sults of this test are specific for tyrosine, tryptophan, and phenylalanine. Hence,  positive results would mean that the sample contains tyrosine, tyrosine, tryptophan, and phenylalanine. To determine a positive result, a dark yellow solution should be perceived. The result of Xanthoproteic Test is presented in Table 6 and can be observed in Figure 7. Only albumin, gelatin, cysteine, and tryptophan have positive results. Moreover, tryptophan turned from yellow solution to a red solution after 20 drops of NaOH. Lastly, tyrosine expressed a rusty orange solution. The rusty orange color tyrosine t yrosine indicates presence of an aromatic acid.

Albumin

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Gelatin

Arginine

Cysteine

Glycine

Histidine

Proline

Serine

Methionine

Tryptophan FIGURE 7. Results of Xanthoproteic Test

Phenylalanine

Tyrosine

When amino acids are tested on it reacts due to its amphoteric nature and the R-group or side chain. An amphoteric means it is capable of reacting to both acids and bases. The side chain or functional groups present in amino acid determines the intensity of the product color. The color intensity is dependent to the number of reacting functional groups. The greater the number, the more saturated the color. Through the following tests conducted, the group was able to determine the amino acids present in albumin and gelatin. TABLE 8. Results for Proteins B

N

HC

LA

S

X

Albumin X X Gelatin *B for Biuret Test. N for Ninhydrin Test. HC for Hopkins -Cole Test. LA for Lead Acetate Test. Te st. S for Sakaguchi Test. X for Xanthoproteic Xantho proteic Test.

The table above shows as to what test did the protein garnered a positive result. The Biuret Test and Ninhydrin Test confirm that albumin and gelatin have at least three peptide bonds and contain amino acids. Hopkins-Cole implies that albumin contains tryptophan. Lead Acetate Test implies that *Assumpta Minette C. Burgos

albumin contains cysteine and methionine. On the other hand, Sakaguchi and Xanthoproteic denote  presence of arginine and tyrosine, tryptophan and phenylalanine. Conclusion Proteins are made of amino acids. When amino acids are tested on, it reacts due to its amphoteric nature and the R-group or side chain. An amphoteric means it is capable of reacting to both acids and  bases. The side chain or functional groups present in amino acid ac id determines the intensity of the product color. The greater number of functional groups that reacted, the more saturated its hue. Specific tests for amino acid would express the amino acid content of a protein.

*Assumpta Minette C. Burgos

References

David, G. L. (2001). Analytical chemistry. c hemistry. India: University Press. Press. Kulkarni, M.V., Rathod, S. S., Thonte, S. S., & Ghiware, N. B. (2008). New Delhi: P ragati Books Pvt. Ltd.

Mohanty, B & Basu, S. (2006). Fundamentals of practical clinical biochemisty. New Delhi: BI Publications Pvt Ltd. Whitford, D. (2013). Proteins: Proteins: Structure and function. US: John Wiley & Sons

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