BioOrganic Chemistry Experiment 8 Characterization of Proteins and Amino Acids

8/18/2019 BioOrganic Chemistry Experiment 8 Characterization of Proteins and Amino Acids

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*Assumpta Minette C. Burgos

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BIOORGANIC

CHEMISTRY

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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 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, tyrosine, and phenylalanine. On the other hand, gelatin is made up of arginine, tyrosine, tryptosan, and

phenylalanine

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

Xanthoproteic 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 andfood. Lipids is for energy reserve, signalling and serves as structural components of cell membranes

Some examples of which are fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E, and K)

monoglycerides, diglycerides, 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 reactswith 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 bedestroyed by the cell. This unfolding of the protein is called denaturation.

https://en.wikipedia.org/wiki/Fathttps://en.wikipedia.org/wiki/Waxhttps://en.wikipedia.org/wiki/Sterolhttps://en.wikipedia.org/wiki/Vitaminhttps://en.wikipedia.org/wiki/Monoglyceridehttps://en.wikipedia.org/wiki/Diglyceridehttps://en.wikipedia.org/wiki/Triglyceridehttps://en.wikipedia.org/wiki/Triglyceridehttps://en.wikipedia.org/wiki/Diglyceridehttps://en.wikipedia.org/wiki/Monoglyceridehttps://en.wikipedia.org/wiki/Vitaminhttps://en.wikipedia.org/wiki/Sterolhttps://en.wikipedia.org/wiki/Waxhttps://en.wikipedia.org/wiki/Fat


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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 sidechain. 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 to identify structural features common to amino acids by conductingspecific 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 Biuret test, Ninhydrin test

Hopkins-Cole test, Lead Acetate test, Sakaguchi test, and Xanthoproteic test. Originally, there wassupposed 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 isthe 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 groupstarted, 10 drops of gelatin and another 10 drops cysteine were collected in different test tubes.

The tests were divided into general tests for proteins and amino acids and specific tests for amino

acids. The general tests for proteins were Biuret and 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 inethanol 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.

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 filledwith 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.


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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 addedto 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, 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 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 HNO340% NaOH (or concentrated NaOH), a red litmus paper, and boiling water bath. 6 pieces of red litmus

paper was provided by the laboratory technician. Next, 10 drops of concentrated HNO3 were added. Thegroup 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 acidThe 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 OBSERVATION1 Albumin Purplish tinted clear solution

2 Gelatin Clear light purple solution

3 Arginine Light blue solution

4 Cysteine Purple solution

(pale brown solution)

5 Glycine Colorless

6 Histidine Clear purple solution

7 Methionine Colorless solution

8 Phenylalanine Light blue solution

9 Proline Clear light blue solution

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


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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 (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 alkaline

medium. Potassium sodium tartrate, on the other hand, stabilizes the chelate complex. As an alternativereagent to the Biuret Test, sodium hydroxide, copper 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 leastthree peptide bonds. A positive result yields a blue- to – 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

http://en.wikipedia.org/wiki/Denticityhttp://en.wikipedia.org/wiki/Denticity


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Proline Serine Tryptophan Tyrosine

FIGURE 1.

Results of Biuret Test

The result of the Biuret Test is expressed in Table 1 and can be observed in Figure 1. In theexperiment, 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. Asobserved 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 bythe 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 solution12 Tyrosine Golden yellow solution

The second test made by the group was the Ninhydrin Test. Ninhydrin Test is for detecting theexistence 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 pigment known as the Rhuemann’s purple. The Rhuemann’s


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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.




FIGURE 2.

Results of Ninhydrin Test

Result of the Ninhyrdin Test is expressed in Table 2 and can be observed in Figure 2. Theexpected positive visible result is a deep blue or purple solution with the exception of proline. Proline’s


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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 testtube could be considered as the cause of error.

TABLE 3. Results of Hopkins-Cole TestSAMPLE OBSERVATION

1 Albumin Clear solution with purplish interphase

2 Gelatin Slightly yellow

3 Arginine Colorless interphase

4 Cysteine Colorless solution

5 Glycine Colorless

6 Histidine Colorless


8 Phenylalanine Colorless solution

9 Proline Clear colorless

10 Serine Colorless11 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 H 2SO4.The protein solution is hydrolysed by H2SO4 whereas reaction of glyoxylic acid

with a free tryptophan will yield a violet product.

Typtophan determines an indole nucleus and causes the formation of a violetring 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.


Figure 3. Indole


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FIGURE 4.

Results of Hopkins-Cole Test

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 thetest 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.

TABLE 4:

RESULTS OF LEAD ACETATE TEST

SAMPLE OBSERVATION

1 Albumin Clear solution turned to clear brown solution

2 Gelatin Colorless solution3 Arginine Colorless solution

4 Cysteine Murky solution

5 Glycine Colorless solution

6 Histidine Light yellow solution without precipitate

7 Methionine Colorless without precipitate


9 Proline Clear colorless solution


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10 Serine Clear light grayish tinted solution

11 Tryptophan Colorless solution

12 Tyrosine 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.




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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 positive result.

TABLE 5: RESULTS OF SAKAGUCHI TEST

SAMPLE OBSERVATION

1 Gelatin Clear grayish solution turned to red solution

2 Albumin Clear red orange solution

3 Arginine Dark red solution

4 Cysteine Saturated yellow solution

5 Glycine Pale brown solution

6 Histidine Clear brown solution

7 Methionine Clear gold solution

8 Phenylalanine Cloudy light orange solution9 Proline Golden yellow solution

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, α- naphthol (1-hydroxy naphthalene) reacts with aguanidine 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-containingsample.


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FIGURE 6.

Results of Sakaguchi Test

The results of the Sakaguchi Test is seen in Table 5 and observed in Figure 6. Positive tesresults 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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TABLE 6. Results of Xanthoproteic Test

SAMPLE OBSERVATION

1 Albumin Cloudy white solution turned to clear solution

after water bath then clear yellow solution

2 Gelatin After 20 drops NaOH clear neon yellow at 15

drops (basic)3 Arginine Colorless solution

4 Cysteine Yellow solution

5 Glycine Colorless solution

6 Histidine Colorless solution



9 Proline Clear colorless solution

10 Serine Colorless solution

11 Tryptophan Yellowish solution turned to red solution after 20drops of NaOH

12 Tyrosine Rusty orange solution

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 results of this test are specific for tyrosine, tryptophan, and phenylalanine. Hence,

positive results would mean that the sample contains 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 orangesolution. The rusty orange color tyrosine indicates presence of an aromatic acid.



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FIGURE 7.

Results of Xanthoproteic Test

When amino acids are tested on it reacts due to its amphoteric nature and the R-group or sidechain. 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 intensityis 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

AlbuminGelatin X X

*B for Biuret Test. N for Ninhydrin Test. HC for Hopkins-Cole Test.

LA for Lead Acetate Test. S for Sakaguchi Test. X for Xanthoproteic 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


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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 determines the intensity of the productcolor. 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.


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References

David, G. L. (2001). Analytical chemistry. India: University 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: Structure and function. US: John Wiley & Sons

BioOrganic Chemistry Experiment 8 Characterization of Proteins and Amino Acids

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