artikel amino acid revisi 2

BIOCHEMISTRY EXPERIMENT

ARTICLE

THE IDENTIFICATION OF AMINO ACID CONTENT IN PROTEIN AND

UNKNOWN SAMPLE SOLUTIONS THROUGH MILLON, HOPKINS-

COLE, NINHYDRIN, PbS AND NITROPRUSIDE TESTS

Created By:

Putu Dian Utami Jelantik (1213031013 / VIA)

CHEMISTRY EDUCATION DEPARTMENT

FACULTY OF MATHEMATICS AND NATURAL SCIENCES

UNIVERSITAS PENDIDIKAN GANESHA

SINGARAJA

2015

Rubrik Penilaian artikel Praktikum

Biokimia S1

Nama mahasiswa : Putu Dian Utami Jelantik

NIM : 1213031013

Tanggal : 9th

March 2015

Mata Acara praktikum : The Identification of Amino Acid Content in Protein and Unknown

Sample Solutions through Millon, Hopkins-Cole, Ninhydrin, PbS

and Nitropruside Tests

No. Aspek yang dinilai Bobot (%) Skor (0-100) Bobot x skor

1 Format 5

2 Abstract 10

3. Introduction 20

4. Materials and Methods 10

5. Result and discussion 30

6. Conclusion 10

7. Acknowledgment 5

8. References 5

9. Clear 5

Total score 100

Singaraja, 9th

March 2015

Penilai,

(Dr. I Nyoman Tika, M.Si)

THE IDENTIFICATION OF AMINO ACID CONTENT IN PROTEIN AND

UNKNOWN SAMPLE SOLUTIONS THROUGH MILLON, HOPKINS-COLE,

NINHYDRIN, PbS AND NITROPRUSIDE TESTS

Putu Dian Utami Jelantik (1213031013)

Chemistry Education Department, FMIPA, UNDIKSHA

Jalan Udayana Singaraja-Bali

Email: [email protected]

ABSTRACT

Protein is as one of the nutrients in the food that has the most important constituent compounds of living cells.

The biological functions of proteins are very diverse, namely as builders, regulators, defense, and as an energy

source. Protein is a polymer of about 20 kinds of -amino acids. Amino acids contain at least one carboxyl group (-COOH) and an amino group (-NH2). In this experiment, it was used egg albumin since one high quality

source of protein or amino acid. The objectives of this experiment were (1) to identify amino acid that contains

in the protein solution through the Millon, Hopkins-Cole, ninhydrin, PbS and nitropruside tests and (2) to

identify the types of amino acids contained in the unknown sample B and D through the amino acids test. This

test was used a qualitative method by observing the color change of solution after the addition of reagent and

after a given of treatment for each test. The result was obtained from this experiment is (1) protein solution

positive toward Millon, Hopkins-Cole, ninhydrin, and PbS test. It means that in protein solution contains of

tyrosine, tryptophan, -amino acid or peptide having free -amino and sulfur element and (2) unknown sample B is positive toward Millon and ninhydrin tests. It means that unknown sample B contains of tyrosine and -amino acid or peptide having free -amino. Unknown sample D is positive toward nitropruside test. It means that unknown sample D contains of cysteine.

Keywords : amino acid, protein solution, Millon test, Hopkins-Cole test, Ninhydrin test, PbS test and

Nitropruside test, unknown sample B, unknown sample D, qualitative analysis.

INTRODUCTION

Every living thing requires adequate

nutrition to sustain their life. Protein is as one

of the nutrients in the food that has the most

important constituent compounds of living

cells. The biological functions of proteins are

very diverse among others, namely as builders,

regulators, defense, and as an energy source.

Proteins are macronutrient materials unlike

other materials macronutrient (carbohydrate or

fat), proteins play a role in the formation of

biomolecules is more important than energy

sources.

No other group of compounds that has

function like proteins. In terms of chemical

composition, proteins are polymers of about

20 types of amino acids (Purba, 2004). There

are 20 amino acids that are divided into two

groups, namely non-essential amino acids and

essential amino acids. The 10 types of non-

essential amino acid produced by the body,

namely alanine, proline, serine, glycine,

cysteine, asparagine, glutamine, tyrosine,

aspartic acid, and glutamic acid. While, the

remaining 10 amino acids are the essential

amino acids that must be obtained from food,

namely arginine, threonine, lysine, valine,

phenylalanine, tryptophan, methionine,

histidine, leucine, and isoleucine (Wade,

2010). All of amino acid is an -amino acids, except proline. Variations that occur between

amino acids located in the R group or the

chain side. Based on its R group would be

predicted properties of an amino acid. Instead,

based on the identified properties will be

known the group R contained in the amino

acid or the amino acid types (Tika, 2010).

The sources of amino acids are from

soybeans, fruits, vegetables, beef, chicken,

eggs, and dairy products. Egg is one of amino

acid source that has high quality of protein.

Eggs are divided into several components or

main parts, namely shells or egg shells, egg

albumin as well as egg yolks. The amount of

protein in the egg yolks is 16.5% and in the

egg albumin is 10.9% (Sudaryani, 2003).

The existence of proteins in egg

albumin can be identified through amino acid

test, because the protein is composed of amino

acids. The identification of amino acids can be

done by two methods namely qualitative and

quantitative methods. Analysis of amino acids

with qualitative method can be done by

methods such as Millon, Hopkins-Cole,

ninhydrin, PbS and nitropruside tests. The

following table testing of amino acids.

Table. 01 Amino Acid Test

(Tika, 2010)

Millon Test

In the Millon test used Millon reagent.

Millon test is used to identify the protein

solution which contains of tyrosine. This test

is carried out on compounds containing

hydroxyl phenyl derivative monofenol. If a

solution containing hydroxyphenyl react with

Millon reagent it will produce a white

precipitate. If the temperature of the mixture is

raised then the color of the solution will

change into a red color. The red color is

formed due to the salt formed from the

nitration of tyrosine. The reaction is as follows

(Tika, 2010).

+H3N CH CH2

COOH

OH Hg22+

HNO3HgO+ +

-H2O +

H3N CH CH2

COOH

NO2 +(aq) (aq) (aq) (aq) (s)

Figure 1. Reaction in Millon Test

Hopkins-Cole Test

Hopkins-Cole test used to identify

protein solution which contains of that has

indole group (tryptophan). Protein that

contains of tryptophan will produce purple

ring. Purple ring that formed in the border

field between the two liquids is the

condensation result of tryptophan with

aldehyde group from glyoxylic acid in sulfuric

acid (Redhana, 2003). The reaction that occurs

is as follows.

C

CHN

H

CH2 CH COOH

NH2

+ OHC

COOH

H2SO

4

NH

COOH

H

NH

HH

H H

+ H2O(aq)

(aq)

(aq) (aq)

Figure 2. Reaction in Hopkins-Cole Test

Ninhydrin Test

Ninhydrin test is used to identify protein

solution which contains of -amino acid or

peptide having free -amino. If ninhydrin (triketohidrindine hydrate) is heated with amino

acid, it will be formed colored complex

compound (Tika, 2010).

Colored complex compound formed

from two molecules of ninhydrin which reacts

with ammonia which is released at amino acid

oxidation. Reaction with ninhydrin showed

positive results for amino acids in general,

except for proline. The reaction as follows.

Type of Test Reagent Amino Acid that detected Color

Millon Test HgNO3 in nitrate acid

with a little bit of nitric

acid

Tyrosine Red

Hopkins-Cole

Test

glyoxylic acid in

concentrated H2SO4

Tryptophan Purple

NinhidrinTest Triketohidrindine

Hydrate -amino acid and peptide having

free -amino

Blue

PbS Test PbS Amino Acid that can release S

element

Black

Nitroprusida

Test

Sodium Nitropruside in

dilute NH3

Cysteine Red

Tryptophan Gyoxylic acid 2,3,4,5-tetrahydro--carboline-4- carboxylic acid

Tyrosine Nitrated Tyrosine

CC

O

O

OH

OH

(aq)

NH2 C COOH

H

R

(aq)

C

C

O

O

OH

H

(aq)

+ NH3(aq)

+ CO2(g)

+ RCHO(aq)

+

NinhydrinNinhydrin Reducted

C

C

O

O

OH

OH

(aq)

NH

H

H+

(aq)

C

C

O

O

OH

H

+ (aq)

C

C

O

O

N

C

C

O

O

Complex Colored Compound

(aq)

Figure 3. Reaction in Ninhydrin Test

PbS Test

PbS test is used to identify protein

solution which contains of amino acid that can

release sulfur element. Amino acid that can

release sulfur is cysteine or methionine in the

presence of NaOH. The presence of NaOH in

this case is to denaturation proteins. Sulfide ion then reacts with Pb

2+ ion to form a black

precipitate (Redhana, 2003). The reaction is as

follows.

S2-

(aq) + 2NaOH(aq) Na2S(aq) + 2OH-(aq)

Na2S(aq) + Pb(CH3COO)3(aq) PbS(s) + CH3COONa

black precipitate

Figure 4. Reaction in PbS Test

Nitropruside Test

Nitropruside test is used to identify

protein solution which contains of cysteine.

Protein contains of cysteine will produce red

solution, it is caused by COOH groups and -NH2 group, R group in amino acid can also

thiol group (sulfhydryl groups) or peptide.

Some of the proteins that give negative results

on this reaction turned out to be positive after

having heated to coagulation or denaturation.

Amino acids can react with sodium nitropruside

in excess ammonia produces a red complex

(Tika, 2010). The reaction is as follows.

Fe3+

(CN)5NO

2-

+ NH3 SH CH2 CH COOH

NH2

+ NH4+

2

CH2 COOHCH

NH2

Fe3+(CN)5NOS

2-

(aq)

(aq) (aq)

(aq)

Cysteine Red Colored Complex Compound

Figure 5. Reaction in Nitropruside Test

Based on these facts, the objective of this

experiment were (1) to identify amino acid that

contains in the protein solution through the

Millon, Hopkins-Cole, ninhydrin, PbS and

nitropruside tests and (2) to identify the types of

amino acids contained in the unknown sample

B and D through the amino acids test.

MATERIALS AND METHODS

The method used in conducting this

experiment was adopted from Praktikum

Biokimia book developed by Dr. I Nyoman

Tika, M.Si (2010:22-29). The experiment was

conducted at Organic Chemistry Laboratory in

Chemistry Education Department, UNDIKSHA

Singaraja on February 23rd

2015 at 07.3012.00 WITA.

Equipment and Material

This identification was conducted

through qualitative testing by using equipment

namely test tube (7 pieces), test tube rack (1

piece), drop pipette (2 pieces), beaker glass 100

mL (2 pieces), graduated pipette 5 mL (1

piece), ball filler (1 piece), spirit burner (1

piece), test tube holder (1 piece), beaker glass

250 mL (1 piece), and heater (1 piece).

Material was used in this experiment is

15 drops of Millon reagent, 18 mL of protein

solution, 18 mL of unknown sample B solution,

18 mL of unknown sample D solution, 6 mL of

Hopkins-Cole reagent, 3 mL of concentrated

H2SO4, 1.5 mL of ninhydrin solution 0.1%, 6

mL of NaOH solution, 6 drops of

Pb(CH3COO)3 solution, 1.5 mL of sodium

nitropruside solution 1%, 1.5 mL of

concentrated NH3 and distilled water as needed.

Preparation of Protein Solution

A total of 5 mL of egg white of an egg

was taken. The next egg whites combined with

25 mL of distilled water. Then, it was stirred

until homogeneous. Protein solution is ready to

be used to test amino acid.

Millon test

Three milliliters of protein solution was

poured into test tube and label was given. Five

drops of Millon reagent was added into protein

solution and it was heated. The color change of

solution was observed. The same treatment was

also conducted for the unknown sample B and

D solutions.

Hopkins- Cole test

Two milliliters of protein solution was

poured into test tube and label was given. Two

milliliters of Hopkins-Cole reagent was added

into protein solution. Drop by drop of

concentrated H2SO4 was added through the side

of test tube until the volume about 5 mL and it

was stirred. The color change of solution was

observed. The same treatment was also

conducted for the unknown sample B and D

solutions.

Ninhydrin test

Three milliliters of protein solution was

poured and label was given. A total of 0.5 mL

of ninhydrin solution 0.1% was added into

protein solution. Then, it was heated until boil

and the color change was observed. The same

treatment was also conducted for the unknown

sample B and D solutions.

PbS test

Five milliliters of protein solution was

poured into test tube and label was given. A

total of 2 mL of NaOH solution was added into

protein solution. After that, two drops of Pb-

acetate was added into protein solution. Then, it

was heated in the water bath and the color

change of solution was observed. The same

treatment was also conducted for the unknown

sample B and D solutions.

Nitropruside test

Five milliliters of protein solution was

poured into test tube. A total of 0.5 mL of

sodium nitropruside solution 1% was added

into protein solution. After that 0.5 mL of

concentrated NH3 was added into protein

solution. The color change of solution was

observed. The same treatment was also

conducted for the unknown sample B and D.

RESULT AND DISCUSSION

Result

Based on the experiment, it can be obtained the result as shown in the table below.

Table 02. The Observation result of Amino Acid in Protein and unknown sample B and D solutions

Millon Test Hopkins-Cole

Test

Ninhydrin

Test

PbS Test Nitroprusde

reaction

Protein

Solution

Red solution

and red

precipitate

2 layer (white in

the up and goldish

yellow in bottom)

and purple ring

between 2 layers

Purplish blue

solution

Black

solution and

black

precipitate

Colorless

Unknown

sample B

solution

Red solution

and red

precipitate

2 layers (yellowish

in the up and

goldish yellow in

the bottom)

Purplish blue

solution

Colorless Colorless

Unknown

sample D

solution

Colorless and

white

precipitate

2 layers (yellowish

in the up and

goldish yellow in

the bottom)

Brownish

solution

Colorless Purplish red

solution

Discussion

Based on experiment result, protein

solution has cloudy yellowish colloidal

solution, the unknown sample B solution has

colorless color and the unknown sample D

solution has white turbid color.

Millon test

In the Millon test used Millon reagent.

Millon test is used to identify the protein

solution which contains of tyrosine. This test

is carried out on compounds containing

hydroxyl phenyl derivative monofenol. If a

solution containing hydroxyphenyl react with

Millon reagent it will produce a white

precipitate. If the temperature of the mixture is

raised then the color of the solution will

change into a red color. The red color is

formed due to the salt formed from the

nitration of tyrosine. The reaction is as

follows.

+H3N CH CH2

COOH

OH Hg22+

HNO3HgO+ +

-H2O +

H3N CH CH2

COOH

NO2 +(aq) (aq) (aq) (aq) (s)

Figure 6. Reaction in Millon Test

Based on the experiment, when protein

solution was added with Millon reagent was

produced colorless solution and white

precipitate. After heated, it was produced red

solution and red precipitate. It was indicated

that in the egg albumin (protein solution)

contains of tyrosine.

This experiment also conducted to

identify amino acid that was contained in the

unknown sample B and D. When unknown

sample B solution was added with Millon

reagent was produced colorless solution and

white precipitate. After heated, it was

produced red solution and red precipitate.

Meanwhile, when unknown sample D solution

was added with Millon reagent was produced

white turbid solution. After heated, it was

produced colorless solution and white

precipitate. After compared with protein

solution, it can be concluded that protein and

unknown sample B solutions contain of

tyrosine, but unknown sample D solution does

not contain of tyrosine.

(a) (b) (c)

Figure 7. In Millon Test (a) Protein Solution Produced Red Solution and Red Precipitate (b)

Unknown Sample B Solution Produced Red Solution and Red Precipitate (c) Unknown

Sample D Solution Produced Colorless Solution and White Precipitate

Hopkins-Cole Test

Hopkins-Cole test used to identify

protein solution which contains of that has

indole group (tryptophan). Protein that

contains of tryptophan will produce purple

ring. Purple ring that formed in the border

field between the two liquids is the

condensation result of tryptophan with

aldehyde group from glyoxylic acid in sulfuric

acid. The reaction that occurs is as follows.

Tyrosine Nitrated Tyrosine

CCHN

H

CH2 CH COOH

NH2

+ OHC

COOH

H2SO

4

NH

COOH

H

NH

HH

H H

+ H2O(aq)

(aq)

(aq) (aq)




solution was added with Hopkins-Cole reagent

was produced two layers (white solution in the

top layer and goldish yellow in the bottom

layer). After added with H2SO4, it was

produced two layers (white solution in the top

layer and goldish yellow solution in the bottom

layer) and there is purple ring between two

layers. It was indicated that in egg albumin

(protein solution) contains of tryptophan.



unknown sample B and D solutions. When

unknown sample B solution was added with

Hopkins-Cole reagent was produced two layers

(colorless solution in the top layer and goldish

yellow in the bottom layer). After added with

H2SO4, it was produced two layers (yellowish

solution in the top layer and goldish yellow

solution in the bottom layer). Meanwhile, when

unknown sample D solution was added with

Hopkins-Cole reagent was produced two layers

(colorless solution in the top layer and goldish

yellow in the bottom layer). After added with

H2SO4, it was produced two layers (yellowish

solution in the top layer and goldish yellow

solution in the bottom layer). After compared

with protein solution, it can be concluded that

unknown sample B and D solutions do not

contain of tryptophan.

(a) (b) (c)

Figure 9. In the Hopkins-Cole Test (a) Protein Solution Produced White Solution in Top Layer and

Goldish Yellow in Bottom Layer and there is Purple Ring (b) Unknown sample B Solution

Produced Yellowish Solution in Top Layer and Goldish Yellow in Bottom Layer (c)

Unknown sample D Solution Produced Yellowish Solution in Top Layer and Goldish

Yellow in Bottom Layer

Ninhydrin Test

Based on the theory, in ninhydrin test is

used to identify protein solution which contain

of -amino acid and peptide having free -amino. If ninhydrin (triketohidrindine hydrate)

is heated with amino acid, so will be formed

colored complex. The formation of blue

complex is occurs because of oxidative

decarboxylation of the amino acid and produce

reduction of ninhydrin, NH3 and CO2. Then it

occurs in the reaction of reduced ninhydrin with

the other ninhydrin molecule as well as with

NH3 released, so it was formed colored blue

complex. The reaction that occurs in this

process is as follows.

Tryptophan Gyoxylic acid 2,3,4,5-tetrahydro--carboline-4- carboxylic acid

CC

O

O

OH

OH

(aq)

NH2 C COOH

H

R

(aq)

C

C

O

O

OH

H

(aq)

+ NH3(aq)

+ CO2(g)

+ RCHO(aq)

+

NinhydrinNinhydrin Reducted

C

C

O

O

OH

OH

(aq)

NH

H

H+

(aq)

C

C

O

O

OH

H

+ (aq)

C

C

O

O

N

C

C

O

O

Complex Colored Compound

(aq)

Figure 10. Reaction in Ninhydrin Test


solution was added with ninhydrin solution was

produced pale yellow solution. After heated, it

was produced purplish blue solution. It was

indicated that egg albumin (protein solution)

contains of of -amino acid and peptide having

free -amino. This experiment also conducted to


unknown sample B and D solution. When


ninhydrin solution was produced turbid

solution. After heated, it was produced purplish

blue solution. Meanwhile, when unknown

sample D solution was added with ninhydrin

solution was produced yellowish solution. After

heated, it was produced brownish solution.

After compared with protein solution, it can be

concluded that unknown sample B solutions

contain of -amino acid and peptide having

free -amino and unknown sample D solution

does not contain of -amino acid and peptide

having free -amino.

(a) (b) (c)

Figure 11. In the Ninhydrin Test (a) Protein Solution Produced Purplish Blue Solution (b) Unknown

Sample B Solution Produced Purplish Blue Solution (c) Unknown Sample D Solution

Produced Brownish Solution

PbS test

Based on the theory, PbS test is used to

identify protein solution which contains of

amino acid that can release sulfur element.

Amino acid that can release sulfur is cysteine or

methionine in the presence of NaOH. The

presence of NaOH in this case is to

denaturation proteins. Sulfide ion then reacts with Pb

2+ ion to form a black precipitate. The

reaction is as follows.

S2-

(aq) + 2NaOH(aq) Na2S(aq) + 2OH-(aq)

Na2S(aq) + Pb(CH3COO)3(aq) PbS(s) + CH3COONa

black precipitate

Figure 12. Reaction in PbS Test


solution was added with NaOH solution was

produced colorless solution and after added

with Pb(CH3COO)3 was produced colorless

solution. After heated, it was produced black

solution and black precipitate. It was indicated

that egg albumin (protein solution) contains of

amino acid that can release sulfur element.





NaOH solution was produced colorless solution

and after added with Pb(CH3COO)3 was

produced colorless solution. After heated, it

was produced colorless solution meanwhile,

when unknown sample D solution was added

with NaOH solution was produced colorless

solution and after added with Pb(CH3COO)3

was produced colorless solution. After heated,

it was produced colorless solution. After

compared with protein solution, it can be

concluded that unknown sample B and D

solutions do not contain of contains of amino

acid that can release sulfur element.

(a) (b) (c)

Figure 13. In the PbS Test (a) Protein Solution Produced Black Solution and Black Precipitate (b)

Unknown Sample B Solution Produced Colorless Solution (c) Unknown Sample D

Solution Produced Colorless Solution

Nitropruside test

Based on the theory, nitropruside test is

used to identify protein solution which contain

of cysteine. Protein contains of cysteine will

produce red solution, it is caused by COOH groups and NH2 group, R group in amino acid can also thiol group (sulfhydryl groups) or

peptide. Some of the proteins that give negative

results on this reaction turned out to be positive

after having heated to coagulation or

denaturation. Amino acids can react with

sodium nitropruside in excess ammonia

produces a red complex (Tika, 2010). The reaction as is follows.

Fe3+

(CN)5NO

2-

+ NH3 SH CH2 CH COOH

NH2

+ NH4+

2

CH2 COOHCH

NH2

Fe3+(CN)5NOS

2-

(aq)

(aq) (aq)

(aq)

Cysteine Red Colored Complex Compound

Figure 14. Reaction in Nitropruside Test


solution was added with sodium nitropruside

solution was produced colorless solution and

after added with NH3 was produced colorless

solution. It was indicated that egg albumin

(protein solution) do not contain of cysteine. In

protein solution does not find cysteine amino

acid because in protein rarely contains of

cysteine because it oxidized to become

cysteine.





sodium nitropruside solution was produced

colorless solution and after added with NH3

was produced colorless solution. Meanwhile,

when unknown sample D solution was added

with sodium nitropruside solution was

produced colorless solution and after added

with NH3 was produced purplish red solution.

After compared with protein solution, it can be

concluded that unknown sample D contains of

cysteine, but unknown sample B solution do

not contain of cysteine.

(a) (b) (c)

Figure 15. In the Nitropruside Test (a) Protein Solution Produced Colorless Solution (b) Unknown

Sample B Solution Produced Colorless Solution (c) Unknown Sample D Solution

Purplish Red Solution

CONCLUSION

Based on this experiment, it can be

concluded that (1) protein solution positive

toward Millon, Hopkins-Cole, ninhydrin, and

PbS test. It means that in protein solution

contains of tyrosine, tryptophan, -amino acid

or peptide having free -amino and sulfur element and (2) unknown sample B solution is

positive toward Millon test and ninhydrin test.

It means that unknown sample B contains of

tyrosine and -amino acid or peptide having

free -amino. Unknown sample D solution is positive toward nitropruside test. It means that

unknown sample D contains of cysteine.

ACKNOWLEDGMENT

Firstly, writer is grateful to the God, for

the unlimited knowledge was created. Then,

thanks for biochemistry experiment lecturer

Mr. I Nyoman Tika, M.Si and the lecturer

assistant Mrs. Kadek Dewi Wirmandiyanthi,

S.Pd, M.Si who guides the writer in

conducting experiment. Thank for the

laboratory assistant, Mr. Dewa who guides the

writer in conducting experiment and give

information how to work safely. Moreover,

the last for all members of VIA class who

give great motivation to the writer.

REFERENCE

L.G.Wade, JR. 2010.Organic Chemistry

Seventh Edition. USA: Pearson Education,

Inc.

Purba, M. 2004. Kimia Untuk SMA Kelas XII.

Jakarta : Erlangga.

Redhana, I. W and Maryam, S. 2003. Penuntun

Praktikum Biokimia. Singaraja: IKIP

Negeri Singaraja.

Sudaryani. 2003. Kualitas Telur. Jakarta:

Swadaya.

Tika, I N. 2010. Buku Penuntun Praktikum

Biokimia. Singaraja: Universitas

Pendidikan Ganesha.

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