Expt 6

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ALON, Jamaica P.

CHEM 133L YB
BALINGIT, Beatriz Mitzi B. July 6, 2019
VILLACOTE, Melvin P. Locker No. 101

Experiment 6: Color Reactions of Proteins and Amino Acids

V. OBSERVATION

SAMPLES (Indicate +/-  and color response)


TEST
Casein Albumin Amino Acid Blank
Test

Ninhydrin + + Gly + (PURPLE) -


(PURPLE) (PURPLE)

Biuret + + Gly + (LIGHT BLUE) -


(PURPLE) (PURPLE)

Trp + (YELLOW)
Xanthoproteic + - Tyr + (YELLOW) -
(YELLOW Phe - 
with precipitate) His -

+
Millon-Nasse (OLD ROSE - Tyr + (YELLOW) -
with precipitate)

+
Hopkins-Cole + (violet ring w/ cloudy Trp + (purple ring above -
(violet ring) white suspension) yellow ring)

Bromine - - Trp + (red violet) -


Water (clear; white ppt) (clear; white ppt) (clear
yellow)

Pauly - - His - -
Tyr -

Lead Acetate - - Cys + (slightly dark color) -


(clear, light (murky brown) pcs. of hair + (dark ppt)
brown w/ ppt)

Sakaguchi - - Arg + (red color)  -


(black color) (black color) (black
color)

1.) Why must the solution to be tested with ninhydrin be neutral?

The NaOAc was added to the Ninhydrin solution in order to neutralize the
samples in the Ninhydrin test. Ninhydrin is a chemical that detects amines
(secondary/primary) and ammonia. At neutral conditions (pH=4), amino acids are
able to react with ammonia which explains the importance of the samples to be
neutralized. This allows the reaction of the Ninhydrin solution to all α- amino acids
(triketohydrindene hydrate) to be detected.

2.) What is the use of the marble in the ninhydrin reaction?

The purpose of the marble in the Ninhydrin reaction is to prevent the evaporation
of the solution inside for it has to be heated to boiling in a water bath for 1-2 minutes.

3.) A very dilute solution of CuSo4 is used in the biuret test. Why?

Peptide bonds which are the basis of protein formation are detected by
performing the Biuret test resulting to a purple solution. Copper will precipitate in
basic solution. By controlling the amount of copper ions present, the amount of
precipitate formed will be minimized, and the copper will react completely with the
peptides.

4.) Are the Xanthoproteic and Millon-Nasse tests satisfactory for use in the urinary
examination for protein? Why?

Both the Xanthoproteic and Millon-Nasse tests are unsatisfactory for the
utilization in the detection of proteins. This is caused by their difference in the type of
amino acids they detect. The Xanthoproteic test detects the presence of phenyl and
aromatic rings in proteins. On the other hand, the Millon-Nasse test detects the
presence of hydroxyphenyls. These tests would fail if these were to be used in Urine
Protein Tests due to failure of measuring all the urine’s complete amount of protein.
On the brighter side, these tests can be utilized and effective it is supported by
several other tests in order to detect a wider range of proteins and amino acids
present

5.) Why does the bromine water be avoided in the test for free tryptophan?

The Bromine water tests free tryptophan in solutions. Free tryptophan interacts
with bromine water and n-amyl alcohol to form a pinkish lavender complex. However,
the presence of excess bromine water will cause the pink color to disappear and it
may be masked by the color of the reagent. The colored complex is soluble at the
alcohol layer.

6.) Which test can be used to show up to what stage the hydrolysis of a protein
proceeds?

The stage that shows us the hydrolysis that a protein proceeds is usually carried
out by the ninhydrin reaction which was first observed by Ruhemann.

7.) Discuss each test to detect groups in proteins and amino acids and write
equations to support results.

Ninhydrin Test:

Ninhydrin is a strong oxidizing agent used in amino acid analysis for the precise
determination of protein quantities. It is mainly used as a detector in liquid
chromatography methods coupled with ninhydrin post-column derivatization
systems. The reagent which is initially yellow reacts with free alpha amino groups
present in all amino acids, proteins, or peptides and forms a deep blue or purple
colored complex known as Ruhemann’s purple.

The samples that were used for the test were neutralized with solid NaOAc with
2-3 drops of ninhydrin solution. It was then covered with marbles and was heated in
a boiling water bath for 2-3 minutes. Colors deep blue and purple were observed in
the solution.
Biuret Test:

The Biuret Test positively identifies the presence of proteins (not less than two
peptides). The reaction in this test involves the complex formation of the proteins
with Cu2+ ions in a strongly alkaline solution. The samples were mixed with 0.5 mL
of 10% NaOH with 1-2 drops of 0.5% CuSO4. The reason why the samples were
mixed with NaOH because the biuret test for proteins solution reacts in a basic
solution to form a deep violet complex.

Xanthoproteic Test:

The Xanthoproteic test uses a nitration reaction to determine the presence of


proteins in a solution. When the sample is treated with a hot, concentrated nitric acid
it reacts with aromatic amino acids such as phenylalanine, tyrosine and tryptophan
and forms a yellow colored product known as Xantho protein. With the addition of
strong base such as NH3 or NaOH, it further changes to deep-orange color. So, this
test gives a positive result in those proteins which contain amino acids that have
aromatic rings in their side chains.

Each of the samples were added with 0.5mL of concentrated HNO 3, it was then
observed if a white precipitated was formed, if present, the solution was then heated
and observed if the precipitate turned yellow. The solutions were cooled and were
added with 10% NaOH but there was no change in the color of the precipitate
observed. The intensity of the yellow color deepens when the reaction occurs in
basic solution. This reaction is one of the reactions that occur if you spill a
concentrated solution of nitric acid onto your skin. The proteins in skin contain
tyrosine and tryptophan, which become nitrated and turn yellow.

MIllon-Nasse

Compounds containing hydroxybenzene radical react with Millon’s reagent to


form red complexes. The only amino acid having hydroxybenzene ring is tyrosine.
Thus, this test is specific for the amino acid tyrosine and the protein containing this
amino acid. Tyrosine when reacted with acidified mercuric sulphate solution gives
yellow precipitate of mercury-amino acid complex. On addition of sodioum nitrate
solution and heating, the yellow complex of mercury-amino acid complex converts to
mercury phenolate which is in red color.

Hopkins-Cole Reaction:

This chemical test is used to identify the presence of tryptophan, the only amino
acid containing in dole group. When the protein solution is mixed with Hopkins-Cole
reagent the in-dole ring reacts with the glyoxylic acid in the presence of concentrated
sulfuric acid and forms a violet or purple colored product, signifying a positive result.
The protein solution is hydrolyzed by the concentrated H 2SO4 at the solution
interface. Once the tryptophan is free, it reacts with the glyoxylic acid to form the
violet product.
Bromine Water Test

It is used to detect the presence of unsaturated compounds of alkene and


alkyne. Such a test for alkenes works via the mechanism of making alkenes or
hydrocarbons, having a minimum of one double bond that undergoes addition
reactions. The alkenes and hydrocarbons combine with bromine to impart a colorless
appearance to this element. As for the change in bromine's color, alkenes are
colorless and therefore, their combination with bromine causes the latter to lose
color, as well as it gets consumed in the reaction process. The color of the alcohol
layer that was observed was colorless. The equation for this reaction is:

EQUATION:
H2 = CH2  H2BrC - CBrH2

Pauly Reaction:

In five test tubes, casein, albumin, histidine, tyrosine and a blank were treated
with sulfanilic acid with NaNO2and was cooled in an ice bath for 3 minutes and
made to alkaline with Na2CO3. The principle behind the Pauly’s reaction is
diazotized. Sulfanilic acid will bediazotized with the addition of NaNO2and
Na2CO3and formed diazotized component.7Thediazonium component reacts with
the imidazole ring of histidine and a phenol group of tyrosine to form dark red
compound. However, in the experiment, instead of forming a red compound, yellow
product was obtained. Thus, a negative result was observed.
Acetate Reaction:

Four test tubes were obtained with casein, albumin, a blank, and an untreated
piece of hair were added with NaOH and Pb (OAc)2. Covered with marble, the
samples were heated to boiling under a water bath for a few minutes. The albumin
and the test tube which contains the hair gave a positive result. Sulphur containing
amino acids upon boiling with NaOH, yields Na 2S. This reaction is due to the partial
conversion of the organic Sulphur into inorganic.

Sakaguchi Reaction:

The Sakaguchi reagent is used to test for a certain amino acid and proteins. The
amino acid that is detected in this test is arginine. Since arginine has a guanidine
group in its side chain, it gives a red color with α-naphthol in the presence of an
oxidizing agent like bromine solution Based on our observation, the casein solution
turned color red after it was mix thoroughly with 0.5mL bromine water.
CONCLUSION:
This experiment was carried out to conduct a general test for proteins and amino
acids as well as a specific test for the amino acids present in albumin and casein.
We therefore conclude that albumin and casein are proteins that consists of several
peptide chains hence both show a positive result for Biuret’s test since Biuret’s test is
a test for detecting at least two peptide bonds. Both albumin and casein show a
violet solution for ninhydrin test hence they contain an alpha-amino acid. Both also
has a presence of unsaturation. By conducting different specific test such as
Xanthoproteic, Millon-Nasse, Hopkin’s-Cole, Lead acetate, Pauly, Bromine water and
Sakaguchi test specific amino acids were determined in albumin and casein. Albumin
contains tyrosine, tryptophan, cysteine, histidine and arginine whereas casein
contains only tyrosine and arginine. Obviously gelatin lacks few of the essential
amino acids hence the term incomplete protein, on the other hand albumin is a
complete protein.
References:

(Kumar, P.) Qualitative and Quantitative Tests for Amino Acids and Proteins
https://vlab.amrita.edu/?sub=3&brch=63&sim=1094&cnt=1
(Kamineni, S., Manepally, M., Kamineni P.) (2016.) Musculoskeletal Protein Analysis
Techniques - A Review
Color Reactions of Proteins. (2015). Retrieved July 6, 2019, from Gopzonfire:
https://gopzonfire.wordpress.com/2015/07/11/color-reactions-of-proteins/amp/

Hunt, I. (n.d.). Chapter 27: Amino Acids, Peptides and Proteins. Retrieved July 6,
2019, from University of Calgary-Department of Chemistry:
http://www.chem.ucalgary.ca/courses/351/Carey5th/Ch27/ch27-3-3.html

Karki, G. (2018, June 29). Adamkiewicz reaction (Hopkin’s-cole test): Objective,


Principle, Reagents, Procedure and Result. Retrieved July 6, 2019, from Online
Biology Notes: https://www.onlinebiologynotes.com/adamkiewicz-reaction-hopkins-
cole-test-objective-principle-reagents-procedure-and-result/

Karki, G. (2018, April 25). Millon’s test: Objective, Principle, Reagents, Procedure
and Result. Retrieved July 5, 2019, from Online Biology Notes:
https://www.onlinebiologynotes.com/millons-test-objective-principle-reagents-
procedure-and-result/

Karki, G. (2018, April 18). Ninhydrin Test: Principle, Requirements, Procedure and
Result. Retrieved July 4, 2019, from One Biology Notes:
https://www.onlinebiologynotes.com/ninhydrin-test-principle-requirements-procedure-
and-result/
Karki, G. (2018, April 25). Sakaguchi test: Objective, Principle, Reagents, Procedure
and Result. Retrieved July 6, 2019, from Online Biology Notes:
https://www.onlinebiologynotes.com/sakaguchi-test-objective-principle-reagents-
procedure-and-result/

Kumar, P. (n.d.). Qualitative and Quantitative Tests for Amino Acids and Proteins.
Retrieved July 5, 2019, from Biology Discussion:
http://www.biologydiscussion.com/proteins/qualitative-and-quantitative-tests-for-
amino-acids-and-proteins/13065

Lesson 19. Qualitative Test for Proteins. (2012, September 19). Retrieved July 5,
2019, from e-Krishi Shiksha: http://ecoursesonline.iasri.res.in/mod/page/view.php?
id=4188

OpenStax. (n.d.). Hydrolysis of Salt Solutions. Retrieved July 6, 2019, from BC Open
Textbooks: https://opentextbc.ca/chemistry/chapter/14-4-hydrolysis-of-salt-solutions/

The Ninhydrin Test. (2011). Retrieved July 5, 2019, from Harper College Chemistry
Department:
http://dept.harpercollege.edu/chemistry/chm/100/dgodambe/thedisk/food/ninhy/ninhy
.htm

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