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Group No. 66
Laboratory Report
Date
RATIONALE
Proteins are the third energy-supplying nutrients and present in every living cell.
Proteins function as structural materials and as enzymes that regulate the countless
chemical reactions taking place in every living organism, including the reactions
involved in the decomposition and synthesis of proteins. All proteins are polymeric
substances that yield amino acids on hydrolysis. Those that yield only amino acids when
hydrolyzed are classified as simple proteins; those that yield amino acids and one or
more additional products are classified as conjugated proteins. Proteins can be also
characterized by their chemical equations. Most proteins are soluble in water, in alcohol,
in dilute base or in various concentrations of salt solutions.
In this activity proteins and amino acids are going to be classified by their color
reactions. And to determine what type of amino acids are present in the every solutions
and how does peptide bonds occur when the reagents was added.
INTERPRETATION:
A. Biuret Reaction
Name of test
A. Biuret
Test Solutions
Reagents Added
Result
1% Albumin
10% NaOH
1% CuSO4
Violet
Reaction
1% Casein
Violet
1% Glycine
Light Blue
1% Peptone
Violet
Urea (s)
Light Blue
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Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University
Biuret test is a chemical test used for detecting the presence of peptide bonds of proteins.
The positive indication for this test is a violet color that is produced when dilute copper
(II) sulfate is added to an alkaline solution of a peptide or protein. At least two peptide
bonds must be present, since amino acids and dipeptides do not give a positive biuret
test. Albumin, casein, and peptone gives a positive result for biuret test it means that
they contain two or more peptide linkages, while glycine and urea (s) do not because
glycine and urea is an amino acid.
INTERPRETATION:
B. Millons Test (Theoretical Result)
Name of test
B. Millons Test
Test Solutions
Reagents Added
1% Albumin
Millons Reagent
[HgNO3 and
Hg(NO3)2 added
with conc. HNO3]
1% Casein
1% Gelatin
Result
1% Phenol
Millons test is to detect the phenolic group in the amino acid tyrosine. Millons test is
given by any compound containing a phenolic hydroxy group. Consequently, any protein
containing tyrosine will give a positive test of a reddish brown color. The millons
reagent is a solution of mercuric and mercurous ions in nitric and nitrous acids. The
positive indication for this test is a reddish brown color due to the mercury salt of the
nitrated tyrosine.
INTERPRETATION:
Name of test
C. Xanthoproteic
Reaction
Test Solutions
Reagents Added
Result
1% Albumin
Conc. HNO3
Conc. NH4OH
Yellow
1% Casein
Yellow
1% Gelatin
Yellow
1% Phenol
Orange
C. Xanthoproteic Reaction
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Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University
OH
OH
HNO3
NH2
HO
NH2
HO
NH2
Tyrosine
O
Yellow Product
O
OH
NH2
NH
H2N
HNO3
OH
NH2
NH
Tryptophan
Yellow Product
O
OH
NH2
+
HNO3
OH
H2N
NH2
Phenylalanine
Yellow Product
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Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University
INTERPRETATION:
D. Test for Sulfur
Test
Solutions
Name of test
D. Test for
1% Albumin
Sulfur
Gelatin (s)
Reagents
Added
Result
Black Precipitate
10% NaOH
5%
Pb(CH3COO)2
White Precipitate
Peptone (s)
Orange Color
The test for sulfur is to determine the sulfur containing amino acids. Cysteine and
cystines in the free state and in proteins give a positive result to the sulfur test. When
sulfur containing amino acids are boiled with an alkali, the sulfyhydryl or disulfide
groups are converted to an inorganic sulfide, NaS. This reacts with lead acetate to form a
black precipitate of PbS. Albumin is the only solution that produce a black precipitate
because the albumin has a sulfur containing amino acids. The gelatin and peptone didnt
produce a black precipitate it remains to its own color.
INTERPRETATION:
E. Hopkins-Cole Reaction
Name of test
E. Hopkins-Cole
Test
Solutions
Reagents
Added
1% Albumin
Conc. H2SO4
Reaction
Result
Violet Ring Formed Between the Two
Layers
1% Casein
1% Gelatin
No Change
Hopkins-Cole Test determines the presence of the amino acid tryptophan. The
indole ring of tryptophan condenses with glyoxalic acid in the presence of sulfuric acid
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Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University
to form a violet colored complex or a violet ring formed between the two layers. This is
the basis of the Hopkins-Cole Test. In this test the albumin and casein forms the violet
ring between the two layers, this confirms that albumin and casein contained glyoxylic
acid and a protein. And there are some products that do not show the reaction, such as
gelatin.
INTERPRETATION:
F. Ninhydrin Reaction
Name of test
Test Solutions
0.2% Albumin
F.
Reagents
Added
Result
No Change
0.1%
Ninhydrin
Solution
Ninhydrin
Ammonia
Water
Reaction
0.2% Urea
No Change
0.2% Glycine
Ruhemanns Purple
No Change
Ninhydrin test is used to detect the presence of the amino group in alpha amino acids.
When amino acids are heated with ninhydrin (triketohydrindenehydrate), ammonia,
carbon dioxide and aldehyde are produced. The NH3 liberated combines with one mole
of the reduced ninhydrin and one mole of the oxidized ninhydrin to form Ruhemanns
purple. The triketohydrindene hydrate, generally known as ninhydrin is an extremely
sensitive reagent for amino acids. All amino acids, except proline and hydroxyproline,
give a deep blue solution with ninhydrin. Proline and hydroxyproline produce a yellow
solution. For ninhydrin test there are two positive visible results: deep blue or
ruhemanns purple. The color varies slightly from acid to acid, because of the unreacted
acids complex with the pigment. In this test only glycine produced ruhemanns purple
because it is the only solution has the presence of amino group in alpha amino acids.
ANSWERS TO QUESTIONS
1. Why do all proteins give a positive result for Biuret test?
Answer: Because they have more than two peptide linkages and hence, they react
with CuSO4.
2. Why does nitric acid stain the skin with a yellow color?
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Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University
Answer: Nitric acid stain the skin with a yellow color due to a reaction with the
protein keratin. These yellow stains turn orange when neutralized.
3. Will methionine give a positive result for the sulfur test? Explain.
Answer: Yes, because it contains sulfur and it is one of only two amino acids that
contains a sulfur group.
4. What grouping in amino acids or proteins is responsible for the ninhydrin
reaction?
Answer: The amino group, NH2.
5. What color will be produced by proline with ninhydrin? Why?
Answer: Ruhemanns purple. The color varies slightly from acid to acid because
unreacted acids complex with the pigment.
CONCLUSION
Proteins function as structural materials and as enzymes that regulate the countless
chemical reactions taking place in every living organism. And proteins are highly
specific in their functions. The amino acid units in a given protein molecule are arranged
in a definite sequence. Proteins ultimately yield amino acids, when hydrolyzed. And they
can be hydrolyzed by boiling in a solution containing a strong acid such as hydrochloric
acid or in a solution containing a strong base such as sodium hydroxide. Amino acids
will produce positive result depending on their peptide bonds. When the two amino
acids are joined together by a peptide bond or peptide linkage. The product is a
dipeptide. The loss of a proteins natural conformation without peptide bond hydrolysis is
due to the denaturation process. And some test produces positive result depending on
what type of amino acids they are and how they will react with the following reagents.
REFERENCES
1. Hein, M., Best, L., & Pattison, S. (1984). College Chemistry. Monterey, California: Brooks/
Cole Publishing Company.
2. Brown, W., Rogers, E. General Organic, and Biochemistry. Monterey, California: Brooks/
Cole Publishing Company.
3. Boikess, R., Breslauer, K., Edelson, E. Elements of Chemistry. General, Organic, and
Biological.
4. Allen, T., Keefer, R. (1817). Chemistry Experiment and Theory. New York: Harper and
Ron, Publishers.
5. Mousse93. (n.d.). Lab Exam 1. San Francisco: Quizlet Inc. Retrieved February 28, 2016,
from https://quizlet.com/privacy
________________________________________________________________________________________________________
Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University
6. The Wall of Biochemistry. (2012, March). Picnonotus. Retrieved February 28, 2016, from
https://fulltimes.wordpress.com/
7. Chemistry- Environment & Ecology- Engineering- Economics- Psychology. (2007). Author.
Retrieved February 28, 2016, from http://www.funqa.com/chemistry/2504-Chemistry4.html
8. Senese, F. (1997). General Chemistry. Amino acid test. Retrieved February 28, 2016 from
http://antoine.frostburg.edu/chem/senese/101/organic/faq/amino-acid-test.shtml
________________________________________________________________________________________________________
Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga
University