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Pacana | Miranda | Dofredo | Rada
Ramos | Pama | PerezBMLS 2A (Summer)
Qualitative Test for Proteins (Color Reaction)
Objectives
Detect proteins using different tests.
Determine significance of tests to protein hydrolysis.
Determine chemical structures in protein that brings out color change.
Determine positive/negative results.
Know the principles involved in each test.
Objectives
Tests
Xanthoproteic Test
1 mL egg albumin
+ 5 drops of conc. HNO3
Heat, then Cool
+ 5 drops NH4OH
Xanthoproteic Test
Principle:It uses concentrated nitric acid.
The test gives a positive result in those proteins with amino acids carrying aromatic groups, especially in the presence of Tyrosine.
Xanthoproteic Test
Present:
Proteins with amino acids
carrying aromatic groups,
especially Tyrosine
Xanthoproteic Test
Color of precipitate:
Dark yellow (+HNO3)
Orange (+NH4OH)
Hopkins-Cole Test
1 mL egg albumin
+ 1 mL H-C reagent
Incline + 10 drops pure conc. H2SO4
Hopkins-Cole Test
Principle:When protein mixed with glyoxylic acid and H2SO4, a violet ring will form beneath the protein mixture.
Due to the presence of an indole nucleus in the tryptophan component.
Tryptophan condenses with the aldehyde to form the colored product.
Hopkins-Cole Test
Present:
Tryptophan (indole group)
Hopkins-Cole Test
Color:
Violet ring between two
liquids
Biuret Test
1 mL egg albumin
+ 1 mL 10% NaOH
1 mL 0.5% CuSO4, drop by drop
Biuret Test
Principle:Alkaline solution of proteins treated with copper sulfate solution results in the production of a rose-pink to violet color.
This is due to the presence of a peptide linkage.
Biuret Test
Present:
Peptide bonds
(--C=ONH2)
Biuret Test
Color:
Purple solution
Ninhydrin Test
1 mL egg albumin
+ 1 mL 0.1% Ninhydrin(aq)
Heat to boiling
Cool
Ninhydrin Test
Principle:When protein is boiled with ninhydrin, a blue color is produced.
Due to the presence of the alpha-amino acid.
All proteins are positive except proline.
alpha-amino acid + 2 ninhydrin ---> CO2 + aldehyde + final complex(BLUE) + 4H2O
alpha-amino acid + ninhydrin ---> reduced ninhydrin + alpha-amino acid + H2O alpha-amino acid + H2O ---> alpha-keto acid +NH3
alpha-keto acid + NH3 ---> aldehyde + CO2
Ninhydrin Test
Present:
All proteins, peptides,
And amino acids
Except Proline
Ninhydrin Test
Color:
Blue solution
Sakaguchi Test
1 mL test solution
+ 1 mL 10% NaOH
+ 6 drops dilute alcoholic alpha-naphthol solution
Mix
+ 10 drops NaBrO solution
Sakaguchi Test
Present:
Arginine (guanidinium group)
Sakaguchi Test
Principle:It is used for the detection of a specific type of protein with the amino acid containing the guanidinium group to form a red color.
Sakaguchi Test
Color:
Red solution
Results
Test Test SpecificityTheoretical
Result
Experimental Result
Remarks
Result Drawing
1. Xanthoproteic Test
Amino acids with a benzene ring
Yellow precipitate Yellow Precipitate (+)
2. Hopkins-Cole Test TryptophanViolet ring between
the two liquidsCloudy white Precipitate
(-)
3. Biuret Test Peptide Bonds Violet Solution Violet Solution (+)
4. Ninhydrin TestAll proteins, peptide
bonds and amino acids except Proline
Blue Solution Blue Solution (+)
5. Sakaguchi Test Arginine Red Solution Red Solution (+)
ResultsTable 2A – 1 Qualitative Test for Ovalbumin
Analysis
The experiment yielded a positive result when a yellow precipitate formed.
Thus, amino acids with benzene rings (Tyosine, Phenylalanine, Tryptohan)
are present.
Xanthoproteic Test - Analysis
The experiment yielded a negative result when a cloudy precipitate formed instead of a violet ring between the two liquids.
Thus, tryptophan is absent.
We accept this as an experimentl error as ovalbumin generally contains a lot of tryptophan.
Hopkins-Cole Test - Analysis
The experiment yielded a positive result when the solution turned blue-violet.
Thus, many peptide bonds are present.
Biuret Test - Analysis
The experiment yielded a positive result when the solution turned blue.
Thus, many proteins, peptide bonds and amino acids are present.
Though it exempts Proline, it is not a guarantee that Proline is not present. It’s just that it cannot detect Proline’s existence.
Ninhydrin Test - Analysis
The experiment yielded a positive result when the solution turned red.
Thus, the amino acid containing the guanidinium group, Arginine, is present.
Sakaguchi Test - Analysis
Conclusion
Conclusion
Ovalbumin in egg white contains many types of amino acids, be it as residues, connected via peptide bonds, or part of long-chain proteins. That is why eggs are important in the human diet, as it provides the essential amino acids required to run our body in the principle of homeostasis.
There are different tests for different types of amino acids and some tests exclude a specific amino acid.
Tryptophan is present in ovalbumin, even if our experimental data showed otherwise. This just means that there are many factors involved in the accuracy of data gathered.