Qualitative Color Reaction for Casein M.A. Llado; *A.T.N Lopez; S.R. Maningas; R.J. Martin; P.C. Medina
Abstract: Our group was assigned to isolate the pr otein Casein from skim milk through isoelectric precipitation precipitation with acetic acid. After precipitating casein, several samples were used for the qualitative color reactions. After which we did the Alkaline Hydrolysis of Intact Protein for Buiret test, Ninhydrin test, Xanthoproteic test, Millons test, Hopkins-Cole test, Sakaguchi test, Nitroprusside test, Fohls test, Test for Amide and Pauly test. The intact protein was negative in Millons Test and Hopkins-Cole Test. However However it showed a positive result only for Fohls Test. The alkaline or basic hydrolysate had negative results in Biuret test, Ninhydrin test, Millons test, Sakaguchi test, and Nitroprusside test.
of the element calcium. It is not susceptible to Introduction
denaturing because of its structure (3).
Proteins are probably the most important class
Isolation of a protein is a method for isolating a
of biochemical molecules, although of course lipids and
single type of protein from a complex mixture. The
carbohydrates are also essential for life. Proteins are
importance of isolating proteins is to characterize their
the basis for the major structural components of animal
solubility, acid-base property, function, structure, and
and human tissue. Proteins are natural polymer
interactions. Proteins can be separated depending on their size,
molecules consisting of amino acid units. The number of
shape,
amino acids in proteins may range from two to several
properties. Some of the methods that are commonly used
thousand (1).
are
The objectives of this experiment are to isolate
charge,
isoelectric
hydrophobicity
precipitation,
and
heat
physiochemical
denaturation,
solu so lubi bililiza zati tion on,, salt-ind salt-induced uced prec precipit ipitatio ation, n, chromato chromatograp graphy, hy, and and
the following proteins like Casein from skimmed milk by isoelectric precipitation. To analyze chemical groups
In isoelectric precipitation, the isoelectric point must be achieved
responsible for color reaction and explain the principle
wherein the net charge of the protein will be equal to zero. It is
involved in each test. To perform acid, alkaline and
done by precipitating a complex mixture until the protein is
enzymatic hydrolysis on the isolated proteins and
precipitated at a certain pH level (5).
enumerate the advantages and disadvantages of each type of hydrolysis (2).
Methodology
Casein is a protein that is found in milk and used independently in many foods as a binding agent. It
A. Q ualitative ualitative Color Reactions of Casein
is part of a group called phosphoproteins, collections of
1. Biuret Test
proteins bound to something containing phosphoric
In the prepared test tube with the intact protein
acid. Casein is a salt, meaning it has no net ionic charge,
solution, 20 drops of 2.5 M NaOH was added and
mixed well. Another 2-3 drops of 0.1 M CuSO 4
A 0.5 ml of NaOH and 10 drops of 0.02% naphthol
solution was added. The test tube was shaken and
solution was added to the sample. Mixed then for 3
the color of the solution was noted.
minutes it was let stand. Three drops of 2% NaOBr was added and mixed. The formation of red
2. Ninhyndrin Test
solution was noted.
The sample was added with 6-10 drops of 0.1% ninhydrin solution and was heated in a boiling water bath. The color of the solution was noted.
8. Fohls Test Five drops of 30% NaOH and 2 drops of 5% (CH3COO)2 Pb to the sample. After which the tube
3. Xanthoproteic Test For this test the sample was slowly added with 10
was placed in a boiling water bath. The appearance of dark (black or brown) sediment was noted.
drops conc. HNO3 and mixed then the color of the solution was noted. After which the sample was
9. Test for Amides
slowly added 10 drops conc. NaOH. It was mixed
A 1 ml of 20% NaOH to 10 drops of the samples was
and the color of the solution was noted.
put in the sample. The tube was placed in a boiling water bath. The evolution of gas was tested during
4. Millons Test The sample was treated with 5 drops of Millons
heating by placing a moistened red litmus paper over the mouth of the tube. The results were noted.
reagent and the change in color was noted. 10. Pauly Test 5. Hopkins-Cole Test
The diazo reagent was prepared first by mixing 1%
In the prepared test the sample was slowly added
sulfuric acid with 3 drops 5% NaNO2 solution. A 5
with 20 drops Hopkins-Cole reagent and mixed well.
drops of the sample was added and 3-5 drops 10%
The test tube was inclined and 20 drops conc. H 2SO4
Na2CO3 to the diazo reagent. A red coloration was
was slowly along the side. The color and interface
noted.
was noted. B. Alkalyne Hydrolysis of Casein 6. Sakaguchi test The sample was treated with 10 drops of 10% NaOH
The intact protein which is casein was hydrolyzed by
and 10 drops of 0.02% naphthol solution and was
adding 10 ml of 4 M NaOH to 0.5 g isolated protein and
mixed. It was left to stand for 3 minutes, after, 3
was placed in a hard glass test tube and was labeled.
drops of 2% NaOBr was added and was mixed. The
The tube was covered with cotton which will act as a
color produced was noted.
stopper and was submitted for autoclaving in 15psi for 5 hours. The appearance was noted and 10 ml of
7. Nitroprusside Test
distilled water was added and mixed well. The mixture was placed into a 250ml beaker. The mixture was
neutralized with 1M HCl and this hydrolysate was used
intact protein produced a violet solution which is a
for
positive indication of the Biuret test.
another
set
of
characterization
test
and
chromatography. Ninhydrin test is for detecting free alpha amino groups. The only amino acid that is negative for the said test is
Discussion
In Table. 1 it shows the results obtained for the Q ualitative
proline. Its principle is oxidative deamination and
Color Reaction of Casein and the Basic
decarboxylation. A positive indication of this test would
Hydrolysis. As seen here amino acids have a variety of
be a blue violet coloration in the solution. Casein and
chemically reactive groups that can be used to
the two other hydrolysates must yield a positive result
characterize both free amino acid and proteins. The
in this test.
following tests are used to detect presence of amino acid and proteins and distinguish between them.
The test for presence of aromatic rings which includes tryptophan and tyrosine is Xanthoproteic test. Although
Table No. 1 Results for the Qualitative Color Reaction of Casein and Basic Hydrolysis Color Intact Protein Basic Reactions (Casein) Hydrolysis Biuret Test
Purple solution
Ninhydrin Test
White precipitate Yellow precipitate White precipitate White precipitate Yellow solution
Xanthoproteic Test Millons Test Hopkins-Cole Test Sakaguchi test Nitroprusside Test Fohls Test Test for Amides Pauly Test A.
Yellow precipitate Brown precipitate Red to blue litmus paper Red solution
Light blue solution Light yellow solution Orange solution Yellow precipitate Light brown solution Light yellow solution Yellow solution Brown solution
phenylalanine is considered one of them, it will not have a positive result because it is inactive. Nitration of the phenyl group is the principle invoved in this test. There should be a positive visible reaction of yellow to orange solution for this test. Intact proteins, acid hydrolysates and basic hydrolysates are positive for this test.
Millons test is a test for the presence of tyrosine. Its principle is the complexation reaction between phenolic group and mercury that is found in the Millons reagent. A positive indication of this test is old rose or red precipitate. Intact proteins and the hydrolysates should
Yellow solution
be positive in this test.
Orange solution
Q ualitative Color Reactions
The Biuret test is a general test for proteins and for detecting peptide linkage. The principle involved is complexation reaction. The intact protein should be positive in this test since its peptide linkage is not broken unlike the two other samples that had undergone hydrolysis. As seen in the results, only the
The test for the presence of tryptophan is the HopkinsCole Test. The condensation of indole group with glyoxylic acid and H2SO4 is the principle involved in this test. The formation of purple ring on the surface of the solution is a positive indication of this test. According to the results, it was only the acid hydrolysate is negative for this test because tryptophan cannot be identified
and was destroyed during acid hydrolysis. It becomes
intact proteins, acidic and basic hydrolysates have
black precipitate which is a humin.
positive results for this test.
Sakaguchi test is a test for the presence of free or intact
The last test performed was Pauly Test which is the test
arginine. Subsequently alkaline or basic hydrolysis
for Histidine and Tyrosine. A dark yellow to orange
destroys arginine and produces ornithine and urea, all
solution is its positive outcome.
the samples are positive but basic hydrolysate should be negative for this test. The reaction of guanido group with napthol and an oxidizing reagent is the primary principle involved for this. A positive indication for this test is a red or orange solution. Only the basic
References
1. Retrieved January 6, 2012, http://www.elmhurst.edu/~chm/vchembook/5 65proteins.html
hydrolysate had a light color.
Nitroprusside test is used for indicating the presence of cysteine. In this test, cysteine is partially destroyed and it produced a red solution. Its principle is complexation. Intact protein is very positive in this test while the acid and basic hydrolysate is only somewhat positive. The cysteine that is partially destroyed is evident in the results of the experiment.
Fohls test is a test for sulfur containing proteins. It also indicates the presence of methionine and cysteine because those two amino acids have sulfur in their structures. The principle involved is the fusion followed by ionic interaction. The formation of black precipitate from lead sulfide is the positive outcome for this test. The dark coloration of the samples caused by the Fohls test indicates that there is sulfur in the intact protein, acid hydrolyste and basic hydrolysate.
The test for amides is used to detect R-groups of asparagine and glutamine. The change in color of litmus paper from red to blue is the positive product for this. Its principle is basic hydrolysis. As seen in the result all
2. Crisostomo, A., et al. Laboratory Manual in General Biochemistry . C & E Publishing, Inc. Q uezon City. 3. Retrieved January 6, 2012, http://www.wisegeek.com/what-is-casein.htm.
