ISOLATION AND CHARACTERIZATION OF PROTEINS

Dedan Angelo B. Samson, Bernadette Mae L. Segismundo, Jane Darylle G. Semilla,

Jason Anthony T. Supea, Francis Ernest R. Talusan and Aniko Carlo S. Tendenilla
Group 7 2B Medical Technology Biochemistry Laboratory

ABSTRACT
Amino acids have different chemically reactive groups. The various reactions for side chains, -amino, and -carboxyl
groups can be used to characterize and identify both free amino acids and proteins. Qualitative reactions are usually
used in order to analyze the said chemically reactive groups in amino acids. The following proteins were subjected to
the qualitative reactions: casein, albumin, gluten and myoglobin. The various qualitative reactions used in this
experiment include, Biuret, Ninhydrin, Xanthoproteic, Millons, Hopkins-Cole, Sakaguchi, Nitroprusside, Fohls, Amide,
and Paulys test. The Biuret test is used to detect the presence of peptide bonds. On the other hand, Ninhydrin test is
a usual test for an -amino acid. The Xanthoproteic test determines the presence of side chains of aromatic amino
acids while the Millons and Hopkins-Cole tests determine tyrosine and tryptophan residues, respectively. The
Nitroprusside test is used to find out if sulfur-containing acids are present. Test for amides is used to detect R-groups
of asparagine and glutamine. Lastly, the Pauly Test, which involves the principle of diazotization, is a test for the
presence of histidine and tyrosine. The Biuret Test was completed through adding 20 drops of 2.5 M NaOH and 2-3
drops of 0.1 M CuSO4 solution. For this test, all the intact proteins yielded a positive results. However, the
hydrolysates showed negative results. The Ninhydrin Test was done by placing 6-10 drops of 0.1% ninhydrin solution
and by heating a boiling water bath. Positive results were found in unhydrolyzed casein and myoglobin, hydrolyzed
casein and gluten, and hydrolyzed albumin, gluten and myoglobin. The Xanthoproteic Test was done by adding 10
drops of concentrated HNO3 and 10 drops of conc. NaOH. Positive results were present in unhydrolyzed casein,
albumin, myoglobin, and all hydrolyzed protein samples. The Millons Test was done by adding 5 drops of Millons
reagent. A positive result was observed in gluten. On the other hand, the Hopkins-Cole Test was done by adding 20
drops of Hopkins-Cole reagent and 20 drops conc. H2SO4. Positive result was observed in unhydrolyzed casein, gluten
and myoglobin, as well as basic hydrolysated myoglobin. The Sakaguchi Test was done by adding 10 drops each of
10% NaOH and 0.02% naphthol solution and 3 drops 2% NaOBr. All samples yielded negative in this test. The
Nitroprusside Test was done by adding 0.5 mL of 3 M NaOH and 0.25 Ml 2% nitroprusside solution. H ydrolyzed casein
and gluten showed positive results. The Fohls Test was done by adding 5 drops of 30% NaOH and 2 drops of 5%
(CH3COO)2Pb and placing into a boiling water bath. A positive result was observed in all intact proteins. The Test for
Amides was done by adding 1 mL of 20% NaOH and placing in water bath. All samples displayed positive results.
Lastly, the Pauly test was done by adding 5 drops of the sample and 3-5 drops 10% Na 2CO3 to the diazo reagent. A
positive results was observed to be present in all of the intact proteins as well as some of the hydrolyzed forms of the
samples.

INTRODUCTION The Biuret test is used to detect the presence

A protein molecule is a long chain of amino of peptide bonds. On the other hand, Ninhydrin
acids linked by peptide bonds. The properties are test is a usual test for an -amino acid. The
determined by the order or sequence of the Xanthoproteic test determines the presence of
amino acids in its molecule, and by the three- side chains of aromatic amino acids while the
dimensional structure of the molecular chain. The Millons and Hopkins-Cole tests determine
chain folds and twists and then forming its tyrosine and tryptophan residues, respectively.
conformational structure which gives its The Nitroprusside test is used to find out if sulfur-
distinctive properties. containing acids are present. Test for amides is
Proteins are large molecules consisting of used to detect R-groups of asparagine and
amino acids which our bodies and the cells in our glutamine. Lastly, the Pauly Test, which involves
bodies need to function properly. Our body the principle of diazotization, is a test for the
structures, functions, the regulation of the body's presence of histidine and tyrosine.
cells, tissues and organs cannot exist without
proteins. Structural proteins such as keratin and EXPERIMENTAL
collagen make up the skin, claws, bones, A. Compounds tested (Samples used)
tendons, and ligaments; muscle proteins produce Intact and hydrolyzed casein (the main protein
movement; hemoglobin transports oxygen; and present in milk andin coagulated formin
membrane proteins regulate the movement of cheese. It is used in processed foods and in
substances into and out of cells. For humans, adhesives, paints, and other industrial products),
protein is an essential part of the diet, and is albumin (a simple form of protein that is soluble
found in greatest quantity in soy beans and other in water and coagulable by heat, such as that
grain legumes, meat, eggs, and cheese. During found in egg white, milk, and in particular, blood
digestion, protein molecules are broken down serum), gluten (a substance present in cereal
into amino acids which arethen easily absorbed grains, especially wheat, that is responsible for
into the body. the elastic texture of dough. A mixture of two
proteins, it causes illness in people with celiac nitroprusside solution was added. The formation
disease) and myoglobin (a red protein containing of a red solution was then noted.
heme that carries and stores oxygen in muscle
cells. It is structurally similar to a subunit of
hemoglobin). 8. Fohls Test
To each sample, 5 drops of 30% NaOH and 2
B. Procedure drops of 5% (CH3COO)2 Pb were added to the
Two sets of test tubes containing mixtures of sample. The test tube was then subjected to
the proteinhydrolyzed and unhydrolyzed heating in a boiling water bath. The appearance
respectivelyand 1 mL distilled water, labeled of dark (black or brown) sediment was then
according to the tests performed on them, were observed.
prepared. 9. Test for Amides
To each sample, 1 mL of 20% NaOH was added
1. Biuret Test to the sample. The test tube was then subjected
To each sample, 20 drops of 2.5M NaOH were to heating in a boiling water bath. Testing for the
added to the sample and then were mixed well evolution of gas during heating was done through
by gently swirling the test tube. After which, 2-3 placing a moistened red litmus paper over the
drops of 0.1M CuSO4 solution were added. The mouth of the tube. The result was then noted.
test tube was then swirled again to ensure 10. Pauly Test
complete mixture with the sample and then the First, the diazo reagent was prepared by mixing
color of the solution was then noted. 3-5 drops of 1% sulfanilic acid with 3 drops of
2. Ninhydrin Test 5% NaNO2 solution. Next, for each set, 5 drops of
To each sample, 6-10 drops of 0.1% ninhydrin the sample and 3-5 drops of 10% Na2CO3 were
solution were added into the diluted sample. The added to the diazo reagent. The appearance of a
test tube was then subjected to heating using a red coloration was then noted.
boiling water bath until an appearance of a blue-
violet color was observed. RESULTS AND DISCUSSION
3. Xanthoproteic Test The Biuret Test positively identifies the
To each sample, 10 drops of concentrated HNO 3 presence of proteins in solution with violet
were slowly added to the diluted sample. The color.Biuret reacts with copper (II) ions in a basic
color of the solution was observed after ensuring solution to form a violet complex. The peptide
complete mixture. After which, 10 drops of linkages in proteins resemble those in biuret and
concentrated NaOH were also slowly added. The also form deep violet complexes with basic
color of the solution was then again observed copper (II) ions in solution.
after mixing. A negative result is shown by a blue solution,
4. Millons Test which is initiated by having fewer than two
To each sample, 5 drops of Millons reagent peptide bonds present in the sample protein. All
were added to the diluted samples. The change in of the intact proteins displayed a positive result
color of the solution was then observed. (See Table 1 and 2). After acid hydrolysis and
5. Hopkins-Cole Test basic hydrolysis, all of the protein hydrolysates,
To each sample, 20 drops of Hopkins-Cole exhibited negative results because of the broken
reagent were added to the sample and the test peptide bonds (See Table 3-6). After enzymatic
tube was mixed well by swirling gently. The test hydrolysis, only albumin exhibited the positive
tube was then inclined and then introduced with result (See Table 7 and 8).
20 drops of concentrated H2SO4, which were
added slowly along the side. For this case, the Table 1. Results of Qualitative Color Reaction for
solution was not mixed. The color at the interface Intact Proteins: Casein and Albumin
was then noted. COLOR INTACT PROTEIN
6. Sakaguchi Test REACTION Casein Albumin
To each sample, 10 drops of 10% NaOH and 10 Biuret light violet light violet
drops of 0.02% naphthol solution were added to Ninhydrin blue violet colorless
the sample. The solution was then mixed and yellow/dark light
allowed to stand for 3 minutes. After which, 3 Xanthoproteic
yellow yellow/yellow
drops of 2% NaOBr were added and the color Millons colorless colorless
produced was noted after ensuring complete Hopkins-Cole purple ring colorless
mixture.
Sakaguchi yellow colorless
7. Nitroprusside Test
Nitroprusside yellow light yellow
To each sample, 10 drops of 3M NaOH were
Fohls brown black brown
added to the sample. After which, 5 drops of 2%
 sediments sediments
red to blue
red to blue Table 3. Results of Qualitative Color Reaction for
litmus paper;
Test for Amide litmus paper; Protein Hydrolysate (Acidic): Casein and Albumin
brown
yellow
suspension COLOR ACIDIC
Pauly red red REACTION Casein Albumin
Biuret light blue blue-gray
The Ninhydrin Test is a test for amino acids and Ninhydrin colorless brown
proteins with a free -NH2 group. When such an yellowish- dark
-NH2 group reacts with ninhydrin, a purple-blue Xanthoproteic brown/light brown/light
complex is formed. The principle behind this is yellow yellow
oxidative carboxylation and deamination. The Millons colorless light yellow
reagent in charge for the reactions is oxidized Hopkins-Cole colorless clear brown
ninhydrin in 95% ethanol. A positive result is Sakaguchi light yellow light orange
shown by a blue to blue-violet to violet color in Nitroprusside dark yellow yellow
the presence of -amino acids such as in dark brown
unhydrolyzed casein and myoglobin. It is Fohls light orange
sediments
indicated by a yellow coloration in the presence red to blue red to blue
Test for Amide
of cyclic amino acids, especially proline, such as litmus paper litmus paper
in basic hydrolysated casein and gluten and Pauly dark orange orange
brown in the presence of asparagine such as in
acidic hydrolysated albumin, gluten, and The Millon's Test shows a positive result of flesh
myoglobin. Hydrolysated proteins exhibit a more precipitate. In this test, the phenol group of the
intense positive result. Such was observed in tyrosine was nitrated by nitric acid. The nitrated
enzymatic-hydrolysated casein and albumin. (See tyrosine complexes mercury (I) and mercury (II)
Tables 1-8). ions into the solution to form old rose/flesh to red
precipitate. Therefore, proteins with tyrosine will
Table 2. Results of Qualitative Color Reaction for show a positive result. Gluten is expected to
Intact Proteins: Gluten and Myoglobin show a positive result. However, this was not
COLOR INTACT PROTEIN observed in the experiment.
REACTION Gluten Myoglobin
Biuret clear violet purple Table 4 Results of Qualitative Color Reaction for
Ninhydrin colorless dark purple Protein Hydrolysate (Acidic): Gluten & Myoglobin
Xanthoproteic white/yellow light yellow COLOR ACIDIC
REACTION Gluten Myoglobin
Millons colorless colorless
Biuret brown brown
Hopkins-Cole purple ring purple ring
Ninhydrin brown dark brown
Sakaguchi turbid colorless turbid solution
Xanthoproteic brown light brown
slightly turbid
Nitroprusside light yellow Millons brown clear yellow
yellow
brownish-black brown Hopkins-Cole brown clear yellow
Fohls
sediments sediments Sakaguchi brown brown
red to blue Nitroprusside reddish-brown brown
red to blue
Test for Amide litmus paper;
litmus paper Fohls black-brown brownish-
yellow
sediments orange
Pauly red-orange red
Test for Amide red to blue red to blue
litmus paper; litmus paper
The production of a yellow colored product upon the brown with
addition of nitric acid is a test for the presence of effervescence
tyrosine or tryptophan in a protein. The addition of Pauly red red
strong base will deepen the color to orange. The yellow
stains on the skin caused by nitric acid are the result of
the xanthoproteic reaction. A positive result was The Hopkins-Cole Test showed a positive result of
exhibited by unhydrolyzed casein, albumin and violet ring at the border. The indole ring reacts
myoglobin, and acid hydrolyzed casein, basic with glyoxylic acid in the presence of a strong
hydrolyzed casein, albumin, gluten and myoglobin, and acid to form a violet ring product. Intact casein,
enzymatic hydrolyzed casein. A positive result with the gluten and myoglobin including hydrolyzed
addition of HNO3 is indicated by an orange coloration myoglobin exhibited the positive result (See Tables
which was not observed in the proteins. It is probable 1-8).
that inaccurate results produced are caused by
experimental errors (See Tables 1-8).
 Ninhydrin purple blue-violet
Xanthoproteic light yellow colorless
Table 5. Results of Qualitative Color Reaction for
Millons colorless colorless
Protein Hydrolysate (Basic): Casein and Albumin
COLOR BASIC Hopkins-Cole colorless colorless
REACTION Casein Albumin Sakaguchi colorless light yellow
Biuret turbid brown turbid brown Nitroprusside dark yellow dark yellow
Ninhydrin yellow yellow Fohls light yellow light brown
sediments
Xanthoproteic yellow yellow
Test for Amide red to blue red to blue
Millons yellow yellow litmus paper; litmus paper;
Hopkins-Cole colorless light yellow colorless light yellow
Sakaguchi light yellow light yellow Pauly red red-orange
Nitroprusside red clear yellow
Fohls brown brown The Fohl's Test (Lead (II) acetate Test) has a
sediments sediments positive result of brown to black precipitate. The
Test for Amide blue to red blue to red principle is about the degradation and
litmus paper; litmus paper; substitution reaction to form PbS. It is observed
colorless clear yellow in all the intact protein samples used, acid
Pauly brown colorless hydrolyzed casein, gluten and myoglobin, basic
hydrolyzed casein, albumin, and enzymatic
The Sakaguchi Test has a positive result of red to hydrolyzed albumin.
red-orange color. The principle around this is
about complexation (base-catalyzed Table 8. Results of Qualitative Color Reaction for
condensation of -naphthol with the guanido Protein Hydrolysate (Enzymatic): Gluten and
group of Arginine). None of the samples Myoglobin
displayed such results due to the absence of the ENZYMATIC
COLOR REACTION
amino acid mentioned or the probably presence Gluten
of extremely small amounts (See Table 1-8). Biuret light blue
Ninhydrin colorless
Table 6. Results of Qualitative Color Reaction for
Xanthoproteic colorless
Protein Hydrolysate (Basic): Gluten & Myoglobin
COLOR BASIC Millons colorless
REACTION Gluten Myoglobin Hopkins-Cole colorless
Biuret blue blue Sakaguchi colorless
Ninhydrin colorless violet Nitroprusside yellow
Xanthoproteic yellow pale yellow Fohls colorless
Millons colorless turbid Test for Amide red to blue litmus
paper; colorless
Hopkins-Cole peach purple ring
Pauly orange-yellow
Sakaguchi colorless pale yellow
Nitroprusside yellow yellow
The test for amide is a test for the presence of a
Fohls colorless colorless
carboxamide or amide group, which is found in
Test for Amide red to blue red to blue amino acids asparagine and glutamine. The Test
litmus paper; litmus paper;
for Amide shows a positive result of yellow-
colorless colorless
orange solution and the red litmus paper turning
Pauly red red
into blue. Such was observed in all of the protein
samples used except the basic hydrolyzed form of
The Nitroprusside Test showed a positive result of casein and albumin (See Table 1-8).
yellow solution because the cysteine group reacts
with nitroprusside in alkaline solution. Hydrolyzed The Pauly test is specific for the detection of
casein and gluten exhibited the positive result Tryptophan or Histidine. The reagent used for this
(See Table 1-8). test contains sulphanilic acid dissolved in
hydrochloric acid. Sulphanilic acid upon
Table 7 Results of Qualitative Color Reaction for
diazotization in the presence of sodium nitrite
Protein Hydrolysate (Enzymatic): Casein &
and hydrochloric acid results in the formation a
Albumin
ENZYMATIC
diazonium salt. The diazonium salt formed
COLOR
REACTION Casein Albumin couples with either tyrosine or histidine in
Biuret light blue purple
alkaline medium to give a red coloured
chromogen (azo dye). Myoglobin, which contains
histidine, showed positive results although ome from
hydrolysed forms of the protein samples also http://www.chem.ucalgary.ca/courses/351/Carey
exhibited the red coloration. 5th/Ch27/ch27-3-3.html

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