Vincent, Bautista

Navarro, Mia Grace G.

Fonbuena, Kate Ashley
BSN1-41
DATE: 09-01-22 RATING:

PHINMA UNIVERSITY OF PANGASINAN

COLLEGE OF ALLIED HEALTH SCIENCES
DEPARTMENT OF NURSING

ACTIVITY NO. 5
COLOR TEST FOR PROTEINS AND SPECIFIC AMINO ACIDS

I. DISCUSSION:
Classification and Structure of Amino Acids
Structure of Proteins (Peptide Bond)
Name of test, purpose of test, composition of reagent, positive results obtained.

II. PROCEDURE:

A. GENERAL PROTEIN COLOR TESTS (20 points)

(Perform the following tests on 1 ml. each of 1% solution of egg albumin, glycine and
phenylalanine)

1. Biuret Test
In separate test tubes containing 1 ml. of each of the above solutions, add 1 ml. of 10%
NaOH solution and drops of 0.1% cupric sulfate solution. To a fourth test tube containing
1 ml. of distilled water which serves as a blank, add the same amount of NaOH and
CuSO4. Describe any color change that occurs. Record your results in the table on the
next page.: The color of the samples turned into violet.

2. Ninhydrin Test
In separate test tubes of the above solutions, add 1 ml. of ninhydrin solution. To a fourth
test tube containing 1 ml. of dilute ammonium hydroxide, add the same amount of
ninhydrin solution. Heat the tubes in a boiling water bath for 3 minutes and observe the
color changes which occur in each tube. Record your results in the table on the next page.

3. Xanthoproteic Test

In separate test tubes containing the above solutions, add an equal volume of conc. nitric
acid (CAUTION!) To a fourth test tube containing 1 ml. of tryptophan, add the same
amount of conc. nitric acid. Heat t for 2 minutes in a boiling water bath. Write your
results in the table on the next page.

Why does nitric acid stains a yellow color?

When proteins and amino acids with phenyl rings are treated with concentrated nitric acid,
they form a yellow compound. The yellow stains on the skin are caused by the reaction of
nitric acid with the protein keratin found in the skin. This is known as the xanthoproteic
reaction.
 Vincent, Bautista
Navarro, Mia Grace G.
Fonbuena, Kate Ashley
BSN1-41

Name of Test Importance of Test Composition of Positive Results/ Group Responsible

Reagents Color Produces
Is used to detect the 1 ml 10% NaOH The samples Peptide bonds
presence of peptide turned purple
bonds in a sample, 1-3 drops of violet which
Biuret Test 0.01MCuSO4
which connect indicates
amino acids to positive result.
form proteins.
It is a qualitative 5 drops of 0.1% The color of the Alpha amino groups of
test for detecting Ninhydrin samples was proteins or free amino
the presence of solution turned into blue acids Xanthoproteic
Ninhydrin Test
alpha amino groups or purple
in proteins or free specifying
amino acids, which positive results.
results in a blue or
purple complex.
It is a test used to 1 ml of The samples Xanthoproteic test is a
detect the presence concentrated turned orange biochemical test for the
Xanthoproteic of the amino acids: nitric acid and yellow, detection of amino
Test tyrosine and (HNO3) indicating that acids containing
tryptophan. the result is phenolic or indolic
Saturated NaOH positive because groups like
the benzene ring phenylalanine, tyrosine,
in tyrosine and and tryptophan
tryptophan
became nitrated,
causing a color
change.
 Vincent, Bautista
Navarro, Mia Grace G.
Fonbuena, Kate Ashley
BSN1-41
B. COLOR TEST FOR SPECIFIE AMINO ACIDS (30 Points)

1. Millon’s Test for Tyrosine

In 3 separate test tube, place 2ml. each of 1% egg albumin, 1% gelatin and
0.02% salicylic acid solutions. Add 3 drops of Millon’s reagent and heat the
tubes in boiling water bath for 2 minutes. Note the color produced. Record
your results in the table below.

2. Hopkins-Cole Test for Tryptophan

In 3 separate test tubes, place 2 ml. each of 1% egg albumin, 1% gelatin and e
few granules of casein suspended n 2 ml. o water. Add 2 ml. of Hopkins-Cole
reagent to each tube and mix thoroughly. Hold each test tube in an inclined
position and carefully allow 3 ml. conc. sulfuric acid (CAUTION!) to slide
down the side of each tube. Let stand and observe the color at the zone of the
two fluids. Record your results in the table below.

3. Sakaguchi Test for Arginine

To 5 ml. of gelatin solution, add 1 ml. of 10% NaOH and 1 ml. of 0.02%
alpha-naphthol solution. After 3 minutes, add 3 drops of sodium hypobromite
solution. A strong red color develops but fades quickly. The color can be
stabilized by adding urea to destroy the excess hypobromite. Write your result
in the table below. Repeat using Arginine.

4. Lead Acetate Test for Labile Sulfur

To 2 ml. of 1% egg albumin in test, add 5 ml. of 5% NaOH solution and a few
crystals of lead acetate. Heat in a boiling water bat for 5-10 minutes, with
occasional mixing of the contents of the tube. Note the color change. Record
your result in the table below.

Name of Test Importance of Test Composition of Positive Results/ Group Responsible

Reagents Color Produces
Its purpose is to 5 drops of fresh The color of the Presence of tyrosine
detect the presence Millon’s reagen samples turned into and proteins
Millon’s Test for of soluble proteins. red and this means containing tyrosine
Tyrosine When tyrosine and that the result is
proteins containing positive Phenol group
tyrosine are
present, they
produce a red
color or
precipitate.
Is a test for 2 ml of Hopkins- The formation of a A positive test
 Vincent, Bautista
Navarro, Mia Grace G.
Fonbuena, Kate Ashley
BSN1-41
detecting the Cole reagent purple-colored ring requires the presence
Hopkins-Cole Test indole ring in at the intersection of tryptophan, while
for Tryptophan proteins and 1 ml of of two layers a negative test
consequently concentrated indicates a positive requires the absence
trytophan. A violet sulfuric acid result. of tryptophan in the
between two protein.
layers indicates a
positive test. Indole group
Is a test for the 1 ml 10% of The color of the Presence of arginine
presence of NaOH samples turned into or arginyl residue
Sakaguchi Test for arginine or arginyl red upon the Guanidine
Arginine residue that 1 ml of 0.02% addition of bromine
produces a red alpha-naphthol water and this
complex means that the
2-4 ml of result is positive.
Bromine water
This is a test to 5 ml of 5% NaOH Presence of cysteine
detect the presence or proteins
Lead Acetate for of cysteine or Few crystals of containing cysteine
Labile Sulfur cysteine- Pb (Ac)2 (lead
containing (II) acetate)
proteins. A
positive test is
indicated by a
black or gray
precipitate.