Proteins Test

BIURET TEST
Objective
The biuret test is a chemical test used for detecting the presence of peptide
bonds.
Base Theory
In the presence of peptides, a copper(II) ion forms a violet-colored complex
in an alkaline solution.Several variants on the test have been developed.
The Biuret reaction can be used to assay the concentration of proteins
because peptide bonds occur with the same frequency per amino acid in the
peptide. The intensity of the color, and hence the absorption at 540 nm, is
directly proportional to the protein concentration, according to the BeerLambert law. In spite of its name, the reagent does not in fact contain biuret
((H2N-CO-)2NH). The test is so named because it also gives a positive
reaction to the peptide bonds in the biuret molecule.
Procedure
An aqueous sample is treated with an equal volume of 1% strong base
(sodium or potassium hydroxide most often) followed by a few drops of
aqueous copper(II) sulfate. If the solution turns purple, protein is present. 5
160 mg/mL can be determined.
NINHIDRINS TEST
Objective
Ninhydrin reaction is chemical reaction to detect existence of amino acids.
Base Theory

Ninhydrin (triketohydrindene hydrate) is an oxidating agent which leads to

the oxidative deamination of alpha-amino groups. It is very important for the
detection and the quantitative analysis of amino acids. Ninhydrin also reacts
with primary amines however the formation of carbon dioxide is quite
diagnostic for amino acids. When reacting with free amines from the amino
acid, blue or purple color
is produced. Most of the amino acids are hydrolyzed and reacted with
ninhydrin except proline. When ninhydrin reacts with amino acids, the
reaction also releases CO2. A ninhydrin solution is commonly used by
forensic investigators in the analysis of latent fingerprints on porous
surfaces such as paper. Amino acid containing fingermarks, formed by
minute sweat secretions which gather on the fingers unique ridges, are
treated with the ninhydrin solution which turns the amino acid finger ridge
patterns purple and therefore visible.
Procedure
Get 5 drops 0,1% ninhidrins solution in test tube and add with 2 ml matter
which will be tested. Warming in boiler approximately in 10 minutes.
MILLONS TEST
Objective
Millons test is chemical test that detect the presence Tyrosine
Base Theory
Millons test is given by any compound containing a phenolic hydroxy group.
Consequently, any protein containing tyrosine will give a positive test of a
pink to dark-red colour. The Millon reagent is a solution of mercuric and
mercurous ions in nitric and nitrous acids (CAUTION: MILLONS REAGENT
IS HIGHLY TOXIC AND HIGHLY CORROSIVE). The red colour is probably
due to a mercury salt of nitrated tyrosine.

Procedure
Place 1 mL of casein, 2% egg albumin, and 0.1 M tyrosine into separate,
labelled, 12 x 75 mm test tubes. Add 3 drops of Millons reagent and
immerse the tubes in a boiling water bath for 5 minutes. Cool the tubes and
record the colours formed.
XANTHOPROTEICS TEST
Objective
Xanthoproteic test is used to determine the presence of tyrosine,
trypthopane, and phenylalanine to the protein.
Base Theory
The test gives a positive result in those proteins
with aminoacids carrying aromatic groups, especially in the presence of
tyrosine. Aromatic groups in the aminoacids will be nitrated by HNO3. The
nitro derivate show an intensely yellow color. Because nearly all proteins
contain aromatics it is taken as a protein-test either.
Procedure
Place 2 ml of matter that will be tested to the test tube, add with 1 ml of
strong HNO3 to the test tube.
HOPKINS COLES TEST
Objective
The Hopkins-Cole test determines the presence of the amino acid
tryptophan.
Base Theory

Some chemists are no longer able to use the Hopkins-Cole test because it is
not completely understood in terms of chemistry. What is known about it is
that the tryptophan that the Hopkins-Cole test determines is defined as an
indole nucleus and is known for creating the violet ring where the two layers
meet. In order to perform the Hopkins-Cole test, scientists or chemists must
first know the proper reagent for it, and this is part of analytical chemistry.
When the violet ring appears after the two layers within an indole nucleus
meet, this confirms that concentrated sulfuric acid was added to a mixture
of some sort that contained glyoxylic acid and a protein. However, there are
some products that do not show the reaction, such as gelatin and zein.
Procedure
Place 2 ml of matter that will be tested to the test tube, add with 2 ml of
Hopkins Coles reactant from the wall of test tube.
PROTEIN COMPOTITIONS TEST
Objective
Some test below are used to detect the element that presence in protein
Base Theory
Most protein consist of Carbon (50-55%), hydrogen (6-7.3%), Oxigen (1924%), and nitrogen (13-19%). Another element that presence as micro
component are S, P, Fe, Mn, I, Cu, and Zn.
Procedure
a. To detect the presence of carbon
Fill the test tube with a little of albumin powder that heated until you smells
burned-hair odor and became sandbank.
b. To detect the presence of nitrogen

Fill the test tube with a half of spoon of albumin powder and a spoon of
NaOH powder. Heated slowly until you can
smells amonias odor and
vapor that forming tested with wet papper of red lacmus.