Protein’s 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 Beer-Lambert 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.

NINHIDRIN’S  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 finger’s 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% ninhidrin’s solution in test tube and add with 2 ml matter which will be tested. Warming in boiler approximately in 10 minutes.

MILLON’S  TEST

Objective

Millon’s test is chemical test that detect the presence  Tyrosine

Base Theory

Millon’s 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: MILLON’S 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 Millon’s reagent and immerse the tubes in a boiling water bath for 5 minutes. Cool the tubes and record the colours formed.

XANTHOPROTEIC’S  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 COLE’S 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 Cole’s reactant from the wall of test tube.

PROTEIN COMPOTITION’S  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 (19-24%), 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 amonia’s odor and vapor that forming tested with wet papper of red lacmus.

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