ABOUT PROTEINS

Proteins are large, complex molecules that play many critical roles in the body. They do
most of the work in cells and are required for the structure, function, and regulation of
the body’s tissues and organs.

Further, they are essential components of muscles, skin, bones and the body as a whole.
Examples of proteins include whole classes of important molecules, among them
enzymes, hormones, and antibodies. Proteins are one of the three types of nutrients used
as energy sources by the body, the other two being carbohydrate and fat.

Type of Test: Biuret Test

Objectives:

• To detect the protein in the given solution.

• To demonstrate the presence of the peptide bond.

Procedure:

1. Take 3 clean and dry test tubes.

2. Add 1-2 ml of the test solution, egg albumin, and deionized water in the respective
test tubes.
3. Add 1-2 ml of Biuret reagent to all the test tubes.
4. Shake well and allow the mixtures to stand for 5 minutes.
5. Observe for any color change.

•Description (test): The Biuret reagent is a solution composed of sodium hydroxide

(NaOH) or potassium hydroxide (KOH), hydrated copper (II) sulfate,
and potassium sodium tartrate. Sodium hydroxide and Potassium hydroxide provide the
alkaline medium and potassium sodium tartrate is added to chelate and thus stabilize
the cupric ions in the solution or to maintain their solubility in alkaline solution.

•Rationale of the Test:

A Biuret test is a chemical test used to determine the presence of a peptide bond in a
substance. It is based on the biuret reaction in which a peptide structure containing at
least two peptide links produces a violet color when treated with alkaline copper sulfate.
In presence of an alkaline solution, blue-colored copper II ion can form a complex with
the peptide bonds since the peptide has unshared electron pairs in nitrogen and oxygen
of water. The colored coordination complex is formed between Cu2+ ion and carbonyl
oxygen (>C=O) and amide nitrogen (=NH) of the peptide bond. Once this complex has
been formed, the solution turns from blue to purple. The deeper the purple color, the
higher is the number of peptide-copper complexes. The reaction occurs in any
compound containing at least two H2N-C, H2N-CH2-, H2N-CS- or similar groups joined
together directly or through a carbon or nitrogen atom. One copper ion is probably
linked to 6 nearby peptide linkages by co-ordinate bonds. The intensity of the color is
directly proportional to the number of the peptide bonds present in the protein molecule
that is reacting and also the number of the protein molecules present in the reaction
system.
•Picture of the test: