Basics biochemistry 1

Practice 4
 ▪ The color reactions of proteins are associated
with their chemical composition.

▪ The abundance of functional groups within

the structure of proteins provides them with
Color reactions the ability to interact with a diverse range of
of proteins compounds.

▪ Color reactions serve as the foundation for

numerous techniques used to identify amino
acids and proteins.
 1. Biuret reaction

2. Lead sulfide reaction

Color reactions
of proteins
3. Ninhydrin reaction
 ▪ In a strongly alkaline environment, the peptide bonds in
the protein molecule react with the divalent copper ion
(Cu2+) to form a complex compound with a violet color. The
1. reaction is positive if the substance being analyzed has at
least 2 peptide bonds. This reaction is named after the
compound biuret (NH2C=ONHC=ONH2), which contains 2
peptide bonds and, like proteins, gives a positive reaction.
Biuret reaction
▪ Due to its high sensitivity, the biuret reaction is the most
Principle widely used method for quantitatively determining total
proteins in various biological materials. The intensity of the
violet color of the complex compound is proportional to the
number of present peptide bonds and the concentration of
total proteins.
 Biuret reaction

https://www.youtube.com/watch?v=ijxMMRljnjs
 ▪ Aqueous solution of protein
▪ Sodium hydroxide solution, NaOH (γ = 100g/L)
Reagents and
▪ Copper (II) sulfate, CuSO4 (γ = 10g/L)
equipment
▪ Test tubes, pipettes
 ▪ In a test tube, add
▪ 2 mL of the protein solution,
▪ 1-2 drops of CuSO4 solution,
▪ and 1 mL of NaOH solution.
Procedure ▪ Be careful not to add an excess amount of the
copper (II) sulfate solution, as the blue color
of this solution masks the violet color of the
complex formed, which is a positive sign for
the biuret reaction.
Positive Biuret
reaction
 ▪ The lead sulfide reaction is characteristic for
detecting the presence of amino acids that
contain sulfur (cysteine, methionine).
2.

Lead sulfide
reaction
Principle
▪ The sulfur contained in the amino acids is
bound in the form of sodium sulfide, which
then reacts with lead (II) acetate to form a
black precipitate of lead sulfide.
Lead sulfide
reaction
 ▪ Aqueous solution of protein;
▪ Solution of sodium hydroxide, NaOH (γ = 100g/L);
Reagents and
▪ Solution of lead (II) acetate, Pb(CH3COO)2 (γ = 5g/L);
equipment
▪ Test tubes, pipettes, spirit lamp.
 ▪ To 2 mL of protein solution,
▪ add about 1 mL of NaOH solution and
▪ several drops of Pb(CH3COO)2.
Procedure ▪ Heat the test tube until a black precipitate
appears.
Lead sulfide
reaction
 ▪ This reaction is used to detect all alpha-amino
acids, as well as some amides and amines.
Alpha-amino acids react with ninhydrin,
which is a strong oxidizing agent and causes
3. oxidative deamination and decarboxylation
of the amino acid. This reaction produces
ammonia, the corresponding aldehyde, and
Ninhydrin carbon dioxide, while ninhydrin is reduced to
reaction hydrindantin.
Principle ▪ The resulting ammonia, which actually comes
from the amino acid, reacts with ninhydrin
and hydrindantin to produce a complex
compound with a violet-blue color
(Ruhemann’s purple).
Ninhydrin
reaction

Ruhemann’s purple
 ▪ Solution of protein;
Reagents and ▪ Solution of ninhydrin in 95% acetone (γ = 1g/L);
equipment ▪ Test tubes, pipettes, spirit lamp.
 ▪ In a test tube,
▪ add 2 mL of protein solution and
▪ 2 mL of ninhydrin solution.
Procedure ▪ Heat for 2 minutes until a violet-blue color
appears.
Ninhydrin
reaction