Qualitative Tests for Proteins and Amino Acids

Qualitative Tests for Proteins/Amino Acids

• Proteins are made up of amino acids arranged in a linear chain.
• They are an essential part of living organisms and participate in various cellular processes.
• Amino acids are the monomer units of proteins.
• They contain an amino group, a carboxyl group, and a side chain.
• The specific pH at which an amino acid has no overall charge is known as its isoelectric point.
• At this point, they are known as zwitterions.
• Nonpolar amino acids have hydrocarbons (C and H atoms).

Biuret Test

• It positively identifies the presence of proteins and peptide bonds.
• It requires at least two peptides to give a positive reaction.
• The test involves the formation of a complex between proteins and Cu²⁺ ions in a strongly alkaline solution.
• While the test is named after Biuret (a chemical derived from urea), the reagent used does not contain Biuret.
• A purplish violet color indicates a positive test.
• A blue color indicates a negative test.

Ninhydrin Test

• Detects the presence of amines or α-amino acids.
• A deep blue color indicates a positive test.
• A purple color indicates the presence of ammonia, primary/secondary amines, or amino acids.
• A yellow color indicates the presence of hydroxyproline and proline.
• A brown color indicates the presence of asparagine.

Xanthoproteic Test

• A qualitative test that determines the amount of protein in a solution using nitric acid.
• It is named after xanthoproteic acid, a yellow compound produced in the reaction, derived from the Greek word "xanthos" meaning yellow.
• A yellow to orange colour is a positive result.
• No color change indicates a negative result.

Millon's Test

• Specific for phenol-containing structures (Tyrosine).
• The reagent contains concentrated HNO₃ with dissolved mercury.
• A brick red colored solution or precipitate indicates a positive test.
• A yellow precipitate of Mercuric (II) oxide (HgO) might indicate the solution is too alkaline.
• No red color indicates a negative result.

Sakaguchi Test

• Based on the reaction of 1-naphthol with guanidinium groups on arginine in the presence of an oxidizing agent.
• The exact mechanism is unknown, but a red-colored complex is formed due to an indole-like structure.
• The Sakaguchi reagent includes sodium hypobromite and 1-naphthol.
• Sodium hypobromite acts as an oxidizing agent.
• A strong red color indicates a positive test and the presence of arginine or guanidinium compounds.
• The absence of red color indicates a negative test and the absence of arginine or guanidinium compounds.

Hopkins-Cole Test

• Determines the presence of tryptophan in proteins based on its heterocyclic side chain (indole group).
• A purple colored ring at the interface indicates a positive test.
• No purple colored ring indicates a negative test.

Lead Acetate Test

• Also known as the Lead Sulfide Test, it detects amino acids like cysteine and cystine.
• It specifically detects sulfur-containing amino acids (S-S (sulfide) group in cysteine and S-H (thiol) group in cystine).
• Methionine does not give a positive result in this test.
• A black precipitate at the bottom of the test tube indicates a positive test and the presence of cysteine or cystine.
• The absence of black residue indicates a negative test and the absence of cysteine or cystine.