BIOCHEMLAB 1-2

• Color reaction tests are a qualitative approach to identifying amino acids in intact proteins and acid/base hydrolyzates
• Five color reaction tests are used to reveal functional groups or chemical compounds attached to amino acids in both intact and acid/base hydrolyzates
• Amino acids consist of an amino group, carboxyl group, a hydrogen, and an R group attached to the alpha-carbon
• Revealing the R groups and peptide bonds present in the sample aids in identifying the possible amino acid present
• The aim is to identify the amino acids present rather than quantifying their composition in both intact proteins and acid/base hydrolyzates
• Materials needed for the experiment include protein suspension, acid and base hydrolyzate, and specific reagents for each test
• Biuret test:

  • Purpose: Detect proteins and peptide bonds
  • Reagents: NaOH and CuSO4
  • Principle: Cu2+ binds to protein peptide nitrogen atoms, yielding a deep purple color if peptide bonds are present
• Sakaguchi test:

  • Purpose: Detect arginine and guanidinium groups
  • Reagents: NaOH, NaOCl, and Napthol Solution
  • Principle: Guanidin group reacts with napthol to produce a red-colored complex product, indicating the presence of arginine or guanidium compound
• Ninhydrin test:

  • Purpose: Detect ammonia, primary or secondary amines, or free-alpha amino groups
  • Reagents: Water and Ninhydrin solution
  • Principle: Leads to decarboxylation and deamination, resulting in a deep purple color if ammonia, amines, or amino acids are present
• Xanthoproteic test:

  • Purpose: Detect amino acids containing phenolic or indolic groups
  • Reagents: Conc. HNO3 and NaOH
  • Principle: Detects aromatic amino acids, giving yellow color nitro derivatives on heating with Conc. HNO3
• Hopkins-Cole test:

  • Purpose: Detect indole rings and tryptophan in proteins
  • Reagents: 2H2O3 (Hopkins-Cole Reagent) and Conc. H2SO4
  • Principle: Detects tryptophan residues in proteins, forming a colored compound
• The presence of tryptophan residues in proteins can be detected by the formation of a colored compound
• In the Hopkins-Cole Test, the indole group of tryptophan reacts with glyoxylic acid in the presence of concentrated H2SO4 to give a purple color
• The positive result in the Hopkins-Cole Test is represented by the formation of a purple-colored ring at the junction of two layers, indicating the presence of tryptophan-containing proteins
• In the Biuret test, the presence of proteins and peptide bonds is detected
• In the Ninhydrin test, ammonia, primary or secondary amines, or free alpha-amino groups present in a compound can be detected
• The Xanthoproteic test detects aromatic amino acids like tyrosine and tryptophan
• The principle of the Ninhydrin test involves decarboxylation and deamination
• The principle of the Xanthoproteic test involves nitration, resulting in a yellow precipitate
• The reagents of the Sakaguchi Test include NaOH, NaOCl, and Napthol
• The reagents of the Ninhydrin test include Ninhydrin solution and water for protein suspension in intact protein
• The reagents of the Hopkins-Cole Test include concentrated H2SO4 and C2H2O3
• The Hopkins-Cole Test detects the presence of tryptophan by detecting the indole group
• The positive result in the Ninhydrin test is a deep purple color for acid and base hydrolyzates
• The positive result in the Xanthoproteic test is a yellow precipitate for intact protein and base hydrolyzate
• The intact protein and acid hydrolyzate give a positive result in the Sakaguchi Test
• Only the protein intact can give a positive result in the Biuret test because it is the only one that can present the peptide bonds
• Dipeptides do not give a positive result in the Biuret test
• The principle behind the Biuret test is complexation, resulting in a deep purple complex
• Proline is considered negative in the Ninhydrin test
• The product produced in the Ninhydrin test is called Ruhemann’s complex
• The minimum number of peptides present should be at least two for a positive result in the Biuret test
• In the base hydrolysis, arginine, cysteine, threonine, and serine are destroyed
• In the acid hydrolysis, tryptophan gets destroyed, resulting in black coloration
• The positive result in the Biuret test is a purple color
• The principle of the Xanthoproteic test is nitration
• The principle of the Hopkins-Cole Test involves dehydration and condensation
• The intact protein and base hydrolyzate give a positive result in the Hopkins-Cole Test
• The intact protein and acid hydrolyzate should have a positive result in the Sakaguchi Test
• The intact protein and base hydrolyzate give a positive result in the Ninhydrin test