Proteins, Peptides and Amino Acids: Lab Experiments
Test
Biuret Test
Reagents
CuSO4 sol'n, diluted
NaOH
Test for
Intact proteins and protein hydrlysates (at least tripeptide, 2 peptide bonds
Result
Pink to violet blue color Ninhyrin Test
1,2,3-indanetrione monohydrate or triketohydintene hydrate, ethanol
Alpha- amino group
(usually a general test for amino acids)
Xanthoproteic
Test
Conc. HNO3, conc.
NaOH
For W,F,Y (aromatic except for H)
Blue to blue-violet
Oxidative decarboxylation color & deamination followed by
(proline:hydroxypr condensation oline gives a yellow color) Yellow sol'n/ppt.
Nitration via SEAr with conc. HNO3, orange with excess
NaOH
Millon's rgt.(Hg2+ &
Hg22+nitrates&
nitriles/Hg(NO3)2 in conc. HNO3, w/ trace of HNO2/HgSO4,H2SO4
,NaNO2)
Hopkins-Cole Test Glyoxylic acid (Mg powder, oxalic acid,
HOAc), conc. H2SO4
Millon's Test
Sakaguchi Test
Fohl's Test
Nitroprusside Test
Test for Amide
Pauly's Test
Alpha-naphthol,
NaOBr, NaOH, urea(to stabilize color & destroy excess OBr ions)
Pb(CH3COO)2
NaOH
Na2Fe(CN)3 in dil.
NH3
NaOH
Diazo rgt. (1% sulfosalicylic acid with 5% NaNO2),
10% Na2CO3
Reaction/Principle
Complexation
Form. Of coordination complex Cu+2 and 4 N atoms (2 from each 2 polypeptide chain)
Phenolic group of Y
Old rose/flesh to red ppt.
Complexation(mercuration
& nitration or nitrosation/complexation of nitrohydroxyphenyl derivatives with Hg2+)
Indole group in W
Pink to violet interface Reduction of oxalic acid to glyoxolic acid and acidcatalyzed condensation of two tryptphans w/glyoxolic acid Un-or monosubstituted guanidines in R
,
Red to red-orange Complexation (basecolor catalyzed condensation of alpaha-naphthol w/ the guanidine group of R)
Sulfur-containing
C-C)
Brown to black ppt.
Degradation & substitution rxn to form PbS
For cys
Red coloration
Complexation rxn
10,20,30 amides and nitriles ; peptide bonds
Detects tyr
Biuret Test
Reagents
CuSO4 sol'n, diluted
NaOH
Test for
Intact proteins and protein hydrlysates (at least tripeptide, 2 peptide bonds
Result
Pink to violet blue color Ninhyrin Test
1,2,3-indanetrione monohydrate or triketohydintene hydrate, ethanol
Alpha- amino group
(usually a general test for amino acids)
Xanthoproteic
Test
Conc. HNO3, conc.
NaOH
For W,F,Y (aromatic except for H)
Blue to blue-violet
Oxidative decarboxylation color & deamination followed by
(proline:hydroxypr condensation oline gives a yellow color) Yellow sol'n/ppt.
Nitration via SEAr with conc. HNO3, orange with excess
NaOH
Millon's rgt.(Hg2+ &
Hg22+nitrates&
nitriles/Hg(NO3)2 in conc. HNO3, w/ trace of HNO2/HgSO4,H2SO4
,NaNO2)
Hopkins-Cole Test Glyoxylic acid (Mg powder, oxalic acid,
HOAc), conc. H2SO4
Millon's Test
Sakaguchi Test
Fohl's Test
Nitroprusside Test
Test for Amide
Pauly's Test
Alpha-naphthol,
NaOBr, NaOH, urea(to stabilize color & destroy excess OBr ions)
Pb(CH3COO)2
NaOH
Na2Fe(CN)3 in dil.
NH3
NaOH
Diazo rgt. (1% sulfosalicylic acid with 5% NaNO2),
10% Na2CO3
Reaction/Principle
Complexation
Form. Of coordination complex Cu+2 and 4 N atoms (2 from each 2 polypeptide chain)
Phenolic group of Y
Old rose/flesh to red ppt.
Complexation(mercuration
& nitration or nitrosation/complexation of nitrohydroxyphenyl derivatives with Hg2+)
Indole group in W
Pink to violet interface Reduction of oxalic acid to glyoxolic acid and acidcatalyzed condensation of two tryptphans w/glyoxolic acid Un-or monosubstituted guanidines in R
,
Red to red-orange Complexation (basecolor catalyzed condensation of alpaha-naphthol w/ the guanidine group of R)
Sulfur-containing
C-C)
Brown to black ppt.
Degradation & substitution rxn to form PbS
For cys
Red coloration
Complexation rxn
10,20,30 amides and nitriles ; peptide bonds
Detects tyr