Proteins (Lab) Flashcards
QUALITATIVE ANALYSIS TESTS OF PROTEINS (9)
Biuret Test
Millon’s Test
Hopkins-Cole Test
Reduces Sulfur Test
Ninhydrin Test
Xanthoproteic Test
Sakaguchi Test
Fohl’s Test
Pauly Test
Test for amines or α-amino acids/free
amino acids
NINHYDRIN TEST
NINHYDRIN TEST
When exposed to ninhydrin (C9H6O4), the amino acid undergoes oxidative deamination then releases?
CO2 and an aldehyde (while simultaneously transferring the amino group to ninhydrin)
NINHYDRIN TEST
The amino-containing ninhydrin then
forms a complex with another
ninhydrin molecule to form?
diketohydrin/Ruhemann’s complex (dark purple color)
Test for peptide bonds
BIURET TEST
Biuret test requires a peptide with at least how many peptide bonds?
2
All proteins should test ___ in the Biuret test
positive
Free amino acids yield ___ result in Biuret test.
negative
form a violet-colored chelate complex with peptide bonds in alkaline conditions (still biuret test)
Cu2+ ions
BIURET TEST
T/F - the greater the peptide
bonds, the greater the color (violet) intensity
T
This test is specific for sulfur-containing
amino acids
REDUCES SULFUR TEST
sulfur-containing amino acids (3)
cysteine
cystine
methionine
REDUCES SULFUR TEST
On boiling the sulfur-containing amino
acids, the sulfur is split off from these
amino acids as?
metallic sulfides.
REDUCES SULFUR TEST
By the addition of ____, they are oxidized to black-colored lead sulfides (PbS
lead acetate (Pb(C2H3O2)2)
Test for phenol groups/derivatives
MILLON’S TEST
phenol groups/derivatives in Millon’s test
tyrosine
(T/F) - Million’s test is not specific to proteins and will test positive for other phenol derivatives.
T
MILLON’S TEST
___ form a red colored complex with mercury
Hydroxybenzene radicals
The only amino acid with a
hydroxybenzene (phenol) group is
tyrosine (the phenol group is nitrated)
MILLON’S TEST
Upon the application of ___ the nitrated tyrosine form as a complex with mercury
heat
Test for aromatic amino acids
XANTHOPROTEIC TEST
aromatic amino acids (2)
tyrosine
tryptophan
XANTHOPROTEIC TEST
Phenylalanine yields a negative result
due to the ____ of its side chain
stability (Phenylalanine doesn’t react with nitric acid in the conditions of this test)
XANTHOPROTEIC TEST
Aromatic compounds react with ____ in a
nitration reaction which results to a yellow
color
HNO3 (Nitric acid)
XANTHOPROTEIC TEST
Upon the addition of a ___, the color
further ____ and turns almost orange-yellow.
base, intensifies
Test for indole groups
HOPKINS-COLE TEST
Indole group (1)
tryptophan
‘glyoxylic acid reaction’
HOPKINS-COLE TEST
HOPKINS-COLE TEST
In the presence of indole-containing
compounds like tryptophan, glyoxylic acid
undergoes a ____ reaction,
forming a purple-colored product.
condensation
HOPKINS-COLE TEST
hydrolyzes the protein and
liberates amino acids hence the
formation of a purple color at the
interface
H2SO4 (Sulfuric acid)
Test for guanidine, arginine
SAKAGUCHI TEST
SAKAGUCHI TEST
Under alkaline conditions,
the guanidine group of
arginine is able to react with
____ and ____, forming
a red complex.
napthol and NaOBr (Sodium hypobromite)
Test for sulfur
FOHL’S TEST
two cys residues bound by a disulfide bond
cysteine and cystine
does not yield a positive
result due to the inert nature of its sulfur
atom
Methionine (sulfur in the thioester bond in methionine is not released by the treatment with NaOH)
Reagents for Fohl’s test
Lead acetate and NaOH
DIFFERENCE OF REDUCES SULFUR TEST TO FOHL’S TEST
Methionine does not yield a positive result in Fohl’s test while in Reduces sulfur test it does
FOHL’S TEST
When heated in alkaline conditions, the sulfur
in sulfur-containing amino acids reacts with
____, resulting to a black precipitate in the
form of lead sulfide (PbS).
lead
Test for imidazole, histidine, and
phenol/hyroxybenzyl groups, tyrosine
PAULY TEST
PAULY TEST
contains an N≡N group)
diazo reagent
PAULY TEST
A diazo reagent (contains an N≡N group)
is first prepared through _____
with _____.
sulfanilic acid, NaNO2 (Sodium nitrite)
PAULY TEST
In alkaline conditions, the diazo
reagent will form a _____ with the imidazole or phenol
groups present in proteins.
red-colored
complex
COLOR REACTIONS FOR POSITIVE RESULTS IN DIFFERENT TESTS (9)
Biuret Test (Violet)
Millon’s Test (Red)
Hopkins-Cole Test (Purple)
Reduces Sulfur Test (Black)
Ninhydrin Test (Dark Purple)
Xanthoproteic Test (Yellow to Orange yellow)
Sakaguchi Test (Red)
Fohl’s Test (Black)
Pauly Test (Red)
COLOR REACTIONS
Biuret test (Violet)
Mix 2mL of egg albumin solution with 1mL of dilute NaOH and 10 drops of dil. CuSO4 solution in a test tube.
Shake well. Note the change in color.
COLOR REACTIONS
Millon’s test (Red)
To 2mL egg albumin, add 10 drops of Millon’s reagent.
Shake well and heat over water bath to boiling. Note the color change
COLOR REACTIONS
Hopkins-Cole test (Purple)
To 5 drops of egg albumin solution, add 5 drops of glyoxylic acid.
Carefully run down 5 drop of conc. H2SO4 on the side of the test tube.
Note the color formed at the point of contact between the two liquids
COLOR REACTIONS
Reduces Sulfur test (Black)
To 2mL of egg albumin solution in a test tube, add 1mL of 20%
NaOH and 10 drops of 10% Lead acetate solution.
Cover the test tube with a cork and boil in a water bath for two minutes. Observe color change and explain.
MODES OF PROTEIN PRECIPITATION (5)
- By heat
- By heavy metal salts
- By strong mineral acids
- By alkaloidal reagents
- By alcohol
PROTEIN PRECIPITATION BY HEAT
Reagents required:
1% acetic acid
PROTEIN PRECIPITATION BY HEAT
Reaction:
When the albumin solution is heated, the
white coagulum is obtained because
albumin is denatured by heat. After the addition
of 1 drop of 1% acetic acid, the coagulum
increases because the pH of the albumin
solution is shifted towards the isoelectric
point. At this pH solubility is minimum
and more protein is precipitated from its
solution
(T/F) - Albumin is a coagulable protein).
T
PROTEIN PRECIPITATION BY HEAVY METAL SALTS
Reagents Required:
Lead acetate (Pb(C2H3O2)2) and ferric chloride (FeCl3)
PROTEIN PRECIPITATION BY HEAVY
METAL SALTS
Reaction:
Proteins are precipitated from their solution by
heavy metal ions. These metal ions
precipitate the protein from their solution. On
the alkaline side of isoelectric pH, Protein
dissociates as a protein anion which
combines with a positive metal ion (cation) to
form an insoluble precipitate of metal
proteinates such as the lead albuminate and
silver albuminate.
PROTEIN PRECIPITATION BY STRONG
MINERAL ACIDS
Reagents required:
Concentrated Nitric acid
PROTEIN PRECIPITATION BY STRONG
MINERAL ACIDS
Reaction:
When native protein solution is
treated with Concentrated HNO3 (Nitric acid), a
white precipitate ring is obtained
due to denaturation of protein.
PROTEIN PRECIPITATION BY ALKALOIDAL REAGENTS
Reagents required:
Tannic acid (C76H52O46) and picric acid (C6H3N3O7)
PROTEIN PRECIPITATION BY
ALKALOIDAL REAGENTS
Reaction:
These organic acids exist as negative ions
when organic acids are added to albumin
solution proteins are precipitated from their
solution because, on the acidic side of
isoelectric pH, proteins dissociate as the
cation (protein +ion ) which combine with
anions (protein - ion ) of organic acids to
form a salt of protein
PROTEIN PRECIPITATION BY ALCOHOL
Reagents Required:
Absolute Alcohol
PROTEIN PRECIPITATION BY ALCOHOL
Reaction:
organic solvent such as alcohol, acetone, and
chloroform when added to albumin solution,
decreases the dielectric constant of solvent &
displaces some of the water molecules
(dehydration) associated with protein, and
decreases the concentration of water.
The mechanism of precipitation, in this case,
is dehydration, denaturation & removal of
charges.
PRECIPITATION OF PROTEIN
By heat:
Heat 5 drops of egg albumin solution to boiling. Observe if coagulation takes place. Add 1 drop of Acetic acid and note the effect.
PRECIPITATION OF PROTEIN
By heavy metal salts:
Prepare 2 clean test tubes and place 2mL of egg albumin to
each test tube. To the first test tube, add 2 drops of 5% Ferric Chloride and to the second test tube, add 1 drop of 10% Lead Acetate. Shake and observe.
PRECIPITATION OF PROTEIN
By strong mineral acids:
To two clean test tubes, place 2mL egg albumin solution.
Using a pipette, add 1 drop of conc. Nitric acid to the first tube allowing the acid to slide down the side of the test tube. To the second test tube, add similarly, 1 drop conc. Sulfuric acid. Note the color change at the junction of the protein-acid layer.
PRECIPITATION OF PROTEIN
By alkaloidal reagents:
To 2mL of egg albumin, add 2 drops of Tannic acid in a
test tube. Shake well. Repeat the test using 2 drops of Picric acid in place of Tannic acid. Compare the results.
PRECIPITATION OF PROTEIN
By alcohol:
Place 2mL of egg albumin in a test tube. Add 1 or 2 drops of Acetic acid and 2mL of 95% Ethyl alcohol. Shake and note the precipitate formed.
EGG ALBUMIN (6)
- Albumin is a simple protein.
- Albumin is denatured by strong mineral acid.
- Albumin is precipitated by heavy metal ions.
- Albumin is precipitated by organic acids.
- Albumin is precipitated by organic solvents (alcohol).
- Albumin is coagulated by heat
Biuret test: Samples, Reagents, Observations
2mL of egg albumin solution
1 ml NaOH
violet-colored solution
Hopkin’s-Cole test: Samples, Reagents, Observations
5 drops of egg albumin solution
5 drops of glyoxylic acid & 5 drops of conc. H2SO4 (Sulfuric acid)
formation of a purple-colored ring at the
junction of two layers.
Millon’s test: Samples, Reagents, Observations
2mL egg albumin
10 drops of Millon’s reagent
any protein containing tyrosine will give a positive test of a pink to dark red color.
Reduces Sulfur test: Samples, Reagents, Observations
2mL of egg albumin solution
1mL of 20% NaOH & 10 drops of 10% Lead acetate solution
Explain why egg whites and milk are used as antidotes for heavy metal poisoning
to precipitate the poisonous metal (from lead or mercury poisoning)
The metals are attracted to the disulfide bonds in proteins. The reaction of the heavy metal and the protein will usually lead to an insoluble metal protein salt. Once this reaction occurs, the metal salt becomes concentrated in the stomach and can be discharged from the body (vomitting)
Do free amino acids give a positive result with the biuret test?
No. A single amino acid and the biuret test works for any peptide or protein that contains two or more peptide bonds.
Because biuret is a test for peptide bonds, minimum of two peptide bonds/3 AAs
After heating albumin at a high temperature, is it still biologically active?
No. The heat causes denaturation, hardening the albumen
and coloring it opaque white. The changes in viscosity and color reflect the denaturation of the egg albumin
protein. Thus, denaturation of the protein leads to the inactivity or
dysfunctionality of the albumin.
Why is silver nitrate used in the
cauterization of a wound?
Silver nitrate is an inorganic chemical with antimicrobial properties and is available as a solution or an
applicator stick. It has been used as a cauterizing agent by delivering free silver ions which bind to tissue, forming an eschar and obstructing vessels.
Why is it important that the chromatography paper not
touch the sides of the beaker?
It can make the solvent front run crookedly
