Experiment 3: Analysis of Proteins

DENATURED

any change that alters the unique three-dimensional configuration of protein molecules without causing a concomitant cleavage of peptide bonds.

PROTEINS

one of the major constituents of living cells.

Macromolecules

that contain amino acids (as building blocks joined together by peptide bonds).

Functions of Proteins

Denatured proteins aggregate and become visible as a precipitate, catalyze biochemical reactions, regulate the activity of various organs in the body, counteract the adverse effects of antigens, transport molecular oxygen, and serve as structural materials of the muscle, skin, and hair.

Denaturation

Collectively refers to reactions such as salting out and heat coagulation due to the colloidal nature of proteins.

Color Tests

Different color tests are used to identify the amino acid or proteins due to the presence of specific chemical groups in the protein molecule.

Denaturation Reaction

Occurs due to the presence of specific chemical reagents such as strong mineral acid, salt of heavy metal, or alkaloidal reagents.

a-helix

The structural pattern of polypeptides at the secondary level maintained by weak hydrogen bonds between amino acids.

Tertiary Structure Stabilization

The linkages responsible for the tertiary structure of protein are a function of the nature of amino acid side chains within the molecule.

Bonds Stabilizing Tertiary Structure

Include salt linkages, hydrogen bonds, disulfide linkages, and hydrophobic interactions.

Denatured Proteins

Proteins that have undergone disruption of secondary and tertiary structure due to various physical and chemical methods.

BIURET TEST

A protein detection test that gives a characteristic purple color, positive for all compounds that contain two or more peptide bonds.

NINHYDRIN TEST

A test to detect and identify the presence of amino acids and amines, forming a blue violet colored complex when heated with amino acids.

XANTHROPROTEIC TEST

A test positive for those amino acids that contain a benzene ring or aromatic ring, involving nitration of the amino benzene ring with concentrated nitric acid.

Positive for Biuret Test

All compounds that contain two or more peptide bonds.

Positive Except for Biuret Test

Threonine and serine.

Positive for Ninhydrin Test

All proteins or protein derivatives, as well as ammonia and amines.

Positive for Xanthoproteic Test

Amino acids that contain a benzene ring or aromatic ring.

Color for Ninhydrin Test

Blue violet for most amino acids, yellow for proline and hydroxyproline.

Color for Xanthoproteic Test

Yellow orange derivatives of nitrobenzene.

Coordination in Biuret Test

Due to the coordination of complex formed by cupric ions (in the reagent) and the amino groups.

A-amino group

Responsible for the positive results in the Ninhydrin Test.

Chemical Reagents for Denaturation

Strong mineral acid, salt of heavy metal, or alkaloidal reagents.

Heat Coagulation

Many proteins are coagulated by heat, strong acids, and alcohol.

HOPKIN'S COLE TEST

This test is due to the presence of the indole ring of the tryptophan.

Violet colored complex

Formed when glyoxylic acid condenses with indole derivatives in the presence of strong acids like sulfuric acid.

MILLON'S TEST

Test for the detection of tyrosine-containing proteins in a given sample.

White precipitate

Produced in the MILLON'S TEST which becomes brick red upon prolonged heating.

Phenolic groups

These groups are easily nitrated by a solution of mercuric and mercurous nitrates and nitrites in concentrated nitric acid.

Mercury complex of nitrophenyl derivatives

The color produced in MILLON'S TEST is due to this complex.

SAKAGUCHI TEST

Test for arginine-containing proteins in a given sample.

Red to an orange colored complex

Formed when compounds containing guanidine or guanido group combine with a-naphthol and sodium hypobromite or hypochlorite.

LEAD ACETATE TEST

Detect the presence of sulfur-containing amino acids, particularly cysteine and cystine, in proteins.

Brownish-black precipitate of lead sulfide, Pbs

Formed when inorganic sulphide reacts with lead acetate in LEAD ACETATE TEST.

BIURET TEST

Test that indicates the presence of proteins.

NINHYDRIN TEST

Test that detects free amino groups in proteins.

XANTHROPROTEIC TEST

Test that detects aromatic amino acids by nitration.

Albumin

Contains proteins and aromatic amino acids like tyrosine and tryptophan.

Gelatin

Lacks or has very low levels of aromatic amino acids.

Tyrosine

Has a phenol ring that is easily nitrated.

Phenylalanine

Benzene ring is less reactive than tyrosine; weak or no reaction.

Cysteine and cystine

Sulfur-containing amino acids detected by LEAD ACETATE TEST.

Urea

Has amino groups, but they are involved in a stable structure and do not react strongly with ninhydrin.

Phenol

Directly reacts with Millon's reagent.

Ammonium Water

Has ammonia/amines; result is blue rather than purple.

Trypthopan

Direct source of tryptophan; strong positive reaction in HOPKIN'S COLE TEST.

Biuret test

A test that detects proteins and produces a violet/purple color when proteins are present.

Ninhydrin test

A test that detects amino acids and produces a blue-violet color.

Xanthroproteic reaction

The nitration of aromatic amino acids like tyrosine.

Lead acetate test

A test that reacts with sulfhydryl groups (-SH) in albumin, forming lead sulfide (PbS), which is insoluble.

Mercuric chloride test

A test that reacts with thiol groups (-SH) of albumin, forming mercury salts that precipitate out of solution.

Silver nitrate test

A test that reacts with sulfide groups in albumin, forming silver sulfide (Ag₂S), which is a black precipitate.

Picric acid test

A test that precipitates albumin by forming complexes with the protein, resulting in a yellow precipitate.

Tannic acid test

A test that reacts with albumin to form an insoluble complex, resulting in a brown precipitate.

Sterilization by heat

A method that denatures proteins and disrupts hydrogen bonds, effective against microorganisms.

Disinfection by alcohol

A method that denatures proteins and disrupts hydrogen bonds, effective against microorganisms.

Heavy metal poisoning antidote

Egg white and milk are used as they bind to heavy metals (Chelation) and prevent toxicity.

Ethanol effect on albumin

Ethanol causes albumin to denature and coagulate by disrupting hydrophobic interactions and hydrogen bonds.

HCl effect on albumin

HCl denatures albumin by disrupting ionic and hydrogen bonds, causing it to coagulate.

Sulfur test colored precipitate

The formula of the colored precipitate obtained in the sulfur test or lead acetate test is PbS (lead sulfide).

Albumin + distilled water + Biuret reagent

Produces a violet/purple color indicating the presence of proteins.

Albumin + HCl

Results in a white precipitate due to denaturation of albumin.

Albumin + ethanol

Results in a white precipitate due to denaturation of albumin.

Gelatin + ethanol

Results in little or no visible change as gelatin is less structured and more heat-stable.

Albumin + picric acid

Results in a yellow precipitate indicating the presence of albumin.

Albumin + tannic acid

Results in a brown precipitate indicating the presence of albumin.

Albumin + lead acetate

Results in a white precipitate indicating the presence of lead sulfide (PbS).

Albumin + mercuric chloride

Results in a white precipitate indicating the formation of mercury salts.

Albumin + silver nitrate

Results in a white precipitate indicating the formation of silver sulfide (Ag₂S).