PROPERTIES OF PROTEIN

Denaturation
y

y y y

Refers to any disruption in the secondary, tertiary or quaternary levels of protein structure. It does not cleave the peptide bonds, therefore the primary level of structure is not altered. (+) precipitation or coagulation Most significant consequences of denaturation loss of biological activity

Reaction with Heat

y y

Gentle heating causes reversible denaturation of protein Vigorous heating denatures protein irreversibly by disrupting several types of bonds. Observation (+) formation of white coagulate (+) formation of colorless coagulate (+) formation of white coagulate

Sample Albumin Gelatin Casein

Reaction with Heat

y y

Egg white, contains high percentage of protein, coagulates on heating Heat coagulates and destroys protein present in bacteria thus, sterilization of instruments and clothing use in operating rooms requires the use of high temperatures.

Reaction with Salts of Heavy Metals

y

Heavy metal salts, such as mercuric chloride (bichloride of mercury) or silver nitrate (lunar caustic), lead, precipitate protein. It also cleaves SH bonds. Denatured protein irreversibly by disrupting the salt bridges and the disulfide bonds present in the protein. Poisonous if taken internally because they coagulate and destroy protein present in the body.

Reaction with Salts of Heavy Metals

y

Egg White - Antidote for mercuric chloride or silver nitrate when these poisons are taken internally and is precipitate out.
Egg white colloid has a charge opposite to that of the heavy metal ion and so attracts it.

Sample Albumin Albumin

Reagent + AgNO3 + Hg2Cl2

Observation
(+) formation of white coagulate more (+) formation of white coagulate less

Reaction with Alkaloidal Reagents

y

Alkaloidal reagents, such as tannic acid and picric acid form insoluble compounds with proteins. Alkaloidal reagents denature protein irreversibly by disrupting salt bridges and hydrogen bond. Reagent
+ sat. picric acid + tannic acid + TCA

Sample
Albumin Albumin Albumin

Observation
Formation of yellow coagulate Formation of flesh coagulate Formation of milky white coagulate

Reaction with Alkaloidal Reagent

y

Tannic acid has been used extensively in the treatment of burns.

When applied to a burn area, it causes the protein to precipitate as a tough covering, thus reducing the amount of water loss from the area and reduces exposure to air.

Reaction with Concentrated Acids

y y

Changes in pH can disrupt hydrogen bonds and salt bridges, causing irreversible denaturation. Proteins are coagulated by strong acids as concentrated HCl, sulfuric and nitric acids. Reagent
+ HNO3 + excess HNO3 + HCl +excess HCl

Sample
Albumin

Observation
Formation of yellow coagulate Yellow coagulate disappears Formation of white coagulate White coagulate disappears

Albumin

Reaction with Concentrated Acids

y

Hellers Ring test

Used to detect the presence of albumin in urine A layer of concentrated nitric acid is carefully placed under a sample of urine in a test tube. If albumin is present, it will precipitate out as a white ring at the interface of the two liquids. If acid or base remains in contact with protein for a long period of time, the peptide bond will break.

Reaction with Alcohol

y y

y y

Alcohol coagulates (precipitates) all types of proteins except prolamines. Alcohol denatures protein by forming hydrogen bonds that compete with the naturally occurring hydrogen bonds in the proteins. Process is not reversible 70% alcohol used to disinfect because of its ability to coagulate the proteins present in the bacteria

Reaction with Alcohol

y

(+) formation of white coagulate

Reagent + 95% ethyl alcohol + 70% ethyl alcohol + 40% ethyl alcohol Observation Greatest amount of white coagulate Moderate amount of white coagulate Least amount of white coagulate

Sample Albumin Albumin Albumin

TEST FOR PROTEINS & AMINO ACID

BIURET TEST
y y

Solution contents:
KOH, CuSO4, Potassium sodium tartarate

Test for:
Peptide bonds. (Proteins and peptones give positive results)

Theory:
Following compound is formed between the C-N-C-back bone of the proteins and the copper(II) ions in the solution which results in a violet colour complex.

BIURET TEST

NINHYDRIN TEST
y y

Solution content:
Ninhydrin

Test for:
Amine group (mainly primary amine groups in proteins, peptones and amino acids)

Theory:
When reacting with these free amines, a deep blue or purple color known as Ruhemann's purple is produced.

NINHYDRIN TEST
y

Ruhemanns Purple
A blue-violet dye formed in the reaction of ninhydrin with amino acids Any primary (C-terminal) amino acid will undergo the Ist reaction and will produce a blue colour Secondary (N-terminal ) amino group containing amino acid, proline will give a yellow/orange colour (II).

NINHYDRIN TEST

XANTHOPROTEIC TEST
y y

Solution contents:
Concentrated HNO3

Test for:
Activated benzene rings (tyrosine and tryptophan)

Theory:
The aromatic groups in the amino acids can undergo nitration with nitric acid and give in yellow coloured products if the benzene ring is activated.

XANTHOPROTEIC TEST
y

Phenylalanine, though a derivative of benzene doesnt undergo nitration because its not activated like tryptophan and tyrosine. If you accidentally spill any nitric acid you may experience yellow stains because of this xanthoproteic reaction because the epidermal cells of the skin may contain proteins with aromatic amino acids.

XANTHOPROTEIC TEST

HOPKINS COLE TEST

y y y

Solution content:
Sodium nitroprusside, NH3(aq)

Test for:
Indol group. (Tryptophan)

Theory:
The indol group reacts with glycoxylic acid in conc. Sulfuric acid to form a purple colour / ring.

HOPKINS COLE TEST

MILLONS TEST
y y y

Solution contents:
Mercury (Hg22+, Hg2+) in nitric acid

Test for:
Phenol groups (Tyrosine)

Theory:
Firstly the phenol ring is nitrated with conc. nitric acid and the nitrated product will react with the mercury ions in the solution to give in either a deep red color solution or precipitate.

MILLONS TEST
y

[Hg2+](Some tyrosine containing proteins will give in a white precipitate [Hg22+] which would turn red when heated.

MILLONS TEST

TEST FOR SULFUR GROUP

y y y

Solution contents:
40% NaOH, Lead Acetate

Test for:
Sulfur. (Cysteine, Methionine)

Theory:
By boiling with NaOH, Sulfur in the amino acid is converted into NaS, which then precipitates as black PbS with the addition of lead acetate.

TEST FOR SULFUR GROUP

y

(+) Black ppt