Experiment 3 - Properties of Proteins

Protein

comes from the Greek word “proteios”

Proteios

“of first importance”

Protein

they play a lot of important physiological functions

Protein

It accounts for about 15% of total cell’s mass making it most abundant macromolecule in the body

a-amino acids

Proteins are naturally occurring long chain of ________

a-amino acids

only considered a protein when it contains at least 50 amino acid residues

Peptide

contains at least 50 amino acid residues, otherwise it is known as ________

R-group

also known as the side chain, gives an amino acid its identity

Standard Amino Acids

There are 20 common amino acids found in human proteins

• Non-polar amino acids

• Polar neutral amino acids

• Polar acidic amino acids

• Polar basic amino acids

Standard Amino Acids classified according to the polarity of side chains:

Color reactions

The Standard Amino Acids contain different functional groups within them, which are differentiated from each other in the laboratory using specific chemical tests, known as ________

Peptide bond

Amino acids in peptides and proteins are connected to each other via

Peptide bond

formed when the carboxyl group of one amino acid reacts with the amino group of another amino acid

Native conformation

Proteins assume a specific three-dimensional structure, known as their

Albumin

Based on its native conformation, a protein may be globular (spherical in shape) like

Gelatin

Based on its native conformation, a protein may be fibrous (elongated) like

Denaturation

A change in a protein’s native conformation will alter a lot of its properties and will have a huge impact in its function. This change is known as ________

• Coagulation of Proteins

• Precipitation of Proteins

I. DENATURATION OF PROTEINS:

Denaturation

refers to the disruption and unfolding of protein, thus, losing its biological activity

Denaturation

Its common manifestation is when a protein loses solubility in water

Denaturation

can be reversible or irreversible

Irreversible denaturation

results to either precipitation or coagulation

Precipitation

sticking of molecules together to form a solid that will come out from the solution

Coagulation

clumping of particles together in a solution

Coagulum

Converts it from a transparent liquid into a white solid that clumps together

• Heavy Metal Salts

• Strong Mineral Acids

• Alkaloidal Reagents

Precipitation of Proteins by:

Heavy metals

metals with relatively high densities, atomic weights, or atomic numbers

• Iron

• Cobalt

• Zinc

Essential nutrients:

• Ruthenium

• Silver

• Indium

Relatively harmless:

• Cadmium

• Mercury

• Lead

Highly poisonous:

Mineral acid (inorganic acid)

acid derived from one or more inorganic compounds

Mineral acid (inorganic acid)

dissociate in water to form hydrogen ions and conjugate base

• Hydrochloric acid

• Nitric acid

• Sulfuric acid

Common example of mineral acids:

Alkaloidal reagents

high molecular weight anions

Alkaloidal reagents

the negative charge of these anions counteracts the positive charge of the amino group of proteins giving a precipitate

Alkaloidal reagents

Most of these reagents are astringent

Astringent

causes constriction or shrinking of body tissues

• Biuret Test for Peptide Bonds

• Ninhydrin Test for Free Alpha-Amino Group

• Xanthoproteic Test for Phenyl-Containing Amino Acids

• Adamkiewicz Reaction for Tryptophan

• Reduced Sulfur Test for Cysteine

• Molisch’s Test for Presence of Carbohydrate Moiety

• Modified Neuman’s Test for Phosphate Moiety

II. COLOR REACTION OF PROTEINS:

Biuret Test

also known as Piotrowski’s test

Biuret Test

a general test for the presence of proteins which is dependent on the presence of peptide bonds

Violet color

Cupric ions (Cu2+) in alkaline medium form a complex with the nitrogen of the peptide bonds which has a ________

Violet color

Positive color for Biuret Test

Biuret Test

Sometimes, the color result is pink or pale violet

Directly proportional

The intensity of the violet color is ________ to the amount of peptide bonds present

• Peptides

• Proteoses

• Peptones

Biuret test will also be answered by other compounds containing peptide bonds, such as _________

2 peptide bonds

A minimum of ________ is required to give a positive result individual amino acids will not answer this test

Ninhydrin Test

also a general test for the presence of proteins which is dependent on the presence of free alpha-amino groups

Oxidative deamination

The free alpha-amino groups of proteins and amino acids undergo an _______ with ninhydrin (triketohydrindene hydrate), resulting in the liberation of carbon dioxide (CO₂), ammonia (NH₃), and an aldehyde along with hydrindantin (reduced form of ninhydrin)

Diketohydrin

The liberated NH3 goes on to react with another ninhydrin molecule to form ________, a colored complex

Ruhemann’s purple

The liberated NH3 goes on to react with another ninhydrin molecule to form diketohydrin, a colored complex, which is also known as

Diketohydrin

has a characteristic deep blue or violet color

Deep blue or violet color

Positive color for Ninhydrin Test

Yellow color

Proline will give a _______ with ninhydrin

Brown color

Amino acids with amide group, asparagine, and glutamine, will give a _______

Xanthoproteic Test

used to indicate the presence of amino acids containing phenyl (benzene) ring in proteins

• Phenylalanine

• Tyrosine

• Tryptophan

Xanthoproteic Test used to indicate the presence of amino acids containing phenyl (benzene) ring in proteins, namely _______

Concentrated nitric acid

The ring systems of the mentioned amino acids undergo nitration on treatment with ________ when heated

Xanthoproteic Test

The end-product is yellow in color, which is intensified in strong alkaline medium

Yellow to Orange color

Positive color for Xanthoproteic Test

Adamkiewicz Reaction

a specific test for the presence of the amino acid tryptophan

Indole group

_______ of tryptophan reacts with glyoxylic acid in the presence of concentrated sulfuric acid (H₂SO₄) to give a purple-colored complex

Glacial acetic acid

which has been exposed to the sunlight also contains glyoxylic acid

Formation of violet ring

Positive test for Adamkiewicz Reaction

Reduced Sulfur Test

a specific test for the presence of the amino acid cysteine

Lead sulfide as a black ppt

When cysteine or cysteine-containing proteins are boiled with strong alkali, organic sulfur splits and forms sodium sulfide, which on addition of lead acetate produces _________

Formation of Black precipitate

Positive test for Reduced Sulfur Test

Simple proteins

contain only amino acid components

Conjugated proteins

contain amino acids and non-amino acid groups

Prosthetic groups

contain amino acids and non-amino acid groups called ______, such as carbohydrates and phosphate

Molisch’s Test

general qualitative test for the presence of carbohydrates in solution

Molisch’s Test

identify if the protein is a conjugated protein having carbohydrate as prosthetic group

Glycoproteins

a protein with carbohydrate prosthetic group

Also Formation of Positive Ring

Positive test for Molisch’s Test

Phosphoprotein

A conjugated protein containing a phosphate group

Ammonium molybdate

added which reacts with phosphate in the presence of nitric acid

Canary yellow precipitate of ammonium phosphomolybdate

Ammonium molybdate is then added which reacts with phosphate in the presence of nitric acid to form a _______