BIOCHEM LAB - Experiment 2: Properties of Proteins

Proteins

Comes from the Greek “Proteios”; they play a lot of important physiological functions. It accounts for about 15% of total cell’s making it the most abundant macromolecule in the body; naturally occurring long chain of amino acids

Of first importance

Meaning of Proteios

a-amino acids

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

50 amino acids

How many amino acids does a-amino acids have?

Peptide

What do you call an a-amino acid that has less than 50 amino acids?

R-group

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

20 amino acids

How many common amino acids are found in human proteins

Standard Amino Acid

There are 20 common amino acids found in human protein, known as____; they are usually classified according to the polarity of the side chains

Non-polar amino acids, polar neutral amino acids, polar acidic amino acids, and polar basic amino acids

What are the polarities of the side chains? there are 4

Color Reactions

The standard amino acids contain different functional groups within them, which are differentiated form each other in the laboratory using specific chemical tests, known as?

Peptide Bond

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?

Globular & Albumin

Spherical in shape

Fibrous & Gelatin

Elongated

Denaturation

A change in a protein’s native conformation; refers to the disruption and unfolding of protein, thus, losing its biological activity

Protein loses solubility in water

The most common manifestation in the denaturation of protein

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

Coagulation of proteins

is a common observation; For example, cooking egg white converts it from a transparent liquid into a white solid that clumps together

Coagulum

It means to clump together

Precipitation of proteins

Very important in the laboratory because this is usually employed when isolating proteins from a tissue samples.

Heavy metals

Defined as metals with relatively high densities, atomic weights, or atomic number

Iron, Cobalt, and Zinc

Essential Nutrients heavy metals

Ruthenium, Silver, and Indium

Relatively harmless heavy metals

Cadmium, Mercury, and Lead

Other heavy metals

Mineral Acid (Inorganic Acid)

An acid derived from one or more inorganic compounds; dissociates in water to form hydrogen ions and conjugate base

Hydrochloric Acid (HCl), Nitric Acid (HNO3), and Sulfuric Acid (HSO3)

Common examples of precipitation of proteins by strong mineral acids, there are 3

Heller’s Test & Robert’s Test

Types of test in precipitation of Proteins by strong mineral acids

Alkaloidal Reagents

Are high molecular weight anions. The negative charge of these anions contracts the positive charge of amino groups of proteins giving a precipitate.

Astringent (Alkaloidal Reagent)

Constriction or shrinking of body tissues

Color Reaction

Used to transform colorless chemical compounds into colored derivatives which can be detected visually

Color Reaction of Proteins

Characteristics of the functional group in amino acid is present

Biuret Test

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

Piotrowski’s Test

Other term for Biuret Test

Cupric ions (Cu2+) in Alkaline Medium

Form a complex with the nitrogen of the peptide bonds which has a violet color

Blue Color

Color of Cupric ion

Violet Color

Color of nitrogen of the peptide bonds

Positive test for Biuret Test

Change from the original blue color of the reagent to violet color, sometimes the color result is pink or pale violet

Intensity of the violet color in Biuret test

Directly proportional to the amount of peptide bonds present

Peptides, Proteose, Peptones

Other compounds containing peptide bonds

2 peptide bonds

Required to give a positive result

Individual amino acid

Will not answer the test for Biuret test

Ninhydrin Test

General test for the presence of proteins which is dependent on the presence of free alpha-amino groups

Free-alpha amino groups of proteins and amino acids

Undergo an oxidative deamination with ninhydrin resulting the liberation of carbon dioxide (CO2), ammonia (NH3), and an aldehyde along with hydrindantin

Hydrindantin

Reduced form of ninhydrin

Triketohydrindene hydrate

Other term for ninhydrin

C9H6O4

Formula of triketohydrindene hydrate

Liberated NH3

React with another ninhydrin molecule to form diketohydrin

Diketohydrin

A colored complex which is also known as Ruhemann’s Purple

Deep blue of violet color

Color of diketohydrin or Ruhemann’s purple

Positive test for Ninhydrin Test

Development of a deep blue color or violet color

Proline

Will give a yellow color with ninhydrin

Amino acids with amide group, asparagine, and glutamine

Brown color with ninhydrin

Xanthoproteic Test

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

Phenylalanine, Tyrosine, Tryptophan

Examples of phenyl (benzene) ring in proteins

Nitration, Concentrated nitric acid

The ring systems of phenylalanine, tyrosine, and tryptophan undergo _______ on treatment with _________ when heated

Yellow in color

End product of amino acids undergoing nitration on treatment with concentrated nitric acid when heated; which is intensified in strong alkaline medium

Positive result for Xanthoproteic Test

Formation of yellow to orange color

Adamkiewicz Reaction

Specific test for the presence of tryptophan

Indole group of tryptophan

Reacts with glyoxylic acid in the presence of in the presence of concentrated sulfuric acid (H2SO4)

C2H2O3

Formula of glyoxylic acid

H2SO4

Formula of concentrated sulfuric acid

Purple-colored complex

What is the color of the result when the indole group of tryptophan reacts with glyoxylic acid and concentrated sulfuric acid

Positive result for Adamkiewicz Reaction

Violet Ring (same as Molisch's Test)

Glacial Acetic Acid

Has been exposed to the sunlight also contains glyoxylic acid and can thus be used for Adamkiewicz test

Reduced Sulfur Test

Specific test for the presence of cysteine

Organic Sulfur Splits and form sodium sulfide

When cysteine or cysteine-containing proteins are boiled with alkali________________________

Lead Sulfide

Sodium sulfide which in addition of lead acetate produces________

Black Precipitate

Color of Lead Sulfide Precipitate

Methionine

Does not answer the Reduced Sulfur Test

Sulfur in methionine

It is in the thioether linkage which is difficult to break

Simple Proteins

Contain only amino acid compounds

Conjugated Proteins

Contain amino acids and non amino acid groups

Prosthetic Group

amino acid and non-amino acid group (such as carbohydrate and phosphate)

Molisch’s Test

General qualitative test for the presence of carbohydrates in solution; to identify if the protein is a conjugated protein having carbohydrate as prosthetic group; test for carbohydrate moiety.

Glycoproteins

A protein with carbohydrate prosthetic group

Phosphoprotein

A conjugated protein containing a phosphate group

the protein solution is heated with NaOH which releases phosphate

To test if a protein contains phosphate_____

Ammonium Molybdate

Reacts with phosphate in the presence of nitric acid

Canary Yellow Precipitate

Color of precipitate in ammonium phosphomolybdate

Positive test for molisch's test

Violet ring