BIOCHEMISTRY 1

Lab (2) Proteins Cont.

 Protein structure
 Each protein is a polymer– specifically a polypeptide – that is

a sequence formed from various amino acids. By convention, a

chain under 40 residues is often identified as a peptide, rather

than a protein.

 To be able to perform their biological function, proteins fold into

one or more specific spatial conformations, driven by a number

of non-covalent interactions such as hydrogen bonding, ionic

interactions, Van der Waals forces, and hydrophobic packing.

 Protein structural levels
 Primary structure
 Amino acid linear sequence of the polypeptide chain.
 Held together by peptide bonds, which are made during the process of
protein biosynthesis or translation.
 The two ends of the polypeptide chain are referred to as the carboxyl
terminus (C-terminus) and the amino terminus (N-terminus).
 Secondary structure
 Highly regular structures.
 Two main types of secondary structure, the alpha helix and the beta
strand or beta sheets.
 These secondary structures are stabilized by patterns of hydrogen
bonds between the main-chain peptide groups.
Tertiary structure
 Three-dimensional structure of a single protein molecule.
 The alpha-helices and beta-sheets are folded into a compact globule.
 The folding is driven by the non-specific hydrophobic interactions, salt
bridges, hydrogen bonds, and disulfide bonds.
Quaternary structure
 Is the assembly of several subunits or monomers forming a multi-
subunit protein.
 Complexes of two or more polypeptides (i.e. multiple monomers) are
called multimers.
 The quaternary structure is stabilized by the same non-covalent
interactions and disulfide bonds as the tertiary structure.
 Specifically, it would be called a dimer if it contains two subunits,
a trimer if it contains three subunits, and a tetramer if it contains four
subunits (e.g. Hemoglobin).
Four levels of Protein
 Protein Denaturation:
Denaturation: the disruption of the native structure
of proteins (secondary, tertiary, and quaternary
structures) with a resulting loss of biological
activity.
Denaturating agents: are agents that cause a disorder
of the structural organization of protein molecules
and produce alterations in physico- chemical (in
particular solubility) and biological properties of
proteins.
Denaturating agents
1) Chemical agents:
e.g. Acids & heavy metal ions.

2) Physical agents:
Temperature and ionizing radiations constitute two most
large groups of denaturants.

3) Biological denaturation may be evoked by

proteolytic enzymes (e.g., trypsin) which destroy the
higher organization levels of protein molecules.
Protein Denaturation
Practical application of protein denaturation in:
(1) Precipitation of proteins in biological materials.

(2) Identification of urinary proteins.

(3) Binding heavy metal salts with proteins in cases

of poisoning.

(4) Disinfection of the skin and mucous membranes.

A) Denaturation of protein by concentrated mineral
:acids
Principle:
The method is based on the property of mineral acids to produce
charge neutralization and destruction of protein spatial structure
causing denaturation and precipitation of protein.
Procedure:
1- Transfer 10 drops of concentrated nitric acid to a test tube.

2- Add 5 drops of egg’s white solution by allowing it to flow down the

inner wall of the tube to form an upper layer.

3- Note the change at the liquid-liquid interface (formation of denatured

protein ring).
B) Denaturation of protein by organic acids:

Principle:
The method is based on the property of organic acids to neutralize
the charges on protein molecule and to disarrange its spatial
structure, which leads to denaturation and precipitation of protein.
Procedure:
1- Transfer 10 drops of egg's white solution to two test tubes.
2- Add 2 drops of trichloroacetic acid (TCA) to one tube and
2 drops of perchloric acid to the other tube and observe the
changes occurring.
C) Denaturation of protein by heavy metal salts:
Principle:
The method is based on the ability of heavy metal
ions to bind to the functional group of the side chain
radicals of amino acids in protein molecules; causing
destruction of protein spatial structure and precipitation of
a denatured protein.

When an excess of a heavy metal salt solution is added,

the initially formed precipitate is re-dissolved due to ion
adsorption and development of positive charges on the
protein molecule.
Procedure:
1- Transfer 10 drops of egg's white solution into two test tubes.

2- Add 1-2 drops of cupper sulphate solution to one test tube

and 1-2 drops of lead acetate solution to the other. Note

the formation of a precipitate in either case.

3- Add more drops of the corresponding denaturant to the

tubes and observe the dissolution of the initially formed

precipitates.
D) Denaturation of protein by organic solvents:
Principle:
The method is based on the ability of organic solvents (ethanol,
chloroform, or acetone) to destroy hydrophobic interactions within
the protein molecule and thereby produce its denaturating effect,
leading to protein precipitation.
Procedure:
1- Transfer 10 drops of undiluted egg's white solution into three
test tubes.
2- Add equal volumes of the following organic solvents: ethanol to
the test tube, acetone to the second, and chloroform to the third tube.
3- Note the protein precipitation in each case.
 :Determination of the Isoelectric Point of Proteins
 Protein molecule carries both positive and negative charges due to
presence of acidic and basic amino acids in its structure. The net charge of
protein macromolecules is affected by the medium pH.

 Each protein is characterized by a pH value at which the sum of positive

and negative charges on the protein is equal to zero. This state of
protein is referred to as “isoelectric”, and the corresponding pH value is
called the isoelectric point (pI).

 At pI, the protein is unstable, and prone to precipitation, especially in

the presence of dehydrating agents (ethanol, acetone, etc.).

 The knowledge of the isoelectric point for individual proteins allows

isolation of a certain protein by precipitation from a biological extract and
purification of a specific protein preparation in pharmaceutical industry.
Effect of pH on protein solubility
Principle:
The method is based on the ability of dissolved protein (casein of milk)
at its pI to transit to an unstable state and to form a precipitate which is
shown as a distinct clouding of the solution. Addition of ethanol (as a
dehydrating agent) accelerates protein precipitation .

Procedure:
1- Into three test tubes labeled A, B, and C, introduce 1 ml of buffers of

pH 2, pH 4.6, and pH 9, respectively.

2- Add 2 ml of milk to each test tube, shake the tube contents, and note

the changes occurring in the three tubes.

 Conjugated Proteins

 Conjugated proteins are composed of two different

chemical moieties linked by covalent bonds or weak

bonds (ionic, hydrogen, and van der Waals).

 They include hemoproteins, flavoproteins,

glycoproteins and proteoglycans, lipoproteins,
metalloproteins, phosphoproteins, and nucleoproteins.
:Experiments on Conjugated Proteins
I) Hemoproteins:
The Chemical Nature of Hemoproteins:
• Hemoproteins include hemoglobin, myoglobin, cytochromes,
and the enzymes catalase and peroxidase.

• They are composed of a protein moiety and heme, which is a

tetrapyrrole heterocycle bound to an iron (II) atom.

• The heme, a part of hemoglobin, has a “peroxidase-like action”

which refers to its ability to accelerate the oxidation of substrates
(SH2) by H2O2
 This property is attributed to the structural similarity
between the prosthetic groups of hemoglobin
(hemin) and peroxidase.

 However, in distinction from the peroxidase enzyme,

hemoglobin retains its catalytic properties
unaffected by boiling or strong acids (factors that
cause protein denaturation).
:Experiments on Hemoglobin
Practical applications
The very sensitive benzidine and guaiacum tests are used in
biochemical, clinical, and medicolegal examinations in trace
analysis for blood.
1) Benzidine Test for Hemin Group of Hemoglobin:

Principle:
The method is based on the property of hemin group of hemoglobin
to catalyze the oxidation of benzidine by hydrogen peroxide to
diphenoquinone di-imine that is coupled with a molecule of non-
oxidized benzidine to yield a green-blue colored complex.
Procedure:
1- Introduce 1 ml of 5000-fold diluted blood into a test

tube, place the tube for several minutes in a boiling

water bath, and let it to cool.

2- Add 5 drops of benzidine solution and 5 drops of

hydrogen peroxide solution. Observe the appearance of
the characteristic color.
:Guaiacum Test for Hemin Group of Hemoglobin )2
Principle:
The method is based on the property hemin group of
hemoglobin to catalyze the oxidation of guaiacum by hydrogen
peroxide to its blue colored compound.
Procedure:
Transfer several drops of diluted blood into a test tube and place for -1
.5 minutes in a boiling water bath; then allow it to cool

Add 5 drops of ethanolic guaiacum solution and 3 drops of -2

hydrogen peroxide. Observe the appearance of a characteristic blue
.color
 II) Phosphoproteins:

 The prosthetic group in phosphoproteins is a phosphoric acid

residue linked through an ester bond to the hydroxyl group of

serine or threonine amino acids of proteins.

 Among phosphoproteins are casein of milk, vitellin of egg yolk,

enzymes (phosphorylase, phosphoglucomutase), and many others.

 Phosphoproteins in a biological material may be detected by testing

for phosphate group.

 Color Reactions for Phosphoproteins:
Principle:
• The method is based on testing the protein component of
casein hydrolysates by the biuret reaction and the
phosphoric acid moiety by its reaction with
ammonium molybdate to yield a phosphomolybdate
complex.

• By the action of reducing agent (e.g. ascorbic acid),

ammonium phosphomolybdate is converted to
molybdenum blue.
Procedure:
1- Introduce about 0.1 g of dried casein powder into a test tube. Add 5
ml of 20% sodium hydroxide solution and place the test tube in a
boiling water bath for 30 minutes.

2- Allow the casein hydrolysate to cool, then sample out 0.5 ml of it

into a clean test tube and perform the biuret test by adding 3-5 drops
of biuret reagent. Observe the appearance of a characteristic coloration.

3- Transfer 1 ml of the casein hydrolysate; then add 10 drops of

ammonium molybdate solution, mix and add 10 drops of
ascorbic acid solution. Observe the appearance of a characteristic
Blue color.