Biochem Lecture Notes 7

PROTEINS
Other than water, proteins are the chief constituents of all cells of the body. Proteins are
much more complex than either carbohydrates or fats. All proteins contain the elements
carbon, hydrogen, oxygen and nitrogen. Most proteins also contain sulfur, some contain
phosphorus, and a few, such as hemoglobin, contain some other element.

SOURCES
Plants synthesize proteins from inorganic substances present in the air and in the soil.
Animals cannot synthesize proteins from materials. Animals must obtain proteins from plants
or from other animals who in turn have obtained them from plants.
Animals excrete waste materials containing many nitrogen compounds. These nitrogen
compounds along with decaying animal and plant matter are converted into soluble nitrogen
compounds by soil bacteria. Plants in turn use these soluble nitrogen compounds to
manufacture more protein, thus completing a cycle.

FUNCTIONS

The word protein is derived from the Greek word proteios, which means “of first
importance.” Proteins function in the body in the building of new cells, the maintenance of
existing cells and replacement of the old cells. Thus, proteins are the most important type of
compound in the body. Proteins are also a valuable source of energy in the body. The oxidation
of 1 g of protein yields 4 kcal --- just as does the oxidation of 1 g of carbohydrate.
Proteins are involved in the regulation of metabolic processes(hormones), in the
catalysis of biochemical reactions(enzymes), in the transportation of oxygen(hemoglobin), in the
body’s defense against infection(antibodies), in the transmission of impulses(nerves), in the
transmission of hereditary characteristics(nucleoprotein) and in muscular activity(contraction).
Proteins are components of skin, nails, hair as well as connecting and supporting tissue.

MOLECULAR MASSES
Proteins have very high molecular masses.

AMINO ACIDS
Proteins are polymers built up from simple units called amino acids. Hydrolysis of
proteins yield amino acids. There are 20 amino acids that can be produced by the hydrolysis of
protein. All these amino acids except glycine, which has no chiral carbon, have the
L-configuration.

COMPOSITION
An amino acid is an organic acid that has amine(-NH2) group attached to a chain
containing an acid group. Although the amine group can be anywhere on the chain, amino
acids found in nature usually have the amine group on the alpha carbon – that is, the carbon
next to the acid group.

COOH CHO NH2

| | |
H2N-C-H HO- C- H R-C- COOH
| | |
R CH2OH H
L-amino acid L-glyceraldehyde
R= hydrocarbon (chain of Carbon atoms) CH3-CH2-CH2-CH2

Amino acids can be divided into two groups, polar and non-polar, depending on the
polarity of the R group attached to the alpha carbon. If the R group is non-polar, then the
amino acid will be less soluble in water than one containing a polar group.
 An R group that is polar, such as -OH, -SH, -NH2, or -COOH, produces an amino acid that
is polar. Such amino acids are soluble in water
The body can synthesize some, but not all, all of the amino acids it needs. Those that
cannot synthesize must be supplied from food consumed. These are called the nutritionally
essential amino acids. OH
|
AMPHOTERIC NATURE - C=0 Carboxylic acid amine
Amino acids contain the -COOH group, which is acidic, and the -NH2 group, which is
basic. In solution, the carboxyl group can donate a hydrogen ion to the amino group, forming a
dipolar ion, called zwitterion.

R-CH-COOH --------------🡪 R-CH-COO-

| |
NH2 NH3+
Amino acid a zwitterion form of an amino acid

Amino acids are amphoteric compounds; that is they can react with either acids or
bases. When an amino acid is placed in a basic solution, it forms a negatively charged ion that
will be attracted to a positively charged electrode. In an acid solution, the amino acid forms a
positively charged ion that will be attracted to a negatively charged electrode.

H+ OH-
R- CH-COOH <-------🡪 R-CH-COO 🡨----------🡪 R-CH-COO-
-

| | |
3+ 3+
NH NH NH3+
positively charged ion zwitterion negatively charged ion
(in acid solution) (in basic solution)

Since amino acids are amphoteric, proteins, which are made up of amino acids are also
amphoteric. This amphoteric nature of proteins accounts for their ability to act as buffers in the
blood; they react with either acids or bases to prevent an excess of either.
At a certain pH (that is, a certain hydrogen ion concentration) amino acids will not
migrate toward either the positive or the negative electrode. At this pH, amino acids will be
neutral; there will be an equal amount of positive and negative ions. This point is called the
isoelectric point.
Proteins which are composed of amino acids, also have an isoelectric point, which is
different for each protein. At its isoelectric point, a protein has a minimum viscosity, and also a
minimum osmotic pressure. At a pH above the isoelectric point, a protein has more negative
than positive charges. At a pH below the isoelectric point, a protein has more positive than
negative charges.
Exercise:

1. Draw the dipolar structure of serine.

COOH
|
H2N-C- H
|
CH2OH
COO-
|
+
So, the dipolar structure of serine would be drawn as H3N -C- H
|
CH2OH

2. Draw the dipolar structure of proline. Proline is COOH

|
H2N- C-H
\ \
H2C CH2
\ /
CH2
3. So, the dipolar structure of proline is: COO-
|
+
H3N - C-H
\ \
H2C CH2
\ /
CH2

DIPEPTIDES a-b-a-b-a-b-a-b
Proteins consist of many amino acids joined together by what is called a peptide linkage
or peptide bond. Suppose that a glycine molecule reacts with an alanine molecule. This
reaction can occur in two different ways. The amine part of the glycine may react with the acid
part of the alanine or the acid part of the glycine may react with the amine part of the alanine.
O O
|| ||
CH3-CH- C - OH + H-NH-CH2-COOH -----------🡪 CH3-CH- C - NH-CH2-COOH + HOH
| |
NH2 NH2 peptide bond

alanine glycine alanylglycine(Ala-Gly)

 When two amino acids combine, the product is a called a dipeptide. When three amino
acids combine, the product is called a tripeptide. When many amino acids join together, the
product is called a polypeptide. For just two amino acids, glycine and alanine, two different
combinations have already been indicated---glycylalanine and alanylglycine, in which the first
member of each group acts as the one furnishing the -OH from the acid group. For three amino
acids --- such as glycine, alanine and valine--- there are six possible combinations(or tripeptide
linkages).

1. Glycylalanylvaline (Gly-Ala-Val)
2. Glycylvalylalanine (Gly-Val-Ala)
3. Alanylglycylvaline (Ala-Gly- Val)
4. Alanylvalylglycine (Ala-Val-Gly)
5. Valylglycylalanine (Val-Gly-Ala)
6. Valylalanylglycine (Val-Ala-Gly)

By convention, peptides are written with the -NH2 end (the N terminal) at the left and
the - COOH (the C terminal) at the right.
Proteins contain a large number of peptide linkages, and the number of possible
combinations of the many amino acids in the formation of a protein is beyond all
comprehension(for 20 amino acids, it is 20 x 19 x 18 x 17 x 16 x 15 x 14 x 13 x 12 x 11 x 10 x 9x 8
x 7 x 6 x 5 x 4 x 3 x 2, an unimaginable number).
Insulin illustrates the peptide linkages. It has an A chain, containing 21 amino acids, and
a B chain which contains 30 amino acids. Note that the two chains are connected by two
disulfide bridges.

Assignment:

1. Write all the possible combinations for a tripeptide formed from two alanines and one
glycine.

2. Write all possible tripeptides formed from an alanine(A), glycine(G), and leucine(L).
2nd Lecture (Proteins)

STRUCTURE
When a protein is hydrolyzed (by acids, bases or certain enzymes), it breaks down into
smaller and smaller units, eventually forming amino acids. Likewise, when amino acids combine
(under the influence of certain enzymes), they first form dipeptides, then tripeptides, then
polypeptides, and so on, until they eventually form a protein.

Protein 🡨-------🡪Proteoses 🡨------🡪 Peptones 🡨-------🡪Polypeptides 🡨------🡪 Tripeptides🡨---------🡪

Dipeptides 🡨-------🡪 Amino Acids

Proteins have a three-dimensional structure that can be considered as being composed

of simpler structures.

1.Primary Structure – of a protein refers to the number and sequence of the amino acids in the
protein. These amino acids are held together by peptide bonds. Example: Insulin
A slight change in the amino acid sequence, such as the replacement of a single amino
acid with another, can change the entire protein. (See normal hemoglobin and sickle cell
hemoglobin)
 The primary protein structure is the order in which amino acids are linked together in a
protein. Every protein has its own unique amino acid sequence. Primary protein structure
always involves more than just the numbers and kinds of amino acids present; it also involves
the order of attachment of the amino acids to each other through peptide bonds.
Insulin, the hormone that regulates blood-glucose levels, was the first primary protein
for which primary structure was determined; the “sequencing” of its 51 amino acid was
completed in 1953, after 8 years of work by the British biochemist Frederick Sanger. Today,
primary structures are known for many thousands of proteins, and the sequencing procedures
involve automated methods that require relatively short periods of time.
 This figure shows the primary structure of myoglobin, a protein in involved in oxygen
transport in muscles, it contains 153 amino acids assembled in the particular, definite order
shown above.
The primary structure of a specific protein is always the same regardless of where the
protein is found within an organism. The structures of certain proteins are even similar among
different species of animals. Ex. The primary structures of insulin in cows, pigs, sheep and
horses are very similar both to each other and to human insulin.
An analogy is often drawn between the primary structure of proteins and words. Words,
which convey information, are formed when the 26 letters of the English alphabet are properly
sequenced. Proteins are formed from proper sequences of the 20 standard amino acids. Just as
the proper sequence of letters in a word is necessary for it to make sense, the proper sequence
of amino acids is necessary to make biochemically active protein. Furthermore, the letters that
form a word are written from left to right, as are amino acids in protein formulas. As any
dictionary of the English language will document, a tremendous variety of words can be formed
by different letter sequences. Imagine the number of amino acid sequences possible for a large
protein. There are 1.55 x 1066 sequences possible for the 51 amino acids found in insulin. From
these possibilities, the body reliably produce only one, illustrating the remarkable precision of
life processes. From the simplest bacterium to the human brain cell, only those amino acid
sequences needed by the cell are produced.

2.Secondary Structure – of a protein refers to the regular recurring arrangement of the amino
acid chain.
a. One such arrangement, called the α helix, occurs when the amino acids form a coil, or
spiral. The coil consists of loops of amino acids held together by hydrogen bonds (between the
-H of the -NH2 of one amino acid and the O of the C=O of the acid part of another amino acid.
Each turn of the helix contains an average of 3.6 amino acids. Such a structure is both flexible
and elastic. Hair and wool are examples of protein in such a helical structure.
 When the amino acids are coiled, they can either form a right or left-handed spiral.
However, since α-amino acids in protein are all of the L- configuration, the coils are always
right-handed.

b. The second type of secondary structure, the β-pleated sheet (also called the pleated sheet)
consists of parallel strands of polypeptides held together by hydrogen bonds. Such a structure is
flexible but not elastic. Silk has such a pleated structure. It is strong, but resistant to stretching.
This type of structure is less common than the α-helix.

c. Tertiary Structure – of a protein refers to the specific folding and bending of the coils into
specific layers or fibers. It is the tertiary structure that gives proteins their specific biologic
activity. Tertiary structures are stabilized by several types of bonds. Salt bridges (a) are formed
between positively and negatively charged groups within the protein molecule. Examples of
such groups are the carboxyl and amino side chains found in glutamic acid, lysine, arginine and
aspartic acid. Hydrogen bonds (b) can form between different segments of the coil. Disulfide
bonds (c) can form between cysteine groups in different parts of the coil. Hydrophobic bonds (d)
can be formed. In general, nonpolar amino acids are folded on the “inside” of the protein, and
polar amino acids are on the “outside”, where they can react with water molecules to form
polar group interactions.
d. Quaternary Structure – occurs when two or more protein units, each with its own primary,
secondary and tertiary structure, combine to form a more complex unit. An example of a
protein with quaternary structure is hemoglobin. It consists of two identical β chains
(dark-colored). Each chain enfolds a heme (Iron-containing) group.

Structures of proteins https://www.youtube.com/watch?v=PPJ7C3hcnPw

PERCENT COMPOSITION
The average percentage of nitrogen present in protein is 16 percent; that is about 1/6 of
a protein is nitrogen. Because protein is the major food that contains nitrogen, the chemist can
determine the amount of protein present in a food substance by determining the amount of
nitrogen present. This amount is about 1/6 of the amount of protein present. Therefore, the
amount of protein in the food can be calculated by multiplying the weight of nitrogen by 6 and
converting this to a percentage of the total. For example, suppose that a 100 g sample of food
yielded 4 g of nitrogen on chemical analysis. Since the amount of nitrogen in protein is 1/6 of
the total amount of nitrogen is 6 x 4 g or 24 g. Then the percentage of protein in the original
100 g is 24 percent.

4 g x total = 6 x 4 = 24 g, thus 24/100 x 100 = 24 %

1/6
By ratio and proportion: 100: 4=150:x
100x = 600
100 100
X=6g
Amt nitrogen = 6 x 6 = 36 g N2
% protein = 36/150 x 100 = 24%
CLASSIFICATION

Proteins are divided into three categories ----- simple, conjugated and derived. On
hydrolysis, simple proteins yield only amino acids or derivatives of amino acids. On hydrolysis,
conjugated proteins yield amino acids plus some other type of compound. Conjugated protein
consists of a simple protein combined with a nonprotein compound. Derived proteins are
produced by the action of chemical, enzymatic, and physical forces on the other two classes of
protein. Derived proteins include proteoses, peptones, polypeptides, tripeptides, dipeptides.
They also can be hydrolyzed to amino acids.
Proteins are classified according to their solubility, composition, function or shape.
According to Solubility denaturation

Type of Protein Solubility Coagulated by Heat Examples

Albumins Soluble in water and Yes Egg albumin; serum
salt solutions albumin; lactalbumin
Globulins Slightly soluble in Yes Serum globulin,
water; soluble in salt lactoglobulin;
solutions vegetable globulin
Albuminoids Insoluble in all No Keratin in hair,
neutral solvents and feathers, collagen
in dilute acid and
alkali
Histones Soluble in salt No Nucleohistone in
solutions; insoluble in thymus gland; globin
very dilute NH4OH in globulin

According to Composition

Type Prosthetic Group (Nonprotein portion of Examples

the Combination)
Nucleoproteins Nucleic Acid Chromosomes
Glycoproteins Carbohydrates Mucin in saliva
Phosphoproteins Phosphate Casein in milk
Chromoproteins Chromophore group (color-producing Hemoglobin, hemocyanin,
group) flavoproteins, cytochrome;
Lipoproteins Lipids Fibrin in blood
Metalloproteins Metals Ceruloplasmin (containing
Cu) and siderophilin
(containing Fe) in blood
plasma

Nucleoproteins – these are proteins that are combined with nucleic acids.
Glycoproteins – they are proteins containing carbohydrates in varying amounts. Glycoproteins
have molar masses from 15,000 to more than 1 million. The carbohydrates present in
glycoproteins include the hexoses, mannose and galactose, the pentoses arabinose and xylose,
sialic acids which are derivatives of neuraminic acid. Glucose is not present in glycoproteins,
except for collagen.
Glycoproteins are present in most organisms, including plants, animals, bacteria, viruses
and fungi. Human cell membranes are about 5 % carbohydrate, which is present as
glycoproteins and glycolipids. Glycophorin is a glycoprotein found in the membranes of human
erythrocytes. Heparin, which inhibits clotting of blood, is also a glycoprotein.
In addition to their functions in membranes, glycoproteins serve in the following ways:
as structural proteins (collagen); as lubricants (mucin and mucous secretions); as transportation
molecules for vitamins, lipids, minerals and trace elements; as immunoglobulins such as
interferon; as hormones such as thyrotropin (TSH); as enzymes such as hydrolases and
nucleases; as hormone receptor sites; and for the specification of human blood types.

Lipoprotein – these are proteins containing lipids; are part of cell membranes.
Lipids such as cholesterol and triglycerides are not soluble in water and thus must be
complexed in a water-soluble carrier protein (lipoprotein). Plasma lipoproteins consist of a
neutral lipid core of triglyceride and cholesterol ester that is surrounded and stabilized by free
cholesterol and phospholipid. The relative proportions of nonpolar lipid, protein, and polar lipid
determine the density, size and charge of the resulting lipoproteins. lipids into the blood plasma
via the thoracic lymph duct. They are removed from the plasma with a half-life of 5 to 15
minutes. They are responsible for the creamed-tomato-soup appearance of blood following a
meal containing fats.
Very low-density lipoproteins (VLDL) transport triglycerides synthesized by the liver to
the other parts of the body. Their breakdown leads to the production of the transient
intermediate density lipoproteins (IDL). LDL provides cholesterol for cellular needs. LDL is
thought to promote coronary heart disease by first penetrating the coronary artery wall and
then depositing cholesterol to form atherosclerotic plaque.

High density lipoproteins are involved in the catabolism of other lipoproteins. They
incorporate the cholesterol and phospholipid released by a lipoprotein. HDLs may also remove
excess cholesterol from peripheral tissue.
A new lipoprotein, lipoprotein (a) which is similar to LDL has been detected recently.
The striking structural similarity of lipoprotein (a) to human plasminogen has stimulated intense
studies as to a possible link between atherosclerosis and thrombosis. Primarily, results have
indicated that lipoprotein (a) is an independent risk factor (similar to cholesterol) for coronary
heart disease.
Elevated LDL levels have been associated with an increased risk of developing coronary
artery disease, whereas elevated HDL levels appear to reduce the risk. Women have higher HDL
levels than men (55 vs 45 mg/100 mL), and this may account for women’s lower rate of heart
disease. Aerobic exercises HDL levels (marathon runners average 65 mg/100mL).

CLASSIFICATION ACCORDING TO FUNCTION

Proteins can also be classified according to their biologic function.

Type of Protein Example Use

Structural Collagen In structure of connective tissue
 Keratin In structure of hair and nails
Contractile Myosin, Actin In muscle contraction
Storage Ferritin In storage of iron needed to make
hemoglobin
Transport Hemoglobin In carrying oxygen
Serum Albumin In carry fatty acids
Hormones Insulin In metabolism of carbohydrates
Enzymes Pepsin In digestion of protein
Protective Gamma Globulin In antibody formation
Fibrinogen In blood clotting
Toxins Venoms Poisons

CLASSIFICATION ACCORDING TO SHAPE

Proteins can also be classified according to their shape and dimensions.

(1) Globular proteins – consist of polypeptides folded into the shape of a “ball”. They have a
length-to-width ratio of less than 10. They are soluble in water or form colloidal
dispersions and have an active function. Proteins classified as globular are hemoglobin,
albumin and the globulins.
(2) Fibrous proteins consist of parallel polypeptide chains that are coiled and stretch out.
They have a length-to-width ratio greater than 10. Fibrous proteins are in soluble in
water. Examples include collagen, fibrin, and myosin.

PROPERTIES
A. COLLOIDAL NATURE
Proteins form colloidal dispersions in water. Being colloidal, Protein will pass through a filter
paper but not through a membrane. The inability of the protein to pass through a
membrane is of great importance in the body. Proteins present in the bloodstream cannot
pass through the capillaries and should remain in the bloodstream. Since proteins cannot
pass through the membranes, there should be no protein material in the urine. The
presence of protein in the urine indicates damage to the membranes in the kidneys ---
possibly nephritis.

B. DENATURATION
Denaturation of a protein refers to the unfolding and rearrangement of the secondary and
tertiary structures of a protein without breaking the peptide bonds. A protein that is
denatured loses its biologic activity. When the conditions for denaturation are mild, the
protein can be restored to its original conformation by carefully reversing the conditions
that caused the denaturation. This is called reversible denaturation. If the conditions that
caused the denaturation are drastic, the process in irreversible; the protein will coagulate or
precipitate from solution. Proteins can be denatured by a variety of agents.
REAGENTS OR CONDITIONS THAT CAUSE DENATURATION

ALCOHOL – Alcohol coagulates (precipitates) all types of protein except prolamines. Alcohol
(70 percent) is used as a disinfectant because of its ability to coagulate the presence of
bacteria. Alcohol denatures protein by forming hydrogen bonds that compete with the
naturally occurring hydrogen bonds in the protein. Such a process is irreversible.

SALTS OF HEAVY METALS – Heavy metal salts, such as mercuric chloride (bichloride of
mercury) or silver nitrate (lunar caustic), precipitate protein. These denature protein
irreversibly by disrupting the salt bridges and the disulfide bonds present in the protein.
They are very poisonous if taken internally because they coagulate and destroy protein
present in the body. The antidote for mercuric chloride or silver nitrate when these poisons
are taken internally is egg white. The heavy metal salts react with the egg white and
precipitate out. (The egg white colloid has a charge opposite to that of the heavy metal ion
and attracts it.) The precipitate thus formed must be removed from the stomach by an
emetic or the stomach will digest the egg white and return the poisonous material to the
system.
Dilute silver nitrate solution is used as a disinfectant in the eyes of newborn infants.
Stronger solutions of silver nitrate are used to cauterize fissures and destroy excessive
granular tissues.

HEAT
Gentle heating causes reversible denaturation of protein, whereas vigorous heating
denatures protein irreversibly by disrupting several types of bonds. Egg white, a substance
containing a high percentage of protein, coagulates on heating. Heat coagulates and
destroys protein present in bacteria. Hence, sterilization of instruments and clothing for use
in operating rooms requires the use of high temperatures. The presence of protein in the
urine can be determined by heating a sample of urine, which will cause the coagulation of
any protein material that is present.

ALKALOIDAL REAGENTS
Alkaloidal reagents, such as tannic acid and picric acid, form insoluble compounds with
proteins. Alkaloidal regents denature protein irreversibly by disrupting salt bridges and
hydrogen bonds.
Tannic acid has been used extensively in the treatment of burns. When this substance
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. It also reduces exposure to air. Newer drugs have
taken place of tannic acid for burns, but an old-fashioned remedy still in use for emergencies
involves the use of wet tea bags (which contain tannic acid).

RADIATION
Ultraviolet or x rays can cause protein to coagulate. The radiation denatures irreversibly
by disrupting the hydrogen bonds and the hydrophobic bonds present in the protein. In the
human body, the skin absorbs and stops ultraviolet rays from the sun so they do not reach
the inner cells. Proteins in cancer cells (rapidly driving cells) are more susceptible to
radiation than those present in normal cells, so x irradiation is used to destroy cancerous
tissue.

pH
Changes in pH can disrupt hydrogen bonds and salt bridges, causing irreversible
denaturation. Proteins are coagulated by such strong acids as concentrated hydrochloric,
sulfuric and nitric acids. Casein is precipitated from milk as a curd when it comes into
contact with the hydrochloric acid in the stomach. Heller’s ring test is 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 bonds will break.

OXIDIZING AND REDUCING AGENTS

Oxidizing agents such as bleach and nitric acid and reducing agents such as sulfites and
oxalates denature protein irreversibly by disrupting disulfide bonds.

SALTING OUT
Most proteins are insoluble in saturated salt solutions and precipitate out unchanged.
To separate a protein from a mixture of other substances, the mixture is placed in a
saturated salt solution (such as NaCl, Na2SO4, or (NH4)2SO4). The protein precipitates out
and is removed by filtration. The protein can then be purified from the remaining salt by
the process of dialysis.