Chapter 1: Levels of Structure

Protein structure can be discussed at four distinct levels

I. Primary Structure

▪ Primary protein structure shows numbers and kinds of amino acids present and the order of attachment of the amino
acids to each other through peptide bonds.
▪ The primary structure of a protein is maintained by covalent, peptide bonds connecting the amino acids together.
▪ Any change of the sequence of amino acids may cause abnormal function of the polypeptide. For example:
o Sickle anemia is caused by the change in amino acid sequence of hemoglobin. The normal shape of RBC’s
is converted to sickle-shaped RBC’s.
o From a sequence, Thr-Pro-Glu-Glu to Thr-Pro-Val-Glu

Notebook Assignment: Rewrite the structure of Ala-Leu-Cys-Met peptide chain. The “backbone” of the peptide
chain has been highlighted. Draw a square on each peptide bond (the first one was done for you) and locate the
N-terminal and C-terminal end.

II. Secondary Structure

▪ Secondary protein structure is the arrangement in space adopted by the backbone portion of a protein.
▪ The type of interaction responsible for both of these types of secondary structure is hydrogen bonding.

What are the two forms of secondary structure?

alpha helix (α-helix)

▪ has a coiled shape held in place by hydrogen bonds between the amide groups (-NH) and the carbonyl (-C=O)
groups attached to the -carbon.
▪ R groups are not involved in this level of structure.

beta-pleated sheet (β-pleated sheet)

▪ a secondary structure that consists of polypeptide chains arranged side by side

▪ They are held together by hydrogen bonding between the amine and the carbonyl oxygen within the amino acid
backbone.
▪ The right-handed alpha helix and beta-pleated sheet are common structural motifs found in most proteins.
▪ In the illustration below, each box represents an amino acid (residue).
 Locate the amino and carboxyl groups, a-carbon, and R group. Study how H-bonds are formed and take note of
the terminal ends.

Notebook Assignment: Now you know that the carbonyl (-C=O) and amide (-NH) groups form hydrogen bonds to
build the secondary structure, draw the B-pleated sheet formed from these peptide sequences (Hint: follow the
picture above):

ala-gly-ala-gly-ala-gly

gly-ala-gly-ala-gly-ala

III. Tertiary Structure

▪ Tertiary protein structure is the overall three-dimensional shape of a protein that results from the interactions between
amino acid side chains (R groups) that are widely separated from each other within a peptide chain.

To avoid confusion from secondary structure:

▪ The secondary structures of proteins indicate the three-dimensional spatial “arrangements” of the polypeptide
chains.
▪ The tertiary structure of a protein gives a specific three-dimensional “shape” to the polypeptide chain including
interactions and cross-links between different parts of the peptide chain (simply the interactions of the R groups).

Intra and intermolecular attractions (bonds) among R groups

The interactions among the R groups or side chains make up the tertiary level

1. Hydrogen bonding (Intra)

▪ Hydrogen bonding in tertiary structures can occur between polar R groups or side chains while
hydrogen bond in secondary structure is between the amino (-NH2) and carboxyl (-COOH)
group from the backbone of the amino acid.

▪ Hydrogen bonds are relatively weak and are easily disrupted by changes in pH and
temperature.

▪ The picture on the right shows a hydrogen bond formed between the R groups of glutamine
(gray) and serine (orange).

Can you name the R groups? Glutamine: _____________ Serine: _____________

When glutamine and serine are in close proximity, they have the tendency to form a hydrogen bond.
Blue beads represent glutamine and serine

2. Hydrophobic interactions (inter)

▪ Nonpolar side-chains are attracted to other nonpolar side-chains through London forces, and
form “water-free pockets” in the interior region of the folded and compacted peptide.

▪ The nonpolar R groups tend to aggregate in the interior portion of the polypeptide chain,
leaving the polar portion in the exterior.

▪ The picture on the right shows a bond formed between the R groups of phenylalanine (gray)
and leucine (orange) in a certain peptide chain.

Can you name the nonpolar R groups? Phenylalanine: __________ Leucine: ___________
 When nonpolar amino acids like phenylalanine and leucine are present in a polypeptide chain, they tend to form
bonds and aggregate in the interior. This leaves the hydrophilic portions in the exterior forming hydrogen bonds
with water.
“Pocket” H2O
H2O

H2O

H2O
H2O

Blue beads represent polar/hydrophilic amino acids; yellow beads represent hydrophobic amino acids

3. Electrostatic interaction/Salt bridges (Intra)

▪ Electrostatic interactions, also called salt bridges, always involve the interaction between an
acidic side chain (R group) and a basic side chain (R group).

▪ The picture on the right shows a bond formed between the R groups of aspartic acid (gray)
and lysine (orange) in a certain peptide chain. Take note of the charges.

Can you name the R groups? Aspartic acid: __________ Lycine: ___________

When acidic and basic amino acids are in close proximity like aspartic acid and lysine, they have the tendency to form a salt
bridge.

Blue beads represent aspartic acid and lysine

4. Disulfide Bridges (Intra)

▪ Disulfide bonds in proteins are called disulfide bridges.

▪ Each cysteine residue contains a thiol group (also termed as sulfhydryl group -SH) in its
side-chain that is capable of forming a disulfide bridge with another cysteine residue.

▪ The picture on the right shows a disulfide bridge formed between the thiol groups of
one cysteine (gray) and another cysteine (orange) in the same peptide chain or, this
time, in different peptide chains too.
 When two cysteines are in close proximity, they have the tendency to form a disulphide bridge. These two
cysteines are now called cystine. Disulphide bridge can also be formed by two cysteines from different chains.

Purple beads represent cysteine amino acids

5. Quaternary structure

▪ Quaternary structure is the highest level of protein organization.

▪ It is found only in multimeric proteins. Such proteins have structures involving

two or more peptide chains that are independent of each other — that is,
are not covalently bonded to each other.

▪ Most multimeric proteins contain an even number of subunits (two subunits

= a dimer, four subunits = a tetramer, and so on).

▪ Example of a protein with quaternary structure is hemoglobin.

Summary of the four levels of structure:
Chapter 2: Physical and Chemical Properties of Proteins

I. General properties

1. Color and Taste

▪ Proteins are colorless and usually tasteless. These are homogeneous and crystalline.

2. Shape and Size

▪ The proteins range in shape from simple crystalloid spherical structures to long fibrillar structures. There is a wide
variation in the protein shape: it may be globular (insulin), oval (albumin) fibrous or elongated (fibrinogen).

3. Solubility
▪ The solubility of proteins is influenced by pH. Solubility is lowest
at isoelectric point and increases with increasing acidity or
alkalinity.
▪ Proteins form colloidal solutions instead of true solutions in
water. This is due to huge size of protein molecules.

4. Molecular weight
Tyndall effect of milk VS water
▪ The proteins vary in their molecular weights, which, in turn, is
dependent on the number of amino acid residues.
▪ Each amino acid on an average contributes to a molecular weight of about 110.
▪ Majority of proteins/polypeptides may be composed of 40 to 4,000 amino acids with a molecular weight ranging
from 4,000 to 440,000.
▪ A few proteins with their molecular weights are listed below:
▪ Example: Insulin-5,700; Myoglobin-17,000; Hemoglobin- 64,450; Serum albumin-69,000

5. Denaturation
▪ Denaturation refers to the changes in the properties of a protein. In other words, it is the loss of biologic activity.
▪ In many instances the process of denaturation is followed by coagulation— a process where denatured protein
molecules tend to form large aggregates; and to precipitate from solution. A very good example of protein
denaturation by heat is the frying of eggs.

6. Amphoteric
▪ Like amino acids, the proteins are amphoteric, i.e., they act as acids and alkali. Each protein has a fixed value
of isoelectric point (pl).

7. Ion binding capacity

▪ The proteins can form salts with both cations and anions based on their net charge.

8. Isoelectric pH (pI)
▪ Isoelectric pH (pI) as a property of amino acids has been described.
▪ Amino acids in their pI are electrically neutral, maximum precipitability and least buffering capacity. This means
that solubility is lowest at isoelectric point and increases with increasing acidity or alkalinity.
o For example: casein pI = 4.6 - at an environment with a pH of 4.5 and below, casein becomes more
soluble. Also, at an environment with a pH of 4.7 and above, casein becomes more soluble
▪ Examples of proteins with their pI: Pepsin-1.1; Casein-4.6; Human albumin-4.7; Urease-5.0; Hemoglobin-6.7;
Lysozyme-11.0.

9. Precipitation of Proteins
o Proteins can be precipitated by dehydration or neutralization of polar groups.
II. Loss of Protein Structure

▪ Protein structure is especially sensitive to environmental factors.

▪ Many physical and chemical agents can disrupt a protein’s native conformation (shape and form with their normal
biologic activity).
▪ The process of structure disruption, which may or may not involve protein unfolding, is called denaturation.
▪ Practical example: The cooking of egg white by high temperature:
o Egg white contains soluble globular proteins called albumins that unfold and coagulate.
o Another is the curdling of milk by lactic acid

In many cases, denaturation is irreversible (meaning it cannot be undone). When egg white is cooked, it does not
become uncooked.

Denaturation may be reversible only if the protein has undergone mild denaturing conditions. For example, a protein
can be salted out of solution by a high salt concentration, which denatures and precipitates the protein. The protein
re-dissolves when the solution is diluted with water.

Means of Protein Denaturation

1. Physical Means: heat, violent shaking, very high pressures, UV radiation

▪ Let us take for example, heat coagulation test. Heat disrupts hydrogen bonds of secondary and tertiary protein
structure while the primary structure remains unaffected. The protein increases in size due to denaturation and
coagulation occurs.

2. Chemical Means: pH, high salt concentrations, heavy metal ions, organic solvents, surface active agents, high
concentrations of urea/guanidine salts and formamide, urea, salicylate, detergents (e.g. sodium dodecyl sulfate)

▪ Let us take for example: Heller's test is a chemical test that shows that strong acids cause the denaturation of
precipitated proteins. Concentrated nitric acid is added to a protein solution from the side of the test tube to form
two layers. A white ring appears between the two layers if the test is positive.

Precipitation at isoelectric point (pI)

▪ The proteins in general are least soluble at isoelectric pH.

▪ Certain proteins (e.g. casein) get easily precipitated when the pH is adjusted to pI (4.6 for casein).
▪ Changes in pH alter the protonation state of certain protein side chain groups, which in turn alters hydrogen bonding
and salt bridge patterns.
▪ As a protein approaches its isoelectric point, it becomes less soluble and may precipitate from solution.
▪ Acids and bases disrupt salt bridges held together by ionic charges.
 The illustration below shows how the salt bridge between the -COO- and -NH3+ of glutamic acid and lysine,
respectively, is disrupted by the presence of HCl in the system. The carboxyl group of glutamic acid becomes
neutral when hydrogen, H+ (from HCl) reacts with it while the amino group of lysine becomes neutral by the
reaction of Cl-:

Precipitation by salting out

▪ The process of protein precipitation by the addition of neutral salts such as ammonium sulfate or sodium sulfate is
known as salting out.
▪ Salt attracts some of the water molecules that interact with the protein’s ionizable groups
▪ As water molecules interact with salts, protein- protein interactions increase. The protein molecules aggregate and
then precipitate.
▪ In general, the higher is the protein molecular weight, the lower is the salt required for precipitation.

Steps in precipitation by salting-out (excerpt from https://www.youtube.com/watch?v=rom85WMAm08)

1st Usually, protein molecules are surrounded by water molecules which keep them soluble.

2nd The layer of water molecules around protein is called the “solvation/hydration layer”. This layer keeps the protein in
its soluble form.
3rd Addition of ammonium sulfate removes the hydration layer around the protein molecule since water molecules have
higher affinity (or more attracted to) to ammonium sulfate than to proteins.

4th As the protein fails to interact with water molecule, the protein now precipitates.

Precipitation by salts of heavy metals

▪ Heavy metal ions like Pb2+, Hg2+, Fe2+, Zn2+, Cd2+ cause precipitation of proteins.
▪ These metals being positively charged (+), when added to protein solution being negatively charged (-), in alkaline
medium or basic solution, results in precipitate formation.
▪ Trivia: Based on the principle of precipitation, raw egg-white (protein-albumin) is sometimes used to overcome the
toxicity of mercury (Hg).

Precipitation by anionic or alkaloid reagents

▪ Proteins can be precipitated by trichloroacetic acid, sulphosalicylic acid, phosphotungstic acid, picric acid, tannic
acid, phosphomolybdic acid etc.
▪ The negative charge (-) of these anions or alkaloidal reagents counteracts the positive charge of the amino group
in proteins giving a precipitate. By the addition of these acids, the proteins existing as cations (positively charged)
are precipitated by the anionic form of acids to produce protein such as:
o sulphosalicylate (protein + sulphosalicylic acid)
o protein-tungstate (protein + phosphotungstic acid)
o protein-picrate (protein + picric acid)
▪ Trivia: Industrial tanning of leather is based on the principle of protein precipitation by tannic
acid. So, whenever you use your belt or touch your wallet, think of it as a precipitated protein.

Precipitation by organic solvents

▪ Organic solvents such as alcohol are good protein precipitating agents.

▪ They dehydrate the protein molecule by removing the water envelope and cause precipitation. The use of surgical
spirit (about 20% alcohol) as a disinfectant is based on the precipitation of proteins and the death of bacteria
(protein is one of the components of the bacterial cell wall).
 What happens when proteins undergo denaturation?

Denaturation results to changes in the protein’s physical and chemical properties and
loss in the protein’s biological activity. This phenomenon may lead to the following:

a. Native helical structure of protein is lost but the primary structure of a protein with peptide linkages remains intact,
that is, peptide bonds are not hydrolyzed

b. Decreased solubility at the isoelectric point - denatured protein becomes insoluble in the solvent in which it was
originally soluble.

c. Increased viscosity of globular proteins – viscosity of denatured protein (solution) increases while its surface tension
decreases (protein to protein).

d. Increased surface tension – protein to water surface tension

e. Increased reactivity of side groups – increase in ionizable and sulfhydryl groups of protein. This is due to loss of
hydrogen and disulfide bonds.

f. Increased susceptibility to hydrolysis by proteases or change in optical rotation (increased levorotation) – in relation
to the exposure of more functional groups as the protein unfolds

g. Change in particle size

h. Protein loses its biological activity

i. Denatured protein is more easily digested. This is due to increased exposure of peptide bonds to enzymes. Cooking
causes protein denaturation and, therefore, cooked food (protein) is more easily digested. Further, denaturation of
dietary protein by gastric HCl enhances protein digestion by pepsin.

j. Denaturation is usually irreversible. For instance, omelet can be prepared from an egg (protein-albumin) but the
reversal is not possible.

k. Careful denaturation is sometimes reversible (known as renaturation). Hemoglobin undergoes denaturation in the
presence of salicylate. By removal of salicylate, hemoglobin is renatured.

l. Denatured protein cannot be crystallized.

m. A protein becomes denatured when its normal shape gets deformed because some of the hydrogen bonds are
broken.
Chapter 3: Protein Denaturation
▪ Denaturation is the phenomenon of disorganization of native protein structure. Denaturation results in the loss of
secondary, tertiary and quaternary structure of proteins.
▪ This involves a change in physical, chemical and biological properties of protein molecules.
▪ Denaturation results to changes in the protein’s physical and chemical properties and loss in the protein’s biological
activity.

Heat Denaturation

Steps: (Watch video)

Positive Result: formation of coagulum
Reactive group: hydrogen bonds in the secondary and tertiary structure

Principle:
▪ Heat disrupts hydrogen bonds of secondary and tertiary protein structure while the primary structure
remains unaffected (again, peptide bonds are too strong to be disrupted by heat).
▪ The protein increases in size due to denaturation and coagulation occurs.
▪ Note: Albumin and globulin are coagulable proteins. Meanwhile, gelatin and peptone are non-
coagulable proteins. They do not form coagulum when heated:

Concentrated Mineral Acids

Steps: (Watch video)
Positive Result: precipitation
Reactive group: salt bridges and hydrogen bonds

Principle:
▪ Concentrated acid like concentrated HCl, H2SO4, HNO3 and alkali alter the ionization of the carboxyl and
amino groups, disrupting the salt linkages and hydrogen bonds.
▪ The salt bridge most often arises from the anionic carboxylate (-COO−) of either aspartic acid or glutamic
acid and the cationic ammonium (-NH3+) from lysine or the guanidinium functional group of arginine.
 Isoelectric Precipitation
Steps: (Watch video)
Positive Result: precipitation
Reactive group: salt bridges and hydrogen bond

Principle:
▪ Concentrated acids like conc HCl, H2SO4, HNO3 and alkali (bases) change the ionization (charges) of
the carboxyl (-COOH) and amino (NH2) groups, disrupting the salt linkages (salt bridges) and hydrogen
bonds.
▪ Altered ionization leads to a change in the isoelectric point of the protein (pH where it precipitates).

Salting Out

Steps: (Watch video)

Positive Result: precipitation
Reactive group: ionizable groups (carboxyl group, amino group, R groups of: aspartic acid, cysteine,
glutamic acid, histidine, lysine and tyrosine)

Principle:
▪ Water molecules bound with proteins are more attracted to salt.
▪ Salting out is usually reversible.
▪ Globulins are precipitated by half-saturation with ammonium sulphate, whereas albumins are
precipitated only on full saturation.
▪ Ammonium Sulfate is commonly used due to its high solubility at low temperature and reversible water
binding process. This process is therefore reversible. The protein salted out during partial saturation may
be dissolved upon addition of water.
1st When salt is added in the solution of protein, water molecules form bonds with salt leaving proteins behind.

2nd As salt concentration increases, more water molecules leave proteins behind.

3rd As a result, protein molecules start to aggregate and then precipitate.

 Salts of Heavy Metals

Steps: (Watch video)

Positive Result: Precipitation
Reactive group: salt bridges (carboxylate group) and sulfhydryl groups

Principle:
▪ These metallic ions (positively charged) combine with the anionic form of proteins (negatively charged):
o They combine with the carboxylate group (-COO-) to form metal proteinates.
o Other complexes may be formed between the metal ions and the free amino, imidazole and other
groups present in the protein.
▪ Other metals that may also give this reaction aside from Hg2+, Cu2+ and Fe3+ are Pb2+ and Ag+

Organic Solvents

Steps: (Watch video)

Positive Result: precipitation
Reactive group: hydrophobic interaction between R groups of amino acids; hydrogen bonds between
water and polar R groups of amino acid

Principle:
▪ Water-soluble organic solvents such as ethanol interfere with hydrophobic interactions
1st Acetic acid in the procedure will lower the dielectric constant of the aqueous medium.
2nd Interaction of protein molecules with water will be reduced, leading to protein-protein interaction then
precipitation.
3rd Adding alcohol coagulates the denatured proteins. The alcohol converts the proteins into suspensoids
(from proteins as emulsoids) which flocculate upon addition of the dehydrating agent (alcohol).
▪ Trivia: This fact is the basis for the use of alcohol as a disinfectant and antiseptic (through protein
coagulation)
 Alkaloidal Reagents

Steps: (Watch video)

1. To 2mL of the sample add drop by drop with shaking a few drops of the following reagents:
Test Tube 1 – Saturated Picric Acid Solution
Test Tube 2 – 10% Trichloroacetic Acid (TCA)
Test Tube 3 – Acidify slightly with 2% acetic acid, then add 5% Potassium Ferricyanide
Reactive group: salt linkages (salt bridges)
Positive Result: precipitation

Principle:

1st Prior to reaction, acidifying the medium must first be ensured:

▪ The low pH of the solutions leads to the protonation of the carboxyl groups present (-COO- →-
COOH), leaving the protein with positive charges.

2nd The respective salt linkages get disrupted and the resulting electrostatic repulsion causes the

unfolding or unwinding of the protein.

3rd The alkaloid reagent (anion) then binds to the protein (cation) and change the overall solubility of
the molecule.
▪ Trivia: Aside from precipitating proteins, alkaloidal reagents are known to precipitate alkaloids.
(Hence, the term alkaloidal reagents) This reaction is the basis of many industrial practices (TCA,
trichloroacetic acid, is used to remove proteins that may interfere in the analysis of biological
materials).
 Pharmacy Corner

The following are knowledge and applications based on the properties of proteins:

1. The use of AgNO3 in cauterization is based on this property. It precipitates the proteins of tissues as Ag-salts. Example:
cauterization of warts.

2. Another application of this property is the use of proteins as antidotes to metallic poisons. Egg white, milk and other
proteins can be used to precipitate metal ions. The metallic protein precipitate must be removed from the stomach by
an emetic or by stomach-tube to prevent the liberation and absorption of the poisonous metal.

3. Trichloroacetic acid, Tungstic acid, are commonly used for the preparation of protein-free filtrate of blood and other
biological materials prior to analysis of few constituents such as sugar, urea by specific methods.

4. Using the “salting out” techniques, three proteins have been separated and they are: albumin, globulins, and
fibrinogen. Globulins are precipitated by half-saturation with ammonium sulphate, whereas albumins are precipitated
only on full saturation. Fibrinogen is best precipitated by 1/5th saturation with ammonium sulphate.

5. Some small peptides which have significant biological activity are formed as a result of hydrolysis of large proteins
while some are formed by synthesis:

Glutathione: This is a tripeptide consisting of glutamic acid, cysteine and glycine. By virtue of easy dehydrogenation, it
gets converted to disulphide form and function in oxidation-reduction systems.

Bradykinin: Bradykinin (9 amino acids) or Kallidin I and Kallidin II (10 amino acids) have relaxant effects on smooth
muscle.

Oxytocin and Vasopressin: Found in pituitary gland, these are cyclic peptide hormones made up of 9 amino acids.

Angiotensins: The enzyme, renin, is released from kidneys and acts on globulin fraction of plasma to release a
peptide Angiotensin I (10 amino acids) which has only a slight effect on blood pressure, Angiotensin I is then converted
to Angiotensin II by splitting off two amino acids which has eight amino acid, has greater effect on BP. Removal of one
residue aspartic acid from Angiotensin II results in formation of Angiotensin III (7 amino acids) which plays role in
pathology of hypertension.

Gastrin, Secretin and Pancreozymin: Are gastrointestinal peptides which act as hormones which stimulate secretion
of bile and other enzymes of digestive juices.

β-Corticotropin (ACTH), and β MSH: Are peptides which are hormones.

Antibiotics: Penicillin, gramicidin, polymyxins, bacitracin, actinomycin, chloramphenicol are all peptides which act as
antibiotics

Brain Peptides: Certain brain cells have receptors that bind opiates like morphine and have been termed endorphins
(endogenous morphine). Dynorphin is a peptide of 13 amino acids which is called super opiate since it is significantly
potent. Peptide fragments from brain that reduce intestinal motility are met-enkephalins and leuenkephalin both
pentapeptides.