Here are the key points from the protein separation methods reading: - There are several methods used to separate proteins including precipitation, electrophoresis, chromatography, and ultracentrifugation. - Precipitation separates proteins based on differences in solubility at various pH, temperatures, or in the presence of salts, organic solvents, or other reagents. It is a crude separation method. - Electrophoresis separates proteins based on their charge and size by applying an electric current through a gel or liquid. Common types are PAGE and SDS-PAGE. - Chromatography separates proteins based on differences in how they interact with a stationary phase compared to a mobile phase as they flow through a column. Key types are ion-exchange

1. PROTEINS
SESSION 3

2. What are proteins?
• Proteins are large, complex molecules that play many critical roles in the
body.
• They do most of the work in cells and are required for the structure,
function, and regulation of the body’s tissues and organs.
• Proteins are made up of hundreds or thousands of smaller units called
amino acids, which are attached to one another in long chains.
• There are 20 different types of amino acids that can be combined to make
a protein.
• The sequence of amino acids determines each protein’s unique 3-
dimensional structure and its specific function.
• Amino acids are coded by combinations of three DNA building blocks
(nucleotides), determined by the sequence of genes.

3. AMINO ACIDS
• Cells produce proteins with strikingly different properties
and activities by joining the same 20 amino acids in many
different combinations and sequences.
• This indicates that the properties of proteins are
determined by the physical and chemical properties of
their monomer units, the amino acids.
• Amino acids are the basic structural units of proteins
consisting of an amino group, (-NH2) a carboxyl (-COOH)
group a hydrogen (H) atom and a (variable) distinctive (R)
group.
• All of the substituents in amino acid are attached
(bonded) to a central α carbon atom.
• This carbon atom is called α because it is bonded to the
carboxyl (acidic) group.

4.  In neutral solution
(PH = 7), both the
α- amino and α
carboxyl group are
ionized resulting
the charged form
of an amino acids
called zwitterion
(dipolar) as shown
in the next slide.
 Out of the 20 amino acids, proline is not an α amino
acid rather an α - imino acid. Except for glycine, all
amino acids contain at least one asymmetric carbon
atom (the α - carbon atom).

6. Stereochemistry
Stereochemistry mainly emphasizes the configuration of amino
acids at the α carbon atom, having either D or L- isomers

7. Classification of Amino Acids
They are classified based on;
• Structure
• Electrochemical classification
• Biological and physiological
• Metabolic fate

8. Structural classification: This classification is based on the side chain
radicals (R-groups)
R groups
with
Aliphatic side chains
e.g glycine, alanine,
valine, leucine,
isoleucine
Sulfur group:
cysteine,
methionine
Aromatic group;
phenylalanine,
tyrosine,
tryptophan,
Imino group;
proline

9. Electrochemical
Amino acids could also be classified based on their acid – base properties
Basic amino acids
1. Lysine
2. Arginine
Neutral amino acids
1. Asparagine
2. Serine
3. Threonine
4. Glutamine
Acidic amino acids
1. Glutamic acid
2. Aspartic acid

10. Biological
This classification is based on the functional property of amino acids for the organism.
• Essential: Amino acids which are not synthesized in the body and must be provided in the
diet to meet an animal’s metabolic needs are called essential amino acids.
• Non essential: These amino acids are need not be provided through diet, because they
can be biosynthesized in adequate amounts within the organism.
• Semi-essential amino acids: they can be synthesized within the organism but their
synthesis is not in sufficient amounts. In that they should also be provided in the diet.
Essential Non-essential Semi-essential
Arginine, histidine,
leucine, isoleucine,
lysine, methionine,
phenylalanine,
Threonine, Tryptophan,
Valine
Alanine, Asparagine,
aspartine, cysteine,
glutamic acid,
glutamine, glycine,
proline, serine, tyrosine
Histidine, Arginine

11. Metabolic fate of each amino acid
Amino acids can be classified here as Glucogenic (potentially be converted to
glucose), ketogenic (potentially be converted to ketone bodies) and both glucogenic
and ketogenic.
Glucogenic amino acids: Those amino acids in which their carbon skeleton gets
degraded to pyrurate, α ketoglutarate, succinyl CoA, fumrate and oxaloacetate and
then converted to Glucose and Glycogen, are called as Glucogenic amino acids.
These include:- Alanine, cysteine, glycine, Arginine, glutamine, Isoleucine, tyrosine
Ketogenic amino acids: Those amino acids in which their carbon skeleton is
degraded to Acetoacetyl CoA, or acetyl CoA. then converted to acetone and β-
hydroxy butyrate which are the main ketone bodies are called ketogenic amino acids.
These includes:- Phenylalanine, tyrosine, tryptophan, isoleucine, leucine, and lysine.
These amino acids have ability to form ketone bodies which is particularly evident in
untreated diabetes mellitus in which large amounts of ketone bodies are produced by
the liver (i.e. not only from fatty acids but also from ketogenic amino acids)

12. Ketogenic and glucogenic amino acids: The division between ketogenic and
glucogenic amino acids is not sharp for amino acids (Tryptophan, phenylalanine,
tyrosine and Isoleucine are both ketogenic and glucogenic).
Some of the amino acids that can be converted in to pyruvate, particularly (Alanine,
Cysteine and serine, can also potentially form acetoacetate via acetyl CoA
especially in severe starvation and untreated diabetes mellitus.

14. Peptides
• Proteins are macromolecules with a backbone formed by polymerization of
amino acids in a polyamide structure.
• These amide bonds in protein, known as peptide bonds formed by linkage of α -
carboxyl group of one amino acid with α- amino groups of the next amino acid
by amide bonds.
• During the formation of a peptide bond, a molecule of water is eliminated as
shown below:-
• A peptide chain consisting of two amino acid residues is called a dipeptide,
three amino acids
tripeptide (e. g Glutathione)

16. Glutathione
• Glutathione is a tripeptide
formed from amino acids
glutamate, cysteine and
Glycine, linked together in
that order. The glutamate is
linked to cysteine through
the γ- carboxyl group and α
- amino group of cysteine.
• Here, the carboxyl group is
first activated by ATP to
form an acyl-phosphate
derivative which is then
attacked by cysteine amino
group then undergoes
condensation with glycine.

17. • The role of GSH as a reductant is extremely important particularly in the highly
oxidizing
environment of the erythrocyte.
• The sulfhydryl of GSH can be used to reduce peroxides formed during oxygen
transport. The resulting oxidized form of GSH consists of two molecules
disulfide bonded together (abbreviated GSSG).
• The enzyme glutathione reductase utilizes NADPH as a cofactor to reduce
GSSG back to two moles of GSH.
• Hence, the pentose phosphate pathway is an extremely important pathway of
erythrocytes for the continuing production of the NADPH needed by glutathione
reductase.
• In fact as much as 10% of glucose consumption, by erythrocytes, may be
mediated by the pentose phosphate pathway.

18. Classification of Proteins

19. PROTEINS
• Proteins are macromolecules with a backbone formed by polymerization of
amino acids in a polyamide structure.
Class Assignment 2:
1. List functions of proteins (3 marks)
2. Using the flow chart on slide 18, describe the
classification of proteins (7 marks)

20. Protein Organization
Primary structure
The simplest level of protein structure, primary structure, is simply the
sequence of amino acids in a polypeptide chain.
For example, the hormone insulin has two polypeptide chains, A and B,
shown in diagram below.
(The insulin molecule shown here is cow insulin, although its structure is
similar to that of human insulin.) Each chain has its own set of amino acids,
assembled in a particular order.
For instance, the sequence of the A chain starts with glycine at the N-
terminus and ends with asparagine at the C-terminus, and is different from
the sequence of the B chain.

22. • The sequence of a protein is determined by the DNA of the gene that
encodes the protein (or that encodes a portion of the protein, for multi-
subunit proteins).
• A change in the gene's DNA sequence may lead to a change in the
amino acid sequence of the protein.
• Even changing just one amino acid in a protein’s sequence can affect
the protein’s overall structure and function.
• For instance, a single amino acid change is associated with sickle cell
anemia, an inherited disease that affects red blood cells.
• In sickle cell anemia, one of the polypeptide chains that make up
hemoglobin, the protein that carries oxygen in the blood, has a slight
sequence change.
• The glutamic acid that is normally the sixth amino acid of the
hemoglobin β chain (one of two types of protein chains that make up
hemoglobin) is replaced by a valine.

23. Secondary Structure
• The next level of protein structure, secondary structure, refers to local folded
structures that form within a polypeptide due to interactions between atoms of
the backbone.
• (The backbone just refers to the polypeptide chain apart from the R groups – so
all we mean here is that secondary structure does not involve R group atoms.)
• The most common types of secondary structures are the α helix and the β
pleated sheet.
• Both structures are held in shape by hydrogen bonds, which form between the
carbonyl O of one amino acid and the amino H of another.

25. Tertiary structure
• The overall three-dimensional structure of a polypeptide is called its tertiary
structure. The tertiary structure is primarily due to interactions between the R
groups of the amino acids that make up the protein.
• R group interactions that contribute to tertiary structure include hydrogen
bonding, ionic bonding, dipole-dipole interactions, and London dispersion
forces – basically, the whole gamut of non-covalent bonds.
• For example, R groups with like charges repel one another, while those with
opposite charges can form an ionic bond.
• Similarly, polar R groups can form hydrogen bonds and other dipole-dipole
interactions.
• Also important to tertiary structure are hydrophobic interactions, in which
amino acids with nonpolar, hydrophobic R groups cluster together on the
inside of the protein, leaving hydrophilic amino acids on the outside to interact
with surrounding water molecules.

26. • Finally, there’s one special type of covalent bond that can contribute to tertiary
structure: the disulfide bond.
• Disulfide bonds, covalent linkages between the sulfur-containing side chains of
cysteines, are much stronger than the other types of bonds that contribute to
tertiary structure.
• They act like molecular "safety pins," keeping parts of the polypeptide firmly
attached to one another.

27. Quaternary structure
• Many proteins are made up of a single polypeptide chain and have only three
levels of structure (the ones we’ve just discussed).
• However, some proteins are made up of multiple polypeptide chains, also
known as subunits. When these subunits come together, they give the protein
its quaternary structure.
• As mentioned earlier, hemoglobin carries oxygen in the blood and is made up of
four subunits, two each of the α and β types.
• Another example is DNA polymerase, an enzyme that synthesizes new strands
of DNA and is composed of ten subunits^55start superscript, 5, end superscript.
• In general, the same types of interactions that contribute to tertiary structure
(mostly weak interactions, such as hydrogen bonding) also hold the subunits
together to give quaternary structure

29. Protein Denaturation (Reading
Assignment)
• The phenomenon of disorganization of native protein structure is known as
denaturation.
• Denaturation results in the loss of secondary, tertiary and quaternary
structureof proteins. This involves a change in physical, chemical and
biological properties of protein molecule
Agents of denaturation
• Physical agents: Heat, violent shaking, X-ravs,UV radiation.
• Chemical agents : Acids, alkalies, organic solvents(ether,alcohol), salts of
heavy metals (Pb, Hg), urea,salicylate.

30. Characteristics of denaturation
a) The native helical structure of protein is lost
b) The primary structure of a protein with peptide linkages remains intact i.e.,
peptide bonds are not hydrolysed.
c) The protein loses its biologicalactivity.
d) Denatured protein becomes insoluble in the solventin which it was originally
soluble.
e) The viscosity ol denatured protein (solution) increaseswhile its surface tension
decreases.
f) Denaturation is associated with increase in ionizable and sulfhydryl groups of
protein. This is due to loss of hydrogen and disulfide bonds.
g) 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

31. h) Denaturation is usually irreversible. For instance, omelet can be prepared from an
egg (protein-albumin) but the reversal is not possible.
i) Careful denaturation is sometimes reversible (known as renaturation). Hemoglobin
undergoes denaturation in the presence of salicylate. By removal of salicylate,
hemoglobin is renatured.
j) Denatured protein cannot be crystallized

32. • Flocculation:
• lt is the process of protein precipitation at isoelectric pH.
• The precipitate is referred to as flocculum. Casein (milk protein) can be
easily precipitated when adjusted to isoelectric pH (4.6 by dilute acetic
acid.
• Flocculation is reversible.
• On application of heat, flocculum can be converted into an
irreversible mass, coagulum
Coagulation:
• The term 'coagulum' refers to a semi-solid viscous precipitate of protein.
reversible denaturation results in coagulation.
• Coagulation is optimum and requires lowest temperature at isoelectric
pH. Albumins and globulins (to a lesser extent) are coagulable proteins.
• Heat coagulation test is commonly used to detect the presence of
albumin in urine.

33. Protein Separation Methods (Reading
Assessment)
Group 1: Lydia, Brian, David (Protein Denaturation)
Group 2: Maxwell, Lennick, Gabriel, Peter, Reuben,
Dawn, Kevin, Eric, Ian (Protein separation methods)
Oral assessment (5 marks)