Lecture Notes on BCM 201 (General Biochemistry I)

Topic Amino Acids/Protein: Chemistry, Properties, Functions, Reactions, isolation,

purification and identification

Introduction to Amino Acids

Proteins are complex molecules composed of amino acids, which serve as their fundamental
building blocks. Present in all living organisms, proteins perform diverse functions, including
structural support, regulation, contraction, protection, transport, storage, membrane integration,
enzymatic activity, and toxin formation. They play essential roles in various life processes, such
as metabolism, movement, immune defense, cellular communication, and molecular
recognition.

The building blocks of proteins are amino acids. Individually, these amino acids too have
diverse roles in metabolism, signaling, and as precursors for biomolecules.

How many amino acids form proteins

In all known life, 22 amino acids are genetically encoded (proteinogenic). Of these, 20 are part
of the standard genetic code, while two additional amino acids, selenocysteine and pyrrolysine,
are incorporated through specialized translation mechanisms;

The standard twenty amino acids are as follows:

1. Glycine (gly)
2. Alanine (Ala)
3. Valine (Val)
4. Leucine (Leu)
5. Isoleucine (ile)
6. Proline (Pro)
7. Glutamate (Glu)
8. Glutamine (Gln)
9. Aspartate (Asp)
10. Asparagine (Asn)
11. Lysine (Lys)
12. Histidine (His)
13. Arginine (Arg)
14. Phenylalanine (Phe)
15. Tryptophan (Trp)
16. Tyrosine (Tyr)
17. Serine (Ser)
18. Cysteine (Cys)
19. Threonine (Thr)
20. Methionine (Met)

1.3 Common Structural features of amino acids

 Core Structure:
 o Amino group (-NH₂)
o Carboxyl group (-COOH)
o Side chain (R-group)
o Hydrogen atom
o All attached to a central (α) carbon.

The above general structural formula is common to all amino acids with each amino acids only
differing in their R groups. However, only PROLINE doesn’t obey this common structural
formular. why? This is because the alpha amino group of Proline is fused in a cyclic ring
structure with its R-group, unlike other amino acids with a stand-alone alpha amino group.
Proline is therefore also known as IMINO acids.

It must also be noted that due to the presence of a central carbon bonded to four different
groups, amino acids are said to exhibit a phenomenon called; CHIRALITY. Therefore, all amino
acids have a chiral carbon except GLYCINE. Glycine has hydrogen as its side chain; therefore it
doesn’t have four different groups attached to the central carbon. Due to their chirality, amino
acids two possible stereoisomers. (L-isomer and D-isomer). The L- denotes LEVOROTATORY;
which means it rotates plane polarized light to the left, while D- denotes
DEXTROROTATORY: which means it rotates plane polarized light to the right. Both D and L
isomers are collectively known as ENANTIOMERS.

NOTE: All amino acid residues are in the L-stereoisomer. However when chiral compounds
are formed by ordinary chemical reactions, they yield RACEMIC mixture which can be
converted into a single enantiomer by enzymes known as RACEMASE.

Amino acids can be classified into five groups based on the properties of their R groups

Knowing the chemical properties of amino acids is very important to understanding their
functions. The different R-groups of amino acids determine their properties in the proteins they
predominate. for example, the amino acid Histatin is a cationic antimicrobial peptide because it
is rich in the positively charged amino acids (Histidine). Below are the different classes of amino
acids and the different amino acids belonging to the different classes.
:
 Nonpolar aliphatic amino acids
These amino acids have nonpolar and hydrophobic R groups. Examples include glycine,
alanine, valine, leucine, isoleucine, methionine, and proline.
 Aromatic amino acids
These amino acids have aromatic side chains and are relatively nonpolar. Examples include
phenylalanine, tyrosine, and tryptophan.
 Polar, uncharged amino acids
These amino acids have R groups that are more soluble in water than nonpolar amino
acids. Examples include serine, threonine, cysteine, asparagine, and glutamine.
 Acidic amino acids
These amino acids have acidic or negatively charged R groups. Examples include glutamic
acid and aspartic acid. Negatively charged amino acids
 Basic amino acids
These amino acids have basic or positively charged R groups. Examples include lysine,
arginine, and Histidine. Basic amino acids are also called positively charged amino acids

Uncommon amino acids

These amino acids are not naturally encoded in the genetic code but may arise in plants or
bacteria through post-translational modifications. They can also be chemically synthesized and
used as pharmacological tools in drug development. Uncommon amino acids are structurally
similar to the 20 standard amino acids used in protein synthesis but are less prevalent in nature.
Despite their rarity, they play crucial roles in various biological processes and are of significant
interest in fields such as biochemistry and molecular biology.

Examples of Uncommon Amino Acids:

 Hydroxylysine and Hydroxyproline

Found in collagen, a fibrous protein, these amino acids require vitamin C for their
conversion to hydroxy forms, which are essential for collagen stability.

 Selenocysteine (Sec) and Pyrrolysine (Pyl)

These amino acids are considered the 21st and 22nd in the genetic code. They are
encoded by the UGA and UAG codons, typically stop signals, but are reassigned in
specific translation mechanisms.

Figure 1: CLASSES OF AMINO ACIDS

AMINO ACIDS ARE CLASSIFIED AS ESSENTIAL and NON-ESSENTIAL DEPENDING ON

THE ABILITY OF THE BODY TO SYNTHESIZE THEM
It is not uncommon to hear students define essential amino acids as those that are important
while non-essential are not important. This definition is not correct as far as biochemistry is
concerned.

These amino acids are merely classified on the basis of their dispensability in diet due to the
ability to produce them endogenously. All amino acids that can be produced by the body are
dispensable in diet. I.e. it is not essential to be consumed via diet. This however doesn’t in
anyway imply that they should never be taken through diet rather it means; not taking in diet
would not result in any deficiency since the body can produce them. Nonessential amino acids
on the other hand are those the body lacks the capability to synthesize therefore it is essential
they are consumed in our diet.

Essential include; Phenyalanine, Valine, Tryptophan, Threonine Isoleucine Methionine,

Histidine Arginine, Leucine and Lysine (PVT TIM haLL).

Note: Some amino acids, like arginine and histidine, may be conditionally essential during
certain periods of growth, such as pregnancy, adolescence, or recovery from trauma

Non-essential amino acids: Gluamate, Glutamine, Aspartate, Asparagine, Glycine, Alanine,

Serine, Cysteine, Proline, Tyrosine

1.5 AMINO ACIDS HAVE DUAL ROLES AS ACIDS AND BASES

Amino acids are amphoteric, meaning they can function as both acids and bases depending on
the pH of their surroundings. This property arises from their structure, which includes an acidic
carboxyl group (-COOH) and a basic amino group (-NH₂).

1.6 Acid-Base Properties of Amino Acids:

 Zwitterions
At physiological pH (approximately 7–7.4), amino acids exist as zwitterions—dipolar ions
with equal positive and negative charges.

 Isoelectric Point (pI)

The isoelectric point is the specific pH at which an amino acid exists in its zwitterionic
form, with no net electrical charge.

 Behavior in Acidic Conditions

In acidic environments, the carboxylate group (-COO⁻) gains a proton, resulting in a
positively charged amino acid.

 Behavior in Basic Conditions

In basic environments, the amino group (-NH₃⁺) loses a proton, resulting in a negatively
charged amino acid.

 Biological Significance
The acid-base properties of amino acids are essential for critical biological processes,
including protein folding, peptide bond formation, and enzyme activity regulation.
Note; Amino acids can exist in 3 state based on net charge. At isoelectric point (net charge:’0),
at low ph (+1) at high ph (-1). A few amino acids can however exist as +2 or -2 due to the
presence of additional amino or carboxyl groups. Glu and Asp are the only two amino acids that
can exist as -2 while Histidine, Lysine and arginine are the only ones that an exist in +2 states.

Physical and Chemical Properties

Physical Properties

1. Optical Activity:
o Most amino acids are chiral (except glycine) and exist as L- and D-isomers.
o Proteins use L-isomers.
2. Solubility:
o Polar amino acids are soluble in water.
o Nonpolar amino acids are less soluble in water.
3. Melting Point:
o High melting points due to ionic interactions in the solid state.

Reactions of Amino Acids

3.1 Reactions with Functional Groups

1. With Ninhydrin:
o Forms a purple complex with primary amino acids; used in qualitative analysis.
(will be discussed later in the latter parts of the note)
2. With Carboxylic Acids:
o Peptide bond formation through condensation.
3. Oxidation of Cysteine:
o Cysteine residues oxidize to form disulfide bonds (cystine).
4. Decarboxylation:
o Produces amines (e.g., histamine from histidine).
5. Deamination:
o Removal of the amino group to form α-keto acids.

Special Reactions

1. Peptide Bond Formation:

o Catalyzed by enzymes during protein synthesis.
o Amide linkage between the α-carboxyl group of one amino acid and the α-amino
group of another.
2. Transamination:
o Transfer of an amino group to α-ketoglutarate to form glutamate.
3. Maillard Reaction:
o Non-enzymatic browning reaction between amino acids and reducing sugars.
4. Post-Translational Modifications:
o Phosphorylation, methylation, acetylation, glycosylation modify amino acid side
chains in proteins.
 Biological Significance of Amino Acids

Amino acids perform numerous essential biological functions, including:

1. Protein Synthesis: Amino acids are the fundamental building blocks of proteins, which
are crucial for growth, development, and maintaining living systems. The sequence of
amino acids determines each protein's specific function.

2. Metabolism: They participate in biosynthetic reactions, contributing to the production of

nucleotide bases, hormones, and neurotransmitters.

3. Body Growth and Repair: Amino acids play a vital role in promoting growth and
facilitating tissue repair.

4. Osmotic Pressure Regulation: They help maintain osmotic pressure stability within
cells and tissues.

5. Neurotransmission: Amino acids are key components in neurotransmission, acting as

precursors or direct signaling molecules.

6. Chemical Messengers: Certain amino acids function as chemical messengers,

facilitating communication between cells.

7. Skin, Hair, and Nail Health: Amino acids like methionine and cysteine contribute to the
strength and flexibility of skin, hair, and nails.

8. Immune Support: They aid in strengthening the immune system, supporting the body’s
defense mechanisms.

9. Digestive Health: Amino acids play a role in sustaining the normal functioning of the
digestive system.

10. Buffering roles: Due to their amphoteric nature, amino acids act as physiological
buffers
o .

Chemistry of Peptides and proteins

The early parts of this notes has discussed amino acids. Now we discuss proteins.
Peptides, oligopeptides, and polypeptides are all chains of amino acids linked by peptide bonds,
but the main difference between them is their length:
 Peptides
Short chains of amino acids, typically 2–50 amino acids long
 Oligopeptides
Peptides with fewer than 20 amino acids, such as dipeptides, tripeptides, and tetrapeptides
 Polypeptides
 Longer, unbranched chains of amino acids, typically 50 or more amino acids long
 Proteins
One or more polypeptides joined together, and are essentially very large peptides

Peptide bonds are formed through a dehydration synthesis reaction, where the amino group of
one amino acid and the carboxyl group of another amino acid combine to form a water molecule
and a peptide bond

Note: though, polypeptides and proteins are used interchangeably, they actually are
different. Polypeptides have molecular weights below 10,000 while proteins have larger
molecular weights ..

NOTE: proteins could either exist as a single polypeptide chain or exist have two or more
polypeptides chains. These are called multisubunit [Link] following are important terms
related to multisubunit proteins

1. Oligomeric Proteins

 :
Oligomeric proteins are multimeric proteins composed of two or more polypeptide chains
(called subunits) that are held together by non-covalent interactions or, less commonly,
covalent bonds. These subunits may be identical (homooligomers) or different
(heterooligomers).
o The quaternary structure of oligomeric proteins defines the spatial arrangement
of these subunits.
o Subunits often work cooperatively or allosterically, influencing each other's
activity.

 Specific Examples:
o Hemoglobin: A heterotetramer composed of two α and two β subunits; involved
in oxygen transport in the blood.
o Insulin: A heterodimer composed of an A-chain and a B-chain linked by disulfide
bonds.
o Lactate Dehydrogenase (LDH): A homotetramer involved in the interconversion
of pyruvate and lactate during glycolysis.

2. Protomers

:
A protomer is the repeating structural unit or subunit within an oligomeric protein.
Protomers can be individual polypeptide chains or groups of polypeptide chains that
combine to form the functional oligomer.

o For example, in a homotetramer, each individual identical subunit is a protomer.

o In a heterotetramer, the protomer may consist of a combination of different
subunits.

 Specific Examples:
 o Hemoglobin: The protomer is an αβ heterodimer, as each α and β subunit pair
together to form functional dimers.
o Chaperonin (GroEL): Each protomer in the GroEL protein complex is a single
polypeptide chain that assembles into a heptameric ring.
o ATP Synthase: The F1 portion of ATP synthase contains protomers like α and β
subunits that form a functional catalytic complex.

Proteins can be classified based on their structure, composition, function, and solubility. Below
is a detailed classification with specific examples

1. Classification Based on Structure

A. Fibrous Proteins

 Definition: These are elongated, insoluble proteins with structural or support roles. They
are often arranged in a repetitive sequence.
 Properties: Strong, flexible, and resistant to stretching.
 Examples:
o Collagen: Found in connective tissues like tendons, ligaments, and skin.
o Keratin: Found in hair, nails, and skin.
o Elastin: Found in elastic tissues like blood vessels and lungs.

B. Globular Proteins

 Definition: Compact, spherical, water-soluble proteins that are involved in dynamic

processes such as catalysis and transport.
 Properties: Folded into a three-dimensional structure.
 Examples:
o Hemoglobin: Transports oxygen in blood.
o Myoglobin: Stores oxygen in muscles.
o Enzymes (e.g., amylase): Catalyze biochemical reactions.

C. Membrane Proteins

 Definition: Embedded in or associated with cell membranes, playing roles in signaling

and transport.
 Examples:
o Ion Channels: Transport ions across membranes.
o Receptors (e.g., G-protein coupled receptors): Mediate cellular signaling.

2. Classification Based on Composition

A. Simple Proteins

 Definition: Proteins made only of amino acids.

 Examples:
o Albumin: Found in egg whites and blood plasma.
 o Globulin: Found in blood plasma and involved in immune responses.

B. Conjugated Proteins

 Definition: Proteins combined with a non-protein component (prosthetic group).

 Subtypes and Examples:
o Glycoproteins: Protein + Carbohydrate
 Example: Mucin in mucus.
o Lipoproteins: Protein + Lipid
 Example: LDL (Low-Density Lipoprotein), involved in cholesterol
transport.
o Metalloproteins: Protein + Metal ion
 Example: Ferritin, stores iron.
o Nucleoproteins: Protein + Nucleic acid
 Example: Histones, associated with DNA in chromosomes.

Flavoproteins: protein +flavin nucleotide

Hemoprotein: Protein+heme (Porphyrin)

Note: The non-amino acid component of a conjugated protein is called prosthetic group

3. Classification Based on Function

A. Structural Proteins

 Function: Provide support and structure to cells and tissues.

 Examples:
o Collagen: Gives strength to connective tissues.
o Actin and Myosin: Components of muscle fibers.

B. Transport Proteins

 Function: Carry molecules across membranes or through the bloodstream.

 Examples:
o Hemoglobin: Transports oxygen.
o Albumin: Transports fatty acids and hormones.

C. Enzymatic Proteins

 Function: Catalyze biochemical reactions.

 Examples:
o Lipase: Breaks down fats.
o DNA Polymerase: Catalyzes DNA replication.

D. Regulatory Proteins

 Function: Regulate biological processes.

  Examples:
o Insulin: Regulates blood sugar levels.
o Growth Hormone: Stimulates growth and cell reproduction.

E. Defensive Proteins

 Function: Protect the body from pathogens or injury.

 Examples:
o Antibodies (Immunoglobulins): Target and neutralize pathogens.
o Fibrinogen: Involved in blood clotting.

F. Storage Proteins

 Function: Store nutrients or molecules for later use.

 Examples:
o Ferritin: Stores iron.
o Casein: Stores amino acids in milk.

4. Classification Based on Solubility

A. Albumins

 Properties: Soluble in water.

 Examples:
o Serum Albumin: Maintains osmotic pressure in blood.

B. Globulins

 Properties: Soluble in dilute salt solutions but insoluble in water.

 Examples:
o Immunoglobulins: Play a role in immune responses.

C. Prolamins

 Properties: Soluble in alcohol.

 Examples:
o Gliadin: Found in wheat; contributes to gluten.

D. Glutelins

 Properties: Soluble in acids or bases.

 Examples:
o Glutenin: Found in wheat; contributes to elasticity of dough.

This classification highlights the diversity of proteins and their roles in biological systems,
illustrating their significance in life processes.
 ISOLATION, PURIFICATION AND IDENTIFICATION OF PROTEINS

Biochemists are able to understand the structure and functions of proteins by studying them
individually. Before a protein could be studied in details, the researcher must first separate the
protein of interest from other proteins in a pure form and use appropriate biochemical
techniques to identify and determine their properties. .

The importance of separation when working with proteins stems from the fact that cells contain
thousands of different kinds of protein. Conventional separating techniques utilize properties
that differ from one protein to another including size, charge and binding properties.

ISOLATION OF PROTEINS

Breaking open cells to extract proteins is a crucial step in various biological research and
applications. The choice of method depends on the type of cells, desired protein yield, and
sensitivity of the proteins to different treatments. Below are some common methods:
Mechanical Methods:
 Manual Grinding: This method involves grinding cells using a mortar and pestle, often with liquid
nitrogen to freeze the tissue, making it more brittle. It's suitable for small-scale and tough
samples like plant tissues.

 Bead Beating: Cells are lysed by vigorous shaking with small, hard beads (glass, ceramic, or
metal) in a specialized machine. This method is efficient for disrupting a wide range of cells,
including bacteria and yeast.


 Sonication: High-frequency sound waves disrupt cell membranes. It's a versatile technique
suitable for various cell types but can generate heat, potentially damaging proteins.

 Homogenization: Cells are forced through a narrow opening at high pressure, shearing them
apart. This method is effective for large volumes of cells but can also generate heat.

 Freeze-Thaw Cycles: Repeated cycles of freezing and thawing disrupt cell membranes as ice
crystals form and expand. This method is gentle but can be time-consuming.
Chemical Methods:
 Detergents: Detergents solubilize cell membranes, releasing proteins into solution. Different
detergents have varying strengths and are chosen based on the type of cells and proteins of
interest.
 Enzymatic Lysis: Enzymes like lysozyme (for bacteria) or cellulase (for plant cells) can
specifically degrade cell wall components, releasing cellular contents.
 Osmotic Shock: Cells are exposed to a sudden change in osmotic pressure, causing them to
lyse. This method is suitable for bacteria with weak cell walls.

Considerations:
 Protein Stability: The chosen method should minimize protein degradation and denaturation.
 Cell Type: Different cell types have varying cell wall and membrane structures, requiring
different lysis methods.
 Protein Yield: The method should maximize protein extraction while minimizing cell debris.
 Downstream Applications: The lysis method can impact subsequent steps like protein
purification and analysis.
By carefully considering these factors, researchers can select the most appropriate method for
their specific protein extraction needs.
The resulting products from all the above processes are known as crude extracts.
PURIFICATION of CRUDE PROTEIN EXTRACTS
After the crude protein has been obtained. Various methods are available for purifying one or
more of the proteins it contains. Generally, the extract is subject to methods that separate the
proteins based on different properties such as size or charge. These processes are collectively
known as FRACTIONATION. Different methods of fractionation are explained below:

Principle and Procedure of Separating Crude Protein by Centrifugation

Principle:

Centrifugation is a technique that uses centrifugal force to separate particles from a solution
based on their size, shape, and density. When a sample is spun at high speed, the denser
particles are forced to the bottom of the tube, forming a pellet, while the lighter components
remain in the supernatant.

Procedure:

1. Sample Preparation:

 Cell Lysis: If working with cells, lyse them using appropriate methods like sonication,
homogenization, or chemical lysis to release cellular contents, including proteins.
 Crude Extract: Collect the cell lysate or other crude protein mixture.

2. Centrifugation:

 Low-Speed Centrifugation:
o Purpose: To remove large cellular debris, such as unbroken cells and nuclei.
o Procedure:
1. Transfer the crude extract to centrifuge tubes.
2. Spin the tubes at a low speed (e.g., 10,000 x g) for a short duration (e.g.,
10 minutes).
3. The pellet will contain cellular debris, while the supernatant will contain
smaller particles, including proteins.
4. Carefully transfer the supernatant to a new tube.
  High-Speed Centrifugation:
o Purpose: To separate smaller particles, such as organelles and membrane
fragments, from soluble proteins.
o Procedure:
1. Centrifuge the supernatant from the previous step at a higher speed (e.g.,
100,000 x g) for a longer duration (e.g., 1 hour).
2. The pellet will contain organelles and membrane fragments, while the
supernatant will contain soluble proteins.

3. Pellet Recovery (Optional):

 If the pellet contains the desired protein, resuspend it in a suitable buffer.

 Further purification steps, such as chromatography, can be applied to isolate specific
proteins.

Additional Considerations:

 Centrifuge Type: Choose a centrifuge with appropriate speed and capacity for your
sample volume.
 Centrifuge Tubes: Use tubes that can withstand the required centrifugal force and are
compatible with your centrifuge.
 Temperature Control: Consider using a refrigerated centrifuge to minimize protein
degradation.
 Buffer Conditions: The buffer used should maintain protein stability and prevent
aggregation.
 Multiple Rounds of Centrifugation: For more precise separation, multiple rounds of
centrifugation at different speeds can be performed.

By carefully controlling the centrifugation parameters, it is possible to effectively separate crude

protein mixtures into different fractions based on their size and density.

Dialysis: A Gentle Method for Protein Purification

Dialysis is a technique used to separate molecules on the basis of size, primarily used to
remove small molecules like salts and buffers from a protein solution while retaining the larger
protein molecules. It's a gentle method that avoids denaturing proteins.
How it Works:

1. Semi-Permeable Membrane: A dialysis membrane, a thin, porous film, is used. The

pores are large enough to allow small molecules like salts and buffers to pass through
but too small for larger molecules like proteins.
2. Diffusion: The protein solution is placed inside a dialysis bag or tube made of this
membrane. The bag is then immersed in a large volume of buffer solution.
3. Concentration Gradient: Due to the concentration gradient, small molecules diffuse out
of the dialysis bag into the surrounding buffer, while the larger proteins remain inside.
4. Equilibrium: Over time, an equilibrium is reached where the concentration of small
molecules inside and outside the bag is equal.

Applications of Dialysis in Protein Extraction:

 Buffer Exchange: Replacing the buffer solution surrounding a protein with a different
buffer. This is crucial for subsequent experiments or storage.
 Salt Removal: Removing excess salts that can interfere with protein activity or
subsequent purification steps.
 Small Molecule Removal: Removing small molecule contaminants like detergents or
reagents that might have been used during protein purification.

Key Considerations:

 Membrane Molecular Weight Cut-off (MWCO): Choose a membrane with an

appropriate MWCO to ensure efficient removal of small molecules while retaining the
protein of interest.
 Dialysis Buffer: Select a buffer that is compatible with the protein and the desired
experimental conditions.
 Dialysis Time: The time required for dialysis depends on the size of the molecules to be
removed, the membrane MWCO, and the volume of the dialysis buffer.
 Dialysis Temperature: Temperature can influence the rate of diffusion.

By carefully selecting the dialysis conditions, dialysis can be a powerful tool for purifying and
preparing proteins for further analysis or experimentation

Chromatography: A Powerful Tool for Protein Purification

Chromatography is a versatile technique used to separate mixtures of substances, including

proteins. It involves the distribution of components between a stationary phase and a mobile
phase. The principle is that different components interact differently with the stationary phase,
leading to their separation.

Here are some common types of chromatography used in protein purification:

1. Size-Exclusion Chromatography (Gel Filtration Chromatography)

  Principle: Separates proteins based on their size and molecular weight.
 Mechanism: A porous gel matrix acts as the stationary phase. Larger proteins cannot
enter the pores and elute first, while smaller proteins are retained and elute later.
 Application: Used to determine the molecular weight of proteins and to remove small
molecules like salts.

2. Ion-Exchange Chromatography

 Principle: Separates proteins based on their net charge.

 Mechanism: The stationary phase is a resin with charged groups. Proteins with
opposite charges bind to the resin, while those with the same charge elute.
 Application: Used to separate proteins based on their pI (isoelectric point).

3. Affinity Chromatography
 Principle: Separates proteins based on their specific binding to a ligand immobilized on
the stationary phase.
 Mechanism: The target protein binds to the ligand, while other proteins pass through
the column. The bound protein can then be eluted by changing the buffer conditions or
adding a competing ligand.
 Application: Highly specific purification of proteins, such as antibodies or enzymes.

5. Hydrophobic Interaction Chromatography (HIC)

 Principle: Separates proteins based on their hydrophobic properties.

 Mechanism: A hydrophobic stationary phase binds hydrophobic proteins. Proteins can
be eluted by decreasing the salt concentration, which reduces hydrophobic interactions.
 Application: Purifying proteins that are stable in high salt concentrations.

6. High-Performance Liquid Chromatography (HPLC)

  Principle: A high-pressure liquid is used as the mobile phase to force the sample
through a packed column.
 Mechanism: The stationary phase can be various types, such as ion-exchange, size-
exclusion, or reverse-phase.
 Application: High-resolution separation of complex protein mixtures.

By combining these techniques and optimizing the conditions, researchers can achieve highly
purified proteins for various applications, including structural studies, functional assays, and
therapeutic develop

Key Terms in Chromatography

Here are some key terms commonly used in chromatography:

Fundamental Terms

 Stationary Phase: The fixed phase in a chromatographic system, usually a solid or a

liquid supported on a solid.
 Mobile Phase: The phase that moves through the stationary phase, carrying the sample
components. It can be a liquid or a gas.
 Analyte: The substance being separated or analyzed.
 Eluent: The mobile phase that carries the analyte through the stationary phase.
 Eluate: The solution that emerges from the column containing the separated analytes.

The retention factor (Rf) is a ratio that measures how far a compound travels in
chromatography compared to the distance the solvent travels:
Explanation

Formula Rf = distance traveled by compound / distance traveled by solvent

What it How far a compound travels compared to the distance it could have traveled if it
measures moved with the solvent front
Electrophoresis in Protein Purification

Electrophoresis is a powerful technique used to separate and analyze proteins

based on their size and charge. It involves applying an electric field to a gel matrix
containing 1 the protein sample. The proteins migrate through the gel at different
rates, allowing for their separation.

Electrophoresis in Protein Purification:

1. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE):

 Principle: SDS-PAGE separates proteins based on their molecular weight.

 Process:
1. Denaturation: Proteins are denatured using SDS, a detergent that
imparts a negative charge to proteins, masking their native charge.
2. Gel Electrophoresis: The denatured proteins are loaded into wells
in a polyacrylamide gel and an electric field is applied.
3. Migration: Smaller proteins migrate faster through the gel pores
than larger ones.

2. Isoelectric Focusing (IEF):

 Principle: IEF separates proteins based on their isoelectric point (pI), the
pH at which a protein has no net charge.
 Process:
1. pH Gradient: A pH gradient is established in the gel.
2. Protein Migration: Proteins migrate until they reach their pI, where
 they stop moving.

3. Two-Dimensional Gel Electrophoresis (2-DE):

 Principle: Combines IEF and SDS-PAGE to separate proteins based on

both their pI and molecular weight.
 Process:
1. First Dimension (IEF): Proteins are separated by IEF.
2. Second Dimension (SDS-PAGE): The IEF gel is placed on top of
an SDS-PAGE gel and electrophoresis is performed.

Applications of Electrophoresis in Protein Purification:

 Protein Separation: Separating proteins based on their size, charge, or

both.
 Protein Identification: Identifying proteins using techniques like mass
spectrometry.
 Protein Purity Assessment: Determining the purity of a protein sample.
 Protein Quantification: Estimating the amount of protein in a sample.
 Protein Characterization: Studying the properties of proteins, such as
their molecular weight and isoelectric point.

By understanding the principles and applications of different electrophoresis

techniques, researchers can effectively purify and analyze proteins for various
biological and biomedical research purposes.

Spectrophotometry in Protein Identification

Principle: Spectrophotometry is a technique that measures the amount of light

absorbed by a substance at a specific wavelength. In the context of protein
identification, it leverages the fact that proteins contain amino acids with aromatic
side chains, such as tryptophan, tyrosine, and phenylalanine, which absorb
ultraviolet (UV) light.

Application:

1. Protein Concentration Determination:

o Direct UV Absorption: By measuring the absorbance of a protein
solution at 280 nm, the concentration can be estimated. This is a
rapid and non-destructive method.
o Colorimetric Assays: Protein assays like the Bradford, Lowry, or
BCA assays involve adding reagents to the protein solution to form
colored complexes. The intensity of the color, measured at a
specific wavelength, is proportional to the protein concentration.
2. Protein Purity Assessment:
o By monitoring the absorbance at 280 nm and 260 nm, the purity of a
protein sample can be assessed. A high 280/260 ratio indicates a
 purer protein sample, as nucleic acids absorb strongly at 260 nm.
3. Enzyme Activity Measurement:
o Spectrophotometry can be used to measure the rate of enzyme-
catalyzed reactions by monitoring the change in absorbance of a
substrate or product.
4. Protein-Ligand Interactions:
o By measuring changes in absorbance upon ligand binding,
information about binding affinity and stoichiometry can be obtained.

Beer-Lambert Law: The Beer-Lambert Law is the fundamental principle

underlying spectrophotometry. It states that the absorbance of a solution is directly
proportional to the concentration of the absorbing species and the path length of
the light through the solution.

Mathematically, it is expressed as:

A = εcl

where:

 A = absorbance
 ε = molar absorptivity (a constant specific to the substance)
 c = concentration of the substance
 l = path length of the light through the solution

By measuring the absorbance of a protein solution at a specific wavelength and

applying the Beer-Lambert Law, the concentration of the protein can be
determined.

Limitations: While spectrophotometry is a valuable tool, it has limitations, such as:

 Sensitivity: It may not be sensitive enough for detecting low

concentrations of proteins.
 Specificity: It may not be specific for a particular protein, especially in
complex mixtures.
 Interference: Other substances in the sample may interfere with the
absorbance measurement.

To overcome these limitations, spectrophotometry is often used in conjunction with

other techniques like chromatography and electrophoresis to provide a more
comprehensive analysis of protein samples.

Mass Spectrometry in Protein Analysis

Mass spectrometry (MS) is a powerful analytical technique that has revolutionized

protein analysis. It enables the precise measurement of the mass-to-charge ratio
(m/z) of ions, providing valuable information about protein identity, structure, and
modifications.
Key Steps in Protein Mass Spectrometry:

1. Sample Preparation:
o Protein Digestion: Proteins are often digested into smaller
peptides using enzymes like trypsin. This increases the efficiency of
ionization and mass analysis.
o Sample Cleanup: Techniques like solid-phase extraction (SPE) or
liquid-liquid extraction are used to remove contaminants and
concentrate the protein sample.
2. Ionization:
o Electrospray Ionization (ESI): A solution containing the protein
sample is sprayed through a fine needle at high voltage. The
droplets evaporate, leaving behind gas-phase ions.
o Matrix-Assisted Laser Desorption/Ionization (MALDI): The
protein sample is mixed with a matrix compound and crystallized on
a target plate. A laser pulse vaporizes the matrix and proteins,
forming ions.
3. Mass Analysis:
o Time-of-Flight (TOF) Mass Spectrometer: Ions are accelerated by
an electric field and their time of flight to a detector is measured.
The time of flight is directly proportional to the mass-to-charge ratio.
o Quadrupole Mass Spectrometer: Ions are filtered based on their
m/z ratio by applying oscillating electric fields to four rods.
o Orbitrap Mass Spectrometer: Ions are trapped in an orbitrap cell
and their oscillation frequency is measured, which is related to their
m/z ratio.
4. Data Analysis:
o Peptide Mass Fingerprinting: The masses of peptide fragments
generated from protein digestion are compared to theoretical
peptide masses from a protein database.
o Tandem Mass Spectrometry (MS/MS): Peptide ions are
fragmented, and the resulting fragment ions are analyzed to
determine the amino acid sequence.
o Bioinformatics Tools: Software tools are used to analyze mass
spectrometry data and identify proteins, modifications, and
interactions.

Applications of Mass Spectrometry in Protein Analysis:

 Protein Identification: Identifying proteins in complex mixtures, such as

those from cells or tissues.
 Protein Quantification: Measuring the relative abundance of proteins in
different samples.
 Post-Translational Modification Analysis: Detecting and characterizing
modifications like phosphorylation, glycosylation, and acetylation.
 Protein Structure Analysis: Determining protein structure and
conformation.
  Protein Interaction Studies: Identifying protein-protein interactions and
protein-ligand interactions.

Mass spectrometry has become an indispensable tool in proteomics research,

enabling scientists to investigate a wide range of biological questions.

Bioinformatics in Protein Analysis

Bioinformatics plays a crucial role in protein analysis, enabling researchers to

extract valuable insights from complex biological data. By combining computational
tools and biological knowledge, bioinformatics empowers scientists to understand
protein structure, function, and interactions at a molecular level.

Key Bioinformatics Techniques in Protein Analysis:

1. Sequence Analysis:
o Homology Search: Identifying similar sequences in databases
using tools like BLAST.
o Domain and Motif Prediction: Identifying functional regions within
protein sequences.
o Multiple Sequence Alignment: Aligning multiple protein
sequences to identify conserved regions and evolutionary
relationships.
o Phylogenetic Analysis: Constructing evolutionary trees to
understand the evolutionary history of proteins.
2. Structure Prediction:
o Ab Initio Prediction: Predicting protein structure from amino acid
sequence without relying on experimental data.
o Homology Modeling: Building protein models based on the
structure of homologous proteins.
o Threading: Fitting a protein sequence onto known protein
structures.
3. Functional Prediction:
o Functional Annotation: Assigning functions to proteins based on
sequence similarity and structural information.
o Protein-Protein Interaction Prediction: Identifying potential
interactions between proteins.
o Enzyme Function Prediction: Predicting the catalytic activity of
enzymes.
4. Proteomics Data Analysis:
o Peptide Identification: Matching peptide mass spectra to
theoretical peptide masses from protein databases.
o Protein Quantification: Estimating the abundance of proteins in a
sample.
o Post-Translational Modification Analysis: Identifying and
characterizing modifications like phosphorylation, glycosylation, and
acetylation.
Bioinformatics Tools and Databases:

 BLAST: Basic Local Alignment Search Tool for sequence similarity

searches.
 ClustalW: Multiple sequence alignment tool.
 HMMER: Profile Hidden Markov Models for protein family identification.
 MODELLER: Homology modeling software.
 I-TASSER: Ab initio protein structure prediction server.
 UniProtKB: A comprehensive protein sequence and function database.
 PDB: Protein Data Bank, containing experimentally determined protein
structures.

Applications of Bioinformatics in Protein Analysis:

 Drug Discovery: Identifying potential drug targets and designing novel

drugs.
 Disease Diagnosis: Developing diagnostic markers for diseases.
 Agricultural Biotechnology: Improving crop yields and resistance to pests
and diseases.
 Environmental Science: Studying the impact of environmental factors on
protein function.

By leveraging the power of bioinformatics, researchers can accelerate the pace of

protein research and contribute to advancements in medicine, agriculture, and
other fields.

Protein Structures and Their Relevance to Function

Proteins are the workhorses of the cell, performing a vast array of functions that
depend on their structure. Protein structure can be organized into four hierarchical
levels:

1. Primary Structure

 Definition: The linear sequence of amino acids in a polypeptide chain.

 Relevance to Function:
o Determines how the protein will fold (via interactions between side
chains).
o Mutations in the sequence (e.g., a single amino acid substitution)
can drastically alter function, as seen in diseases like sickle-cell
anemia.

2. Secondary Structure
  Definition: Localized folding patterns stabilized by hydrogen bonds
between the backbone atoms. The two main types are:
o α-Helices: Coiled structures stabilized by hydrogen bonds (e.g.,
found in DNA-binding proteins like transcription factors).
o β-Sheets: Extended, pleated structures that can be parallel or
antiparallel (e.g., seen in structural proteins like silk fibroin).
 Relevance to Function:
o Provides scaffolding and contributes to protein stability.
o Misfolding of secondary structures can lead to diseases, such as β-
sheet-rich amyloid plaques in Alzheimer’s disease.

3. Tertiary Structure

 Definition: The overall 3D shape of a protein, stabilized by interactions

such as:
o Hydrophobic interactions, hydrogen bonds, ionic bonds, van der
Waals forces, and disulfide bridges.
 Relevance to Function:
o Determines the active site in enzymes, critical for catalytic activity
(e.g., the "lock-and-key" or "induced-fit" models).
o Enables interactions with other molecules, such as substrates,
ligands, or other proteins.
o Structural changes in the tertiary conformation can regulate activity,
such as allosteric regulation in hemoglobin.

4. Quaternary Structure

 Definition: The assembly of multiple polypeptide chains (subunits) into a

functional complex.
o Examples: Hemoglobin (tetramer) and ATP synthase (multi-subunit
complex).
 Relevance to Function:
o Allows cooperative binding (e.g., hemoglobin's oxygen transport).
o Increases stability and functionality (e.g., collagen fibers provide
tensile strength).
o Mutations affecting subunit interfaces can disrupt function (e.g.,
certain inherited anemias).

Folding and Misfolding

 Proper folding is essential for function and is facilitated by chaperones.

 Misfolded proteins can form aggregates, leading to diseases such as:
o Alzheimer’s (amyloid plaques).
o Parkinson’s (Lewy bodies).
 o Prion diseases (misfolded infectious proteins).

Based on the more complex staructure of proteins. They can be classified

into fibrous and globular proteins

A. Fibrous Proteins

 Description: Elongated and provide structural support.

 Examples: Collagen, Keratin.

B. Globular Proteins

 Description: Compact and spherical, usually water-soluble.

 Examples: Hemoglobin, Enzymes.

Conclusion

The structure of a protein at all levels determines its functionality. Any disruption in
structure—whether due to genetic mutations, environmental factors, or misfolding
—can impair or entirely abolish its function, often leading to disease.
Understanding protein structure is thus fundamental to fields like biochemistry,
molecular biology, and drug design.

Basic Principles of Tests for Amino Acids and Proteins

Tests for amino acids and proteins rely on their unique chemical and
structural properties. These tests are commonly used in laboratories to
identify, quantify, or confirm the presence of proteins or specific amino
acids.

1. General Principles

 Chemical Reactions: Tests exploit functional groups in amino acids (e.g.,

amine, carboxyl, hydroxyl, or sulfhydryl groups) and specific bonds in
proteins (e.g., peptide bonds).
 Color Changes: Many tests involve reactions that produce a distinct color,
allowing for qualitative or quantitative detection.
 Specificity: Some tests are general for all proteins or amino acids, while
others target specific functional groups or types of amino acids.

2. Common Tests for Proteins

a) Biuret Test
  Principle: The test detects the presence of peptide bonds. In an alkaline
environment, copper(II) ions react with peptide bonds to form a violet-
colored complex.
 Procedure: Add Biuret reagent (a solution of copper sulfate and sodium
hydroxide) to the sample.
 Positive Result: Appearance of a violet color.
 Relevance: General test for proteins but does not detect free amino acids.

b) Xanthoproteic Test

 Principle: Aromatic amino acids (e.g., tyrosine, tryptophan, phenylalanine)

undergo nitration with concentrated nitric acid, forming yellow nitro
compounds.
 Procedure: Add concentrated nitric acid to the sample and heat. Neutralize
with sodium hydroxide.
 Positive Result: Yellow color changes to orange when neutralized.
 Relevance: Detects proteins containing aromatic amino acids.

c) Ninhydrin Test

 Principle: Ninhydrin reacts with free amino groups in amino acids or the N-
terminal amino group in peptides, producing a purple-blue complex
(Ruhemann’s purple).
 Procedure: Heat the sample with ninhydrin reagent.
 Positive Result: Purple-blue color.
 Relevance: Used for detecting free amino acids or small peptides.

d) Millon’s Test

 Principle: Tyrosine reacts with mercuric ions in Millon’s reagent to form a

red complex.
 Procedure: Add Millon’s reagent (mercury nitrate in nitric acid) to the
sample and heat.
 Positive Result: Red precipitate or solution.
 Relevance: Specific for tyrosine-containing proteins.

e) Lead Acetate Test

 Principle: Sulfur-containing amino acids (e.g., cysteine) react with lead(II)

acetate in an alkaline medium, forming a black precipitate of lead sulfide.
 Procedure: Add sodium hydroxide and lead acetate solution to the sample
and heat.
 Positive Result: Black precipitate.
 Relevance: Specific for proteins with sulfur-containing amino acids.
 f) Hopkins-Cole Test

 Principle: Tryptophan reacts with glyoxylic acid in the presence of

concentrated sulfuric acid to form a violet ring.
 Procedure: Add glyoxylic acid reagent to the sample, followed by
concentrated sulfuric acid.
 Positive Result: Violet ring at the interface.
 Relevance: Specific for tryptophan.

g) Sakaguchi Test

 Principle: Guanidinium groups in arginine react with α-naphthol and an

oxidizing agent (e.g., bromine water), forming a red color.
 Procedure: Add α-naphthol and bromine water to the sample.
 Positive Result: Red color.
 Relevance: Specific for arginine.

h) Pauly’s Test

 Principle: Histidine or tyrosine reacts with diazonium salts to produce a red

or yellow color.
 Procedure: Treat the sample with sulfanilic acid and sodium nitrite.
 Positive Result: Red (for histidine) or yellow (for tyrosine) color.
 Relevance: Detects histidine and tyrosine.

Quantitative Protein Tests

 Lowry Assay: Combines the Biuret reaction and Folin-Ciocalteu reagent to

detect proteins with high sensitivity.
 Bradford Assay: Coomassie Blue dye binds to proteins, producing a color
change from brown to blue.
 UV Absorption: Proteins absorb UV light at 280 nm due to aromatic amino
acids (tryptophan, tyrosine).

Bicinchoninic Acid (BCA) Test

The BCA test is a widely used assay to determine the total protein concentration in a
solution.
Principle:

 Reduction of Copper Ions: The assay relies on the Biuret reaction, where proteins
reduce Cu²⁺ ions to Cu⁺ under alkaline conditions.
 Color Development: Bicinchoninic acid (BCA) chelates the reduced Cu⁺ ions, forming a
purple-colored complex. The intensity of the color is directly proportional to the protein
concentration.

Procedure:

1. Mix the protein sample with the BCA reagent (a combination of bicinchoninic acid and
alkaline copper sulfate).
2. Incubate the mixture at 37°C for 30 minutes (or according to the protocol).
3. Measure the absorbance at 562 nm using a spectrophotometer.

Positive Result:

 A purple color develops in the presence of proteins, with intensity proportional to the
protein concentration.

Relevance:

 Used to estimate protein concentration in biological samples.

 Sensitive and compatible with many detergents and buffers used in protein preparation.

Kjeldahl Test

The Kjeldahl test is a classic method for determining nitrogen content in organic
compounds, particularly proteins.
Principle:

 Digestion: Proteins are digested with concentrated sulfuric acid in the presence of a
catalyst (e.g., selenium or copper sulfate). This converts the organic nitrogen in proteins
into ammonium sulfate.
 Neutralization: The digest is neutralized with a strong base (sodium hydroxide),
releasing ammonia gas.
 Ammonia Quantification: The released ammonia is distilled into a solution of boric acid
or a standard acid, where it is titrated to determine nitrogen content.

Procedure:

1. Digest the protein sample with sulfuric acid and a catalyst, heating it until the solution
becomes clear.
2. Add sodium hydroxide to the digest to liberate ammonia.
3. Distill the ammonia into a boric acid solution.
4. Titrate the boric acid solution with a standard acid to determine the amount of ammonia.
Positive Result:

 Nitrogen content is calculated, which can then be used to estimate protein content using
a conversion factor (commonly 6.25, assuming proteins are ~16% nitrogen).

Relevance:

 Used in food chemistry and agricultural sciences to determine protein content in food,
feed, and other organic materials.
 Highly accurate but labor-intensive and requires specialized equipment.

Protein Denaturation and Determination of 3D Protein Structures

Protein Denaturation

Definition:

Protein denaturation is the process by which a protein loses its native three-dimensional
structure due to the disruption of non-covalent interactions (e.g., hydrogen bonds, ionic
bonds, hydrophobic interactions) and, occasionally, covalent disulfide bonds. This often
results in loss of biological function.

Causes of Denaturation:

1. Physical Factors:
o Heat: Increased temperature disrupts hydrogen bonds and hydrophobic
interactions (e.g., cooking an egg).
o Mechanical Agitation: Vigorously shaking proteins (e.g., in foaming) disrupts
their structure.

2. Chemical Factors:
o pH Extremes: Alter charges on amino acid side chains, disrupting ionic and
hydrogen bonding (e.g., gastric acid denatures dietary proteins).
o Organic Solvents: Alcohol and acetone disrupt hydrophobic interactions.
o Detergents: SDS (sodium dodecyl sulfate) disrupts hydrophobic regions and
unfolds proteins.
o Chaotropic Agents: Urea and guanidinium chloride interfere with hydrogen
bonding, destabilizing the native structure.
Consequences of Denaturation:

 Loss of biological function (e.g., enzyme activity).

 Increased exposure of hydrophobic regions, which may lead to aggregation.
 Sometimes reversible (renaturation), depending on the protein and conditions.

PRACTICE QUESTIONS
1. Classify amino acids based on the ability of the body to synthesize them or
not
2. Explain the principle of chromatography in purification of proteins
3. What is denaturation of protein
4. Explain the relevance of spectrophotometry in protein study
5. Define Zwitterions, Chirality, retention factor
6. Discuss the differences among the following structures of protein; Primary,
secondary, tertiary and quaternary structures
7. Discuss the relevance of protein analysis in clinical diagnosis.
8. Differentiate between a simple protein and conjugated protein and give
specific examples of each
9. What are ampholytes. Explain why amino acids are good ampholytes
10. Show how ph affects the net charges of the following : Arginine, Histidine,
Gluatamate, Aspartate and Alanine.