BT-345

Separation and Analysis of Biomolecules

Prof. Shailly Tomar

Dept. of Biosciences and Bioengineering
shailly.tomar@bt.iitr.ernet.in

4 Credits

L-3
T-1
 Marks Distribution
25% CWS
(Presentation 12.5 points:
3 in a group
Research paper during tutorial class)

(Quiz = 12.5 points)

25 % Mid Term Exam

50 % End Term Exam
 Course Material

Class handouts/power point slides

Book for reference:

Principles and Techniques of Biochemistry
and Molecular Biology
by Keith Wilson and John Walker

Chapter 3 Centrifugation
Chapter 8 Protein structure, purification etc
Chapter 10 Electrophoretic techniques
Chapter 11 Chromatographic techniques

Biochemistry book : Leninger / Voet and Voet

 What is Biotechnology?

Is applied sciences in which biological processes,

biological knowledge, organisms, biological
molecules (such as enzymes, proteins, DNA, RNA)
are used to manufacture products intended to
improve the quality of human life.
 Bioengineering of
biomolecules/tissue/organs
Bioengineering means modifications in the genetic material
through in vitro recombinant deoxyribonucleic acid (rDNA)
techniques and for which the modification could not
otherwise be obtained through conventional breeding or
found in nature.

Also deals with: the use of artificial tissues, organs, or

organ components to replace damaged or absent body
parts.
•GMO: Bt-Corn, Bt-cotton
•Artificial heart
(research article of above- tutorial)
https://medschool.duke.edu/news/new-generation-artificial-
heart-implanted-patient-duke-first-us
 Why isolate/purify
cells/molecules?

Analysis of purity?
Purified biomolecules have various applications
•Food industry
•Pharma industry
•Biotech industry
•Agriculture industry
•Cosmetic industry
•Health sector
 Food industry
• Lactose intolerance : allergen

β-glucosidase enzyme
• Lactose free milk, milk products, ice cream

Enzyme has application in biofuel production

https://biotechnologyforbiofuels.biomedcentral.com/articles/10.1186/s13068-018-1125-9

Role and significance of beta-glucosidases in the hydrolysis of cellulose for bioethanol production.
 Food industry
• Gluten
Gluten is a mixture of proteins found in wheat and
related grains, including barley, rye, oat, and all
their species and hybrids.

Many people allergic to it.

Research: Find enzymes (mixture of

proteases/Flavourzyme) that specifically breaks
peptide bonds in Gluten.
3D structure imp of allergies…
Purified viruses??

Vaccine development? Inactivated viruses?

VLPs (no genome) - vaccine development
Virus based gene therapy?

Ab based therapy – research epitopes ?

Structure of viruses: CryoEM need pure samples for it
Biotechnology and pharmaceutical companies:
Manufacture biological products:
Eg:
1. Vaccines (bacterial/viral proteins vaccine)
2. Antibodies (used in cancer treatment/to suppress
organ rejection in transplant surgery)
3. Therapeutic uses: Insulin for diabetes, Interferon (IFN)
for anticancer chemotherapy
4. Organic acids, vitamins, alcohols, sterols, surfactants,
amino acids.

How to purify these products?

Separation techniques are used

 Molecular separation techniques
exploit
the properties of biological molecules
 Biomolecules
A biomolecule is any organic molecule that is produced by a living
organism, including large polymeric molecules such as proteins,
polysaccharides, and nucleic acids as well as small molecules such as
vitamins, lipids, hormones, ethanol, glycerol, amino acids etc

• Classification of biomolecules
• Properties of biomolecules
• Types of chemical bonds
• Chemical basis of molecular
interactions
Chemical basis of molecular interactions

• Polar and nonpolar molecules

– Water
• Universal solvent
• O-H bonds are polarized
– Partial positive and negative charges

~70% is water inside a cell

Chemical basis of molecular interactions
• Polar and nonpolar molecules
– Water is polar blank

H-bond 1.5 to 2.5 Å : a donor atom donates its covalently bonded

hydrogen atom to an electronegative acceptor atom.
Chemical basis of molecular interactions
• Polar and nonpolar
blan
molecules
– Hydrocarbons are
nonpolar
– Properties are conferred
by functional groups
– A functional group
• Behaves as a unit
• Confers physical
properties, chemical
reactivity, & solubility
 Biomolecules: functional groups

The functional groups shown here are found in many different biological
molecules, where “R” is the organic molecule.
How the functional groups of biomolecules are exploited for
separation and analysis?
DNA charged?

Agarose gel electrophoresis - n=moves in electric field

DNA - Separated based on weight (Why?)

becoz- DNA has a uniform mass/charge ratio
 Types of chemical bonds
• Covalent bonds (peptide bond/phosphodiester
bond/glycosidic bond/disulfide bonds)

• H-bonds
• Ionic bonds/salt bridges
• Van der waal interactions
 Types of chemical bonds

• Covalent bonds
– Based on electron sharing
– Strongest Bonds
– Eg: C – C bond,
Single bond
Bond length 1.54 Å
Bond energy ~348 kcal/mol

1 angstrom = 1.0 × 10-10 meters

CHIKV virions are spherical, enveloped particles of ∼70 nm in diameter.

 Types of chemical bonds
• Covalent bonds
– Eg: C = C bond,
Double bond
Stronger than single bond
Bond length 1.34 Å
Bond energy ~ 614 kj/mol
 Types of chemical bonds
• Covalent bonds
– Eg: C≡C bond,
triple bond
Stronger than double bond
Bond length 1.20 Å
Bond energy ~ 839 kj/mol
Nitrogenous bases in nucleic acid
 Types of chemical bonds
Purine base = Adenine (A)

Delocalization of electrons
 Types of chemical bonds

• Noncovalent bonds
– Not based on electron sharing
– Based on electric charges on atoms
– Attractive forces – readily broken and
reformed
– Differ in geometry, strength and specificity
– Affected by the presence of water
Types of chemical bonds
 Types of chemical bonds

Water has weaken

Hexane theDinteraction:
D ~1.8..lesser does same
higher is the interaction in are
…so salts biomolecules
not soluble.
Electrostatic interaction
Types of chemical bonds
 3A-6A

Core of the soluble protein

 Biophysics
The branch of knowledge that applies the principles of
physics and chemistry and the methods of mathematical
analysis and computer modeling to biological systems, with
the ultimate goal of understanding at a fundamental level the
structure, dynamics, interactions, and ultimately the function
of biological systems.
Biophysics seeks to explain biological function in terms of
the physical properties of specific molecules.
>The size of bio-molecules varies from small fatty acids and sugars (~1
nm = 10–9 m), to macromolecules like proteins (5–10 nm), starches
(>1000 nm), and the enormously elongated DNA molecules (over
10,000,000 nm = 1 cm long but only 20 nm wide).

>These building blocks of living organisms, assemble into cells, tissues,

and whole organisms by forming complex individual structures with
dimensions of 10, 100, 1000, 10,000 nm and larger.
Classification of Biomolecules