PROTEINS

M.PRASAD NAIDU
Msc Medical Biochemistry,
Ph.D Research scholar.



Amino Acids, Peptides, and Proteins
1 . Amino Acids Share Common Structural Features
1. 20 Amino Acids and Classification
2. Amphoteric Properties and Titration curve
3. Isoelectric Point(pI)
2. Peptides and Proteins
1. Peptide Bond : Oligopeptide, Polypeptide
2. Characteristic Amino Acid Composition
3. Conjugated Proteins
4. Protein Structure : Primary, Secondary, Tertiary
Quaternary Structure


3. Working With Proteins
1. Protein Purification : Crude Extract, Fractionation,
Column Chromatography, HPLC, Electrophoresis
4 . Covalent Structure of Proteins
1. Amino Acid Sequencing : Edman Degradation
N-terminal, C-terminal determination
2. Breaking disulfide bond, Cleaving polypeptide chain
Sequencing of peptide, Ordering peptide fragments
Locating disulfide bonds
3. Peptides can be chemically synthesized
Some Functions of Proteins
1 . Light : the result of reaction involving the protein luciferin
and ATP, catalyzed by the enzyme luciferase.
2. Oxygen transport function : Red blood cell, hemoglobin
3. Structural Proteins : Hair , horn, wool
General Structure of Amino Acid
1 . Amino Acids
Lysine : Basic Amino Acid
Stereoisomerism in -Amino Acids
Enantiomers : Nonsuperimposable mirror image
Steric Relationship of The Stereoisomers of Alanine to The
Absolute Configuration of L- and D-Glycelaldehyde
Properties of aromatic amino acids
1. Characteristics of UV absorption
2. Wave length; A280
3. Phe : phenyl-,
Tyr : phenol-,
Trp : indole-
** DNA, RNA.. A260
(purine, pyrimidine base)
Disulfide bond formation
1. Bridge formation between proteins
2. Oxidation-reduction reaction
3. Insulin 2 interdisulfide bridges, one intradisulfide bridge
Nonstandard amino acids in proteins
Amino Acid Can Act as Acid and Base
** Zwitterion . dipolar ion
** Can act as acid (proton donor) and base (proton acceptor)
** Amphoteric (ampholytes)
Absorption of light by molecules
Spectrophotometer
Wave length of light. Ultrviolet 200-350nm
Visible 400-700
Infra red 700-
Titration Curve of Amino Acid
1. First COOH group titrated,
then NH
3
group
2. Tow buffer zones
3. Amino acid is amphipatic
4. Isoelectric point (pI)
5. Below pI positive charge,
6. Above pI negative charge
Effect of the chemical environment on pKa
** The pKa of any functional groups is greatly affected by its chemical environment.
Similar effects can be observed in the active site of enzymes.
Glutamic Acid
pI= pK
1
+ pK
R
/ 2
= 2.19 + 4.25 /2
= 3.22

Histidine
pI = pK
2
+ pK
R
/ 2
= 9.17 + 6.0
= 7.59

2 . Peptides and Proteins
Oligopeptide :a few amino acids
Polypeptide : many amino acids
Amino terminal-
N-terminal-
Carboxyl terminal-
C-terminal
Pentapeptide
Ser-Gly-Tyr-Ala-Leu
Tetrapeptide
1. Acid-base behavior of a peptide:
N-terminal, C-terminal, R-groups
2. Peptides have a characteristic
titration curve and a characteristic
pI value
Levels of structure in proteins
Primary structure of protein : amino acid sequence
Secondary structure of protein : local structure
Tertiary structure of protein : three dimensional
structure
Quaternary structure of protein : subunits
Protein Separation and Purification

Why Purification? : to understand the structure and functions of proteins
Purification Procedure : 1. Crude extract 2. Subcellular fractionation
3. Fractionation of proteins---- Size, Charge, pH,
Solubility, Salt concentration, Dialysis
Methods of Protein Purification and Identification:
1. Column Chromatography ---- Ion exchange chromatography
Size-exclusion chromatography
Affinity chromatography
2. Gel Electrophoresis ------- SDS gel electrophoresis
Isoelectric focusing
Two dimensional electrophoresis
(purification)
(Identification)
3. Working with Proteins
1. Column Chromatography
(a) Ion Exchange Chromatography
1. Anion Exchanger--- matrix with
cation(+)
Cation Exchanger--- matrix with
anion(-)
2. Buffer pH is very important (pI)
3. Salt Effect


(b) Size-exclusion Chromatography(Gel Filtration)
1. Protein size
2. Buffer pH, Salt --- No effect
3. Polymer beads---- no charged
(c) Affinity Chromatography
1. Binding specificity
2. Ligands
3. Salt concentration
4. Polymer beads---- ligand
attached
2. Gel Electrophoresis
1. Use electricity
2. Use polyacrylamide gel (polymer)
3. Based on the migration of charged
proteins in electric field
4. pI of proteins are very important
5. Charge , mass, and shape of
protein are importnat

Visualization of Proteins after Electrophoresis
1. Staining with dye(Coomassie blue,
BPB)
2. Destaining with acetic acid solution
3. Smaller and larger charge proteins
move faster
1. Bind to proteins by hydrophobic interaction
2. Make proteins as negatively charged mass
3. So, separated on bases of mass (size)
(a) Estimation of Molecular Weight of Proteins
( SDS Gel Electrophoresis)
(b) Isoelectric focusing
1. Determine the pI value of proteins
2. Use ampholyte solution
3. Proteins are distributed along pH
gradient according to their pI values
4. pI value of protein---- R-group
(c) Two Dimensional Electrophoresis
Isoelectric focusing SDS gel electrophoresis
Two Dimensional Electrophoresis of E. coli Proteins
- more than 2,000 proteins were visualized
Unseparated Proteins (Enzyme) can be Quantified
Quantitating of Proteins (Enzyme Activity):
1. Overall enzymatic reaction 2. Analytical procedures
3. Cofactors or coenzymes 4. Substrate concentration
5. Optimum pH and temperature
1 Unit of enzyme: 1mol/min/at 25C
Specific Activity:
number of enzyme units/mg protein
Specific activity increased
4. Covalent Structure of Proteins (Primary Structure)
Primary structure Amino acid sequence
Different amino acid sequence different function
Genetic disease single amino acid change
Similar function protein of different species
similar sequence of amino acids
Bovine
Insulin
Bovine Insulin : 51 amino acid,
3 disulfide bonds
Frederick Sanger
Steps in Sequencing a Polypeptide
Steps : Determination of amino acid composition
Identification of N-terminal residue(Sangers reagent)
Entire sequence (Edman degradation)

Sangers reagent
Edman reagent
Large Proteins must be Sequenced in Smaller
Segments
1. Breaking disulfide bonds
2. Cleaving the Polypeptide Chain
3. Sequencing of Peptides
4. Ordering Peptide Fragments
Correspondence of DNA and Amino Acids
Proteome : to describe the entire proteins complement
encoded by an organisms DNA