PROTEINS

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PROTEINS

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

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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
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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
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Some Functions of Proteins
1 . Light the result of reaction involving the
protein luciferin and ATP,
catalyzed by the enzyme luciferase.
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2. Oxygen transport function Red blood cell,
hemoglobin
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3. Structural Proteins Hair , horn, wool
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1 . Amino Acids
General Structure of Amino Acid
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Lysine Basic Amino Acid
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Stereoisomerism in a-Amino Acids
Enantiomers Nonsuperimposable mirror image
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Steric Relationship of The Stereoisomers of
Alanine to The Absolute Configuration of L- and
D-Glycelaldehyde
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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)
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Disulfide bond formation

1. Bridge formation between proteins
2. Oxidation-reduction reaction
3. Insulin 2 interdisulfide bridges, one
   intradisulfide bridge

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Nonstandard amino acids in proteins
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Amino Acid Can Act as Acid and Base
Zwitterion . dipolar ion Can act as acid
(proton donor) and base (proton acceptor)
Amphoteric (ampholytes)
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Absorption of light by molecules

• Spectrophotometer
• Wave length of light. Ultrviolet 200-350nm
• Visible
  400-700
• Infra
  red 700-

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Titration Curve of Amino Acid

1. First COOH group titrated, then NH3 group
2. Tow buffer zones
3. Amino acid is amphipatic
4. Isoelectric point (pI)
5. Below pI ? positive charge,
6. Above pI ? negative charge

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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.
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Glutamic Acid
pI pK1 pKR / 2 2.19 4.25 /2 3.22

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Histidine
pI pK2 pKR / 2 9.17 6.0 7.59
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2 . Peptides and Proteins
Carboxyl terminal- C-terminal
Amino terminal- N-terminal-
Oligopeptide a few amino acids Polypeptide
many amino acids
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Pentapeptide
Ser-Gly-Tyr-Ala-Leu
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Tetrapeptide

• Acid-base behavior of a peptide
• N-terminal, C-terminal, R-groups
• 2. Peptides have a characteristic titration
  curve and a characteristic pI value

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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
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3. Working with Proteins
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)
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1. Column Chromatography
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(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

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(b) Size-exclusion Chromatography(Gel Filtration)

1. Protein size
2. Buffer pH, Salt --- No effect
3. Polymer beads---- no charged

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(c) Affinity Chromatography

1. Binding specificity
2. Ligands
3. Salt concentration
4. Polymer beads---- ligand attached

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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

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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

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1. Bind to proteins by hydrophobic interaction
2. Make proteins as negatively charged mass
3. So, separated on bases of mass (size)

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(a) Estimation of Molecular Weight of Proteins
( SDS Gel Electrophoresis)
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(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

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(c) Two Dimensional Electrophoresis
Isoelectric focusing
SDS gel electrophoresis
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Two Dimensional Electrophoresis of E. coli
Proteins
- more than 2,000 proteins were visualized
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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 1µmol/min/at 25ºC Specific
Activity number of enzyme units/mg
protein
Specific activity increased
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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 51 amino acid,
3 disulfide bonds
Bovine Insulin
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Frederick Sanger
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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
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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

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Correspondence of DNA and Amino Acids
Proteome to describe the entire proteins
complement encoded by an organisms DNA
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