proteins
DESCRIPTION
goodTRANSCRIPT
![Page 1: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/1.jpg)
PROTEINS
M.PRASAD NAIDUMsc Medical Biochemistry,Ph.D Research scholar.
![Page 2: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/2.jpg)
Amino Acids, Peptides, and Proteins1 . 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
![Page 3: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/3.jpg)
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
![Page 4: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/4.jpg)
Some Functions of Proteins
1 . Light : the result of reaction involving the protein luciferin
and ATP, catalyzed by the enzyme luciferase.
![Page 5: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/5.jpg)
2. Oxygen transport function : Red blood cell, hemoglobin
![Page 6: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/6.jpg)
3. Structural Proteins : Hair , horn, wool
![Page 7: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/7.jpg)
General Structure of Amino Acid
1 . Amino Acids
![Page 8: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/8.jpg)
Lysine : Basic Amino Acid
![Page 9: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/9.jpg)
Stereoisomerism in α-Amino Acids
Enantiomers : Nonsuperimposable mirror image
![Page 10: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/10.jpg)
Steric Relationship of The Stereoisomers of Alanine to The Absolute Configuration of L- and D-Glycelaldehyde
![Page 11: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/11.jpg)
![Page 12: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/12.jpg)
![Page 13: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/13.jpg)
![Page 14: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/14.jpg)
![Page 15: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/15.jpg)
![Page 16: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/16.jpg)
Properties of aromatic amino acids
1. Characteristics of UV absorption2. Wave length; A2803. Phe : phenyl-, Tyr : phenol-, Trp : indole-
** DNA, RNA….. A260 (purine, pyrimidine base)
![Page 17: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/17.jpg)
Disulfide bond formation
1. Bridge formation between proteins
2. Oxidation-reduction reaction
3. Insulin…… 2 interdisulfide bridges, one intradisulfide bridge
![Page 18: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/18.jpg)
Nonstandard amino acids in proteins
![Page 19: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/19.jpg)
![Page 20: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/20.jpg)
Amino Acid Can Act as Acid and Base
** Zwitterion …. dipolar ion** Can act as acid (proton donor) and base (proton acceptor)** Amphoteric (ampholytes)
![Page 21: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/21.jpg)
Absorption of light by molecules
• Spectrophotometer
•Wave length of light…. Ultrviolet 200-350nm
Visible 400-700
Infra red 700-
![Page 22: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/22.jpg)
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
![Page 23: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/23.jpg)
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.
![Page 24: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/24.jpg)
Glutamic Acid
pI= pK1 + pKR / 2
= 2.19 + 4.25 /2
= 3.22
![Page 25: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/25.jpg)
Histidine
pI = pK2 + pKR / 2
= 9.17 + 6.0
= 7.59
![Page 26: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/26.jpg)
2 . Peptides and Proteins
Oligopeptide :a few amino acids
Polypeptide : many amino acids
Amino terminal-
N-terminal-
Carboxyl terminal-
C-terminal
![Page 27: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/27.jpg)
Pentapeptide
Ser-Gly-Tyr-Ala-Leu
![Page 28: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/28.jpg)
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
![Page 29: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/29.jpg)
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
![Page 30: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/30.jpg)
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
![Page 31: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/31.jpg)
1. Column Chromatography
![Page 32: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/32.jpg)
(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
![Page 33: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/33.jpg)
(b) Size-exclusion Chromatography(Gel Filtration)
1. Protein size
2. Buffer pH, Salt --- No effect
3. Polymer beads---- no charged
![Page 34: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/34.jpg)
(c) Affinity Chromatography
1. Binding specificity
2. Ligands
3. Salt concentration
4. Polymer beads---- ligand attached
![Page 35: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/35.jpg)
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
![Page 36: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/36.jpg)
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
![Page 37: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/37.jpg)
1. Bind to proteins by hydrophobic interaction
2. Make proteins as negatively charged mass
3. So, separated on bases of mass (size)
![Page 38: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/38.jpg)
(a) Estimation of Molecular Weight of Proteins
( SDS Gel Electrophoresis)
![Page 39: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/39.jpg)
(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
![Page 40: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/40.jpg)
![Page 41: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/41.jpg)
(c) Two Dimensional Electrophoresis
Isoelectric focusing SDS gel electrophoresis
![Page 42: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/42.jpg)
Two Dimensional Electrophoresis of E. coli Proteins
- more than 2,000 proteins were visualized
![Page 43: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/43.jpg)
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
![Page 44: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/44.jpg)
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
![Page 45: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/45.jpg)
Frederick Sanger
![Page 46: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/46.jpg)
Steps in Sequencing a Polypeptide
Steps : Determination of amino acid composition
Identification of N-terminal residue(Sanger’s reagent)
Entire sequence (Edman degradation)
Sanger’s reagent
Edman reagent
![Page 47: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/47.jpg)
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
![Page 48: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/48.jpg)
Correspondence of DNA and Amino Acids
Proteome : to describe the entire proteins complement encoded by an organism’s DNA
![Page 49: Proteins](https://reader034.vdocuments.us/reader034/viewer/2022042700/554eaf1cb4c905977e8b4ed6/html5/thumbnails/49.jpg)