Proteins

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Proteins

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

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