1. Advanced Biochemistry 高等生化學 Amino Acids, Peptides and Proteins 陳威戎 2011. 03. 14

2. 1.Amino Acids 2.Peptides and Proteins 3.Working with Proteins 4.Covalent Structure of Proteins Amino Acids, Peptides and Proteins

3. Proteins are polymers of amino acids, with each amino acid residues joined to its neighbor by a specific covalent bond. Twenty different amino acids are commonly found in proteins. First: asparagine (1806) ; last: threonine (1938). Names derived from the sources: Asparagine – asparagus Glutamate – wheat gluten Tyrosine – cheese Glycine – sweet taste Amino Acids

4. 1. Amino acids share common structural features. General structure of an amino acid

5. Two conventions used to identify the carbons in an amino acid The  -carbon atom is a chiral center.

6. The absolute configuration of substituents of the asymmetric carbon atom Stereoisomerism in  -amino acids

7. 2. The amino acids residues in proteins are L stereoisomers Steric relationship of the stereoisomers of alanine to the absolute configuration of glyceraldehyde

8. 3. Amino acids can be classified by R group.

12. Absorption of ultraviolet light by aromatic amino acids

14. Reversible formation of the disulfide bond

17. 4. Uncommon amino acids also have important functions

19. 5. Amino acids can act as acids and bases

20. Beer-Lambert Law Absorbance (Optical density) = log I 0 /I =  cl

21. 6. Amino acids have characteristic titration curves

22. Effect of the chemical environment on pK a

23. 7. Titration curves predict the electric charge of amino acids Isoelectric point, Isoelectric pH, pI

24. ■環境影響分子的帶電性質：■環境影響分子的帶電性質： + Net Charge of a Protein Buffer pH Isoelectric point, pI - 3 4 5 6 7 8 9 10 0

25. 8. Amino acids differ in their acid-base properties

27. Peptides and Proteins 1. Peptides are chains of amino acids Formation of a peptide bond by condensation

28. 1. Peptides are chains of amino acids The pentapeptide serylglycyltyrosylalanylleucine, or Ser-Gly-Tyr-Ala-Leu

29. 2. Peptides can be distinguished by their ionization behavior

30. 3. Biologically active peptides and polypeptides occur in a vast range of sizes Many small peptides exert effects at very low concentrations: Aspartame (2 a.a.): artificial sweetener Oxytocin (9 a.a.): secreted by posterior pituitary stimulate uterine contractions Bradykinin (9 a.a.): inhibits inflammation of tissues Thyrotropin-releasing factor (3 a.a.): formed in hypothalamus stimulates the release of thyrotropin from the anterior pituitary Amanitin: toxic mushroom poison Antibiotics

31. 3. Biologically active peptides and polypeptides occur in a vast range of sizes

32. 4. Peptides have characteristic amino acid compositions

33. 5. Some proteins contain chemical groups other than amino acids

34. 6. There are several levels of protein structure

35. Working with Proteins 1. Proteins can be separated and purified Column chromatography Crude extract Fractionation Ammonium sulfate (salting out) Dialysis

36. Column chromatography Ion-exchange chromatography

37. Column chromatography Size-exclusion (Gel filtration) chromatography

38. Column chromatography Affinity chromatography

39. Purification Table

40. 3. Unseparated proteins can be quantified Activity versus specific activity

41. 2. Proteins can be separated and characterized by electrophoresis

42. SDS 在蛋白質表面均勻附上一層負電荷：

43. ■ 單元體分子量的測定： SDS-PAGE 0.5 1.0 kD 330 220 67 60 36 18.5 kD 94 67 43 30 20.1 14.4 Mol mass kD 300 200 100 80 60 50 40 30 20 10 Migration (R f ) Pharmacia: Molecular Markers for electrophoresis

44. Isoelectirc focusing

45. Two-dimensional electrophoresis

46. Covalent Structures of Proteins 1. The amino acid sequences of millions of proteins have been determined. Frederick Sanger

47. 2. Short polypeptides are sequenced using automated procedures.

48. 3. Large proteins must be sequenced in smaller segments. 1. Breaking disulfide bonds 2. Cleaving the polypeptide chain: proteases 3. Sequencing of peptides 4. Ordering peptide fragments 5. Locating disulfide bonds

49. Breaking disulfide bonds in proteins

50. Cleaving the polypeptide chain: proteases

51. Cleaving proteins and sequencing and ordering the peptide fragments

52. 4. Amino acid sequences can also be deduced by other methods 1.New methods based on mass spectrometry permit sequencing of short polypeptides (20-30 a.a.) in just a few minutes. 2. Development of rapid DNA sequencing methods.