1. Amino Acids, Peptides, and Proteins
Chapter 3
Amino Acids, Peptides,
and Proteins

2. Amino Acids

3. Amino Acids 20 Residues Numbering of carbons 1,2,3... from C of COO-
a , b, g… from C bonded to NH3+ and COO-
Absolute configuration of amino acids
L amino acids in biological system 1 2 a

4. Classification of Amino Acids
Nonpolar
UV absorption at 280 nm

5. Classification of Amino Acids
Nonpolar
Structural role

6. Classification of Amino Acids

7. Uncommon Amino Acids Collagen Myosin Cell wall Collagen A few proteins
Incorporation during translation
Prothrombin
Ca2+ binding proteins
Elastin

8. Amino Acids as Acids and Bases
Zwitterion
Acts as either an acid or a base
Ampholytes
Amphoteric substances
Substances with dual nature
Amino acid is a diprotic acid

9. Titration of Amino Acids
Two pKa and two buffering regions
pI: ioselectric point or isoelectric pH
The point with zero electric charge
Above pI : negative charge
Below pI : positive charge
pI = (pK1 + pK2)/2 = 5.92

10. Effect of Chemical Environment on pKa

11. Amino Acids with Ionizable R Group
pI = (pK1 + pKR)/2 = 3.22
pI = (pKR+ pK2)/2 = 7.59

12. Peptides and Proteins

13. Peptides and Proteins Peptide bond Dehydration reaction
Polypeptide vs. protein
Polypeptide: Mr<10,000
Amino-terminal (N-terminal)
Carboxyl-terminal (C-terminal)

14. Ionization of Peptide
Ionization of peptide
One free a-amino group
One free a-carboxyl group
Inonizable R groups
pKa of R groups in peptide
Different from pKa of free amino acid

15. Biologically Active Peptides and Polypeptides
Size
Small peptide
Vertebrate hormones
Oxytocin (9), thyrotropin-releasing factor (3), insulin ( )
Antibiotics
Large proteins
Titin: vertebrate muscle protein (27,000 a.a.)
Most of the proteins
< 2,000 a.a.
Oligomeric status
Single polypeptide chain
Multisubunit proteins
Oligomeric : at least two subunits are identical
Protomers : identical units
Calculation of the number of amino acid residues
Mr / 110
Average Mr of 20 a.a. : 138
Average Mr of protein a.a : 128
Removal of water during peptide bond formation : =110

16. Conjugated Proteins Conjugated proteins
Proteins with permanently associated chemical components
Prosthetic group
Non-amino acid part of a conjugated protein

17. Working with Proteins

18. Protein Purification Cell lysis Crude extract Fractionation
Use differences in protein solubility
Depending on pH, temperature, salt concentration etc.
Salting out
Addition of ammonium sulfate ((NH4)2SO4) for differential precipitation of proteins
Dialysis
Exchange of salts and buffer using semipermeable membrane
Column chromatography
Separation of proteins based on charge, size, binding affinity etc.
Stationary phase and mobile phase containing protein

19. Ion-exchange chromatography
Cation-exchange chromatography
Solid matrix : negatively charged group
Positive charged proteins migrate slowly
Anion-exchange chromatography
Solid matrix : positively charged group

20. Size-Exclusion Chromatography
Solid matrix : Beads with engineered pores of cavities
Small proteins enter the pores
Slow migration

21. Affinity Chromatography
Beads with covalently attached chemical group
Binding of proteins with affinity for the chemical group

22. Protein Purification HPLC (high-performance liquid chromatography)
Use high pressure pump that speed the movement of the protein molecules
Limited diffusion  High resolution
Determining the methods for protein purification
Mostly empirical

23. Separation of Protein by Electrophoresis
Separation of charged proteins in an electric field
Electrophoretic mobility of proteins
Depending on size and shape of proteins
SDS gel electrophoresis (SDS PAGE)
SDS (sodium dodecyl sulfate) binds to proteins roughly proportional to the molecular weight of the protein
Binding of 1 SDS/ 2 amino acids
Similar chare to mass ratio for all the proteins
Similar shape for all the proteins
Separation of proteins depending on the mass
Useful to determine molecular weight
Visualization of the bands by staining (e.g. Goomassie blue)

24. Determining Molecular Weight of a Protein
SDS PAGE
(polyacrylamide gel electrophoresis)

25. Procedure to determine the pI of a protein
Isoelectric Focusing
Procedure to determine the pI of a protein
Establishment of pH gradient
Gel containing a mixture of low molecular weight organic acids and bases (ampholytes)
Application of electric field
Each protein migrates until it reaches the pH corresponding to its pI

26. Two-Dimensional Electrophoresis
1o : Isoelectric focusing
2o : SDS-PAGE

27. Quantification of Proteins During the Purification
Enzyme assay
1 unit of enzyme activity
The amount of enzyme causing transformation of 1.0 mmol of substrate/min at 25oC under optimal conditions
Activity
Total units of enzyme in solution
Decrease during purification
Specific activity
The number of enzyme units/ mg of total proteins
Measure of enzyme purity
Increase during purification
Other assays
Biological activities
DNA binding, alteration of cell growth
Detection of the presence of the target protein
Western blotting