Page 25

Structural Analysis of Proteins by Biochemical Methods

1. Analysis of QUATERNARY structure
   1. Molecular weight versus concentration

   2. Molecular weight versus polyvalent cation concentration (e.g.: Mg⁺⁺)

   3. Other methods - generally biophysical (e.g.: fluorescence)
2. Analysis of TERTIARY structure
   1. Sedimentation, solubility, etc., versus denaturing conditions

   2. Effects of Disulfidryl bond disruption
      1. Oxidation via O₂, O₃ (ozone), Na₂O₂ (sodium peroxide), HCOOOH (performic acid)

      2. Reduction via mercaptoethanol or glutathione
3. Analysis of SECONDARY structure (amount of alpha-helix)
   1. Tritium Exchange versus urea concentration

   2. Other generally biophysical methods such as optical rotatory dispersion vs λ

4. Analysis of PRIMARY structure

   

   1. Looking at the terminal amino acids
      1. FDNB (Sanger method; page 10) regarding the N-terminal

      2. Amino Peptidase - like PacMan, chews in from the N-terminal one a.a. at a time. (an exopeptidase)

      3. Carboxy Peptidase - chews in from the C-terminal one a.a. at a time (another exopeptidase)

   2. Making fragments with known cision points
      1. Trypsin liberates the carboxyl of basic amino acids (an endopeptidase)

      2. Chymotrypsin liberates the carboxyls of aromatic amino acids (another endopeptidase)