Lecture 3 Flashcards
Life cycle of a protein
-10 steps
- Synthesis
- Folding
- Processing
- Covalent modification
- Translocation
- Activation
- Catalysis
- “Aging”
- Ubiquitination
- Degradation
Highly purified proteins are essential for….
Examination of its physical and functional properties
Selective precipitation
Exploits differences in relative solubility of individual proteins as a function of:
- pH
- Polarity
- Salt concentration
Column Chromatography
- Stationary phase: consists of small beads loaded into a cylindrical container.
- Mobile phase: liquid.
Liquid from the mobile phase is collected into fractions.
High pressure liquid chromatography
Pumps a sample mixture or analyte in a solvent (mobile phase) at high pressure through a column with chromatographic packing material (stationary phase).
Gel filtration (size exclusion) chromatography
- Based on their size.
- Funnel, column (gel beads, consists of a hydrated polymer).
- Small proteins: fit into the gel beads, they will emerge last at the bottom.
- Large proteins: do not fit inside the gel beads, emerge first at the bottom.
ONLY USE this method if there is a large size difference between protein.
Ion- exchange chromatography
- Based on their net charge.
- Special gel beads, can have positive or negative charge (carbohydrate polymer).
- Proteins interact with the stationary phase.
- Proteins with a net positive charge will adhere to beads with negatively charged functional groups and vice- versa
- Bound proteins are then displaced by raising the ionic strength of the mobile phase, thereby weakening charge–charge interactions.
Hydrophobic interaction chromatography
- Based on the reversible absorption of bio molecules according to their hydrophobicity.
- 4 main stages - equilibration, sample application and wash, elution, regeneration.
- Polarity of the mobile phase is decreased by gradually lowering the salt concentration of the flowing mobile phase.
Affinity Chromatography
- Based on a protein’s affinity to bind to specific molecules.
- Gel beads are modified, specific group immobilized ligands are attached to it.
- Only proteins that interact with the immobilized ligand adhere.
Gel Electrophoresis (SDS-PAGE)
- In the presence of sodium docecyl sulfate (SDS).
- Based on the rates at which they migrate in an applied electrical field.
- SDS binds at a ratio of ONE MOLECULE OF SDS PER TWO PEPTIDE BONDS.
- SDS breaks the non- covalent interactions between the proteins denaturing it.
- SDS has a charge of -1, after many the protein net charge will be -1.
- Top: larger proteins band.
- Bottom: smaller proteins band.
Isoelectric focusing
- Based on isoelectric point.
- pH gradient is created and connected to a voltage source.
- Left side: low pH, acidic.
- Right side: high pH, basic.
- Proteins move until they reach a pH at which the net charge is 0.
Who was the first to determine the sequence of a polypeptide?
Frederick Sanger
- Reduced the disulfide bonds.
- Cleaved each chain into smaller peptides.
- Resulting peptides were isolated.
- Each peptide was reacted with Sanger’s reagent.
- Then the amino acid was identified.
Edman’s reagent and Sanger’s reagent
Edman’s reagent: to label the amino-terminal residue of a peptide.
Sanger’s reagent: used to generate a new amino- terminal residue.
Edman reaction
- React the protein with a special molecule called phenyl isothiocyanate.
- Molecule will attach the proteins uncharged alpha amino terminal.
- First peptide bond will cleave without breaking any other peptide bonds.
- PTH + amino acid and peptide with one less amino acid.
Name of the method that replaced Edman technique
Mass spectrometry
-ionizes chemical species and sorts the ions based on their mass-to-charge ratio.
