1. Chapter 5. Protein Purification and Characterization Techniques

2. Protein Purification and Characterization
Chapter 5
Protein Purification and Characterization
Unseparated Proteins Can Be Quantified
- The amount of enzyme in the extract can be quantified in terms of the catalytic effect it produces
 One unit of enzyme activity is defined as the amount of enzyme causing transformation of 1 mol of substrate per minute at 25 oC under optimal conditions
 Activity: the total units of enzyme in a solution
 Specific activity: the number of enzyme units per milligram of total protein A B
Activity: A = B
Specific activity: A < B

3. Extracting Pure Proteins from Cells
Chapter 5
Extracting Pure Proteins from Cells
How do we get the proteins out of the cells?
- Given that cells contain thousands of different kinds of proteins, how can one protein be purified?
 The first step is to break open the cells, releasing their proteins into a solution called a crude extract
 The extract is subjected to treatments that separate the proteins into different fractions based on a property such as size or charge, a process referred to as fractionation
- Homogenization:
 The cells are broken open by grinding with a blender, homogenizer, sonication, or cycles of freezing and thawing

4. Extracting Pure Proteins from Cells
Chapter 5
Extracting Pure Proteins from Cells
Extracting Pure Proteins From Cells
- Differential centrifugation
- Early fractional steps in a purification utilize differences in protein solubility, which is a complex function of pH, temperature, salt concentration, and so on.
e.g., The solubility of proteins is generally lowered at high salt (e.g., ammonium sulfate) concentrations (“salting out” effect)

5. Column Chromatography
Chapter 5
Column Chromatography
- Dialysis a procedure that separates proteins from solvents by the protein’s size
 Partially purified extract is placed in a tube made of semipermeable membrane
- The most powerful methods is use of column chromatography, which takes advantage of differences in protein charge, size, binding affinity, and other properties
 A porous solid material (stationary phase) is held in a column, and a buffered solution (the mobile phase) percolates through it
 The protein-containing solution percolates through the matrix, and the proteins migrate faster or more slowly through the column depending on their properties

6. Column Chromatography
Chapter 5
Column Chromatography

7. Column Chromatography
Chapter 5
Column Chromatography
- Size-exclusion chromatography
(gel-filtration chromatography)
 Based on size differences
 Stationary phase: Cross-linked gel particles
 Carbohydrate polymer: Dextran or agarose
(Trade name: Sephadex and Sepharose)
 Polyacrylamide (Trade name: Bio-Gel)

8. Column Chromatography
Chapter 5
Column Chromatography
- Affinity chromatography
 Uses the specific binding properties
 The beads in the column have a covalently attached chemical group (ligand)
 A protein with affinity for this particular chemical group will bind to the beads in the column

9. Column Chromatography
Chapter 5
Column Chromatography
- Ion-exchange chromatography
 Based on net charge: Cation(anion)-exchange chromatography
 Proteins with a net positive chargemigrate through the matrix more slowly

10. Column Chromatography
Chapter 5
Column Chromatography
Ref: High-performance liquid chromatography (HPLC)
 Use high-pressure pumps that speed the movement of the protein down the column

11. Column Chromatography
Proteins Can Be Separated and Purified
- Chromatographic methods are impractical at early stages, because the amount of chromatographic medium needed increases with sample size
 As each purification step is completed, the sample size generally becomes smaller

12. Chapter 5
Electrophoresis
- Electrophoresis is based on the motion of charged particles in an electric field toward an electrode of opposite charge
 Not generally used to purify but useful as an analytical method
 Permit to estimate quickly the number of proteins in a mixture, the degree of purity, isoelectric point and approximate MW.
- Electrophoresis is carried out in gels of the cross-linked polyacrylamide (PAAm)
 PAAm slows the migration of proteins in proportion to their charge-to-mass ratio
 The protein shape also affects the migration of the proteins

13. Chapter 5
Electrophoresis

14. Electrophoresis Proteins can be characterized by electrophoresis
Chapter 5
Electrophoresis
Proteins can be characterized by electrophoresis
- Sodium dodecyl sulfate (SDS) is commonly employed for estimation of purity and MW
 About one molecule of SDS is bound to every two amino acid residues
 SDS contributes a large net negative charge, rendering the intrinsic charge of the protein insignificant
 The native conformation of a protein is altered and most proteins assume a similar shape
 Electrophoresis in the presence of SDS separates proteins based on MW
 If the protein has two or more different subunits, they will be separated by the SDS

15. Chapter 5
Electrophoresis

16. Chapter 5
Electrophoresis
- Isoelectric focusing is a procedure used to determine the isoelectric point (PI) of a protein
 A pH gradient is established by allowing a mixture of low MW organic acids and bases (ampholytes)

17. Chapter 5
Electrophoresis
- Two-dimensional electrophoresis separates proteins of identical MW that differ in pI, or proteins with similar pI values but different MWs

18. Determining the Primary Structure
Chapter 5
Determining the Primary Structure
- Step 1 is to establish which amino acids are present and in what proportions
 After hydrolysis of protein (6 M HCl, 100 oC), the product is characterized by an amino acid analyzer
- Step 2 is to determine the identites of the N-terminal and C-terminal amino acids
 Useful to check whether a protein consists of one or two polypetides chains
- In step 3, the protein is cleaved into smaller fragments and the amino acid sequence is determined
 Edman degradation procedure: Stepwise modification starting from the N-terminal end, followed by cleavage of each amino acid in the sequence and the subsequent identification of each modified amino acid as it is removed.

19. Determining the Primary Structure
Chapter 5
Determining the Primary Structure
HPLC

20. Determining the Primary Structure
Chapter 5
Determining the Primary Structure
- The large polypeptides found in proteins must be broken down into smaller pieces to be sequenced efficiently
1) Breaking disulfide bonds: Disulfide bonds interfere with the sequencing procedure

21. Determining the Primary Structure
Chapter 5
Determining the Primary Structure
2) Cleaving the polypeptide chain
 Enzymes, called proteases, catalyze the hydrolytic cleavage of peptide bonds
 Some proteases cleave only the peptide bond adjacent to particular AA residues
e.g., Trypsin catalyzes the hydrolysis of only those peptide bonds in which the carbonyl group is contributed by either a Lys or an Arg residue

22. Determining the Primary Structure
Chapter 5
Determining the Primary Structure
3) Sequencing of peptides
 Each peptide fragment is sequenced separately by the Edman procedure
 This method labels and removes only the N-terminal residue from a peptide,
leaving all other peptide bonds intact
 Phenylisothiocyanate + polypeptides  phenylthiocarbamoyl (PTC) adduct
 Repeated procedure provide inform on the complete AA seqence
 The Edman degradation is now carried out on a machine, called a sequencer
4) Ordering peptide fragments
 The amino acid sequences of each fragment obtained by the two or more cleavage procedures (e.g., trypsin and cyanogen bromide) are examined
e.g., trypsin (R & K) + cyanogen bromide (M)

23. Determining the Primary Structure
Chapter 5
Determining the Primary Structure

24. Determining the Primary Structure
Chapter 5
Determining the Primary Structure
Amino Acid Sequences can also be deduced by other methods
- Mass spectrometry
- The sequence of a nucleotides in the gene

25. The Covalent Structure of Proteins
Small Peptides and Proteins can be chemically synthesized
- Many peptides are potentially useful as pharmacologic agents
- There are three ways to obtain a peptide
1) Purification from tissue; (2) Genetic engineering; or (3) Direct chemical synthesis
- R. Bruce Merrifield in 1962
 He synthesized a peptide using a solid support (polymer resin)
 The most important limitation of the process is the efficiency of each chemical cycle
 The synthesis of proteins of 100 amino acid residues takes a few days, while the same protein would be synthesized in about 5 seconds in a bacterial cell

26. The Covalent Structure of Proteins

27. The Covalent Structure of Proteins