1. Protein Purification BL

2. The basic techniques Electrophoresis (size/charge) Immunological
Concentration (size)
precipitation
ultrafiltration
dialysis
centrifugation
Chromatography (size/charge/chemistry)
ion exchange
size exclusion
affinity
hydrophobic interaction
Electrophoresis (size/charge)
"native"
denaturing
isoelectric focusing
2-dimensional
Immunological
chromatography
in situ imaging
immunoblotting

3. Electrophoresis (SDS-PAGE)
Tris-glycine buffer
10% SDS

4. Electrophoresis
“Prestained” markers have dyes covalently bound BEFORE electrophoresis - increased MW

5. Electrophoresis Protein detection using dyes Coomassie blue Sypro
Cybergreen
Silver staining
coomassie brilliant blue A595
Staining with dyes AFTER electrophoresis - no change in MW
non-covalent interaction

6. Western blotting Separate proteins by electrophoresis
Transfer to membrane (e.g. nitrocellulose)
Bind primary antibody
Bind secondary antibody
Detection

7. Immuno-Affinity Chromatography
antibody fixed to matrix
protein binds to antibody
wash unbound and loosely bound proteins off column
elute protein with change in salt/pH

8. Hydrophobic interaction chromatography
Hydrophobic group bound to solid phase
Binding
high salt (increases water surface tension, decreases available water molecules, increases hydrophobic interactions)
Elution
decrease salt
add detergent
decrease polarity
of mobile phase

9. Assay and Specific Activity
Fraction
Volume (ml)
Total
protein (mg)
Total activity
Specific Activity
(activ./mg)
Percent Recovery
(ratio t.a.)
Fold
Purificat'n
(ratio s.a.)
Crude extract
3,800
22,800
2460
0.108
100
Salt ppt.
165
2,800
1190
0.425 48
3.9
IEC 65
720
7.2 29 66
SEC 40
14.5
555
38.3 23
355
Affinity 6
1.8
275
152 11
1407

10. When is protein pure or pure enough?
Criteria for purity
When is protein pure or pure enough?
homogeneity
protein complexes?
constant specific activity
Practical: further attempts at purification are futile since the only material left in the fraction is the material that actually is responsible for the activity being assayed.

11. Protein purification simuation

12. Enzymes BL

13. Objectives What is an enzyme? How do enzymes work?
energetics
underlying general mechanism
components (prosthetic groups, coenzymes)
specific mechanisms
Ch.13.1, 13.2, 14.1, 14.2, 14.3, 14.4, 14.5

14. What is an enzyme? Macromolecular biological catalyst
Can be protein or RNA

15. What is an enzyme? Macromolecular biological catalyst
What is a catalyst?
is not altered by reaction
participates but emerges unchanged
increases the rate at which substrates and products reach equilibrium
does not alter equilibrium

16. Why enzymes?
Why invest energy and resources into creating a large catalyst?
Enzymes endow cells with the remarkable capacity to exert kinetic control over thermodynamic potentiality
Fine tune selectivity (substrate binding specificity)
Fine tune catalytic rate
Additional regulatory control (e.g. allostery, signalling networks)

17. Enzymes are good catalysts
Enzymes can accelerate reactions as much as 1016 over uncatalyzed rates!
Urease is a good example:
Catalyzed rate: 3x104/sec
Uncatalyzed rate: 3x10 -10/sec
Ratio is 1x1014 !

18. Enzymes are selective catalysts
Enzymes selectively recognize proper substrates over other molecules
Enzymes produce products in very high yields - often much greater than 95%
Specificity is controlled by structure - the unique fit of substrate with enzyme controls the selectivity for substrate and the product yield

19. How do enzymes work? How do catalysts in general work?
catalysts lower the activation
energy of a reaction

20. Understand the difference between G and G‡
The transition state
Understand the difference between G and G‡
The overall free energy change for a reaction is related to the equilibrium constant
The free energy of activation for a reaction is related to the rate constant
It is extremely important to appreciate this distinction!

22. How do enzymes work?
Enzymes accelerate reactions by lowering the free energy of activation
HOW?

23. Four contributing factors to enzyme catalysis NO ONE MECHANISM ACCOUNTS FOR CATALYSIS ALONE!
Specific substrate binding
local concentration of reactants
productive orientation of reactants
binding energy used to offset loss of entropy
Control over solvent interactions
desolvation (binding energy offsets)
ordered solvent in binding pocket
Induction of strain on reactants
Alternate reactive pathway
transient involvement of enzyme functional groups

24. How do enzymes work?
Enzymes accelerate reactions by lowering the free energy of activation
Enzymes do this by binding the transition state of the reaction better than the substrate