protein purification bl4010 10.10.05 the basic techniques

Protein Purification

BL4010 10.10.05

The basic techniques

• 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

Electrophoresis (SDS-PAGE)

• Tris-glycine buffer

• 10% SDS

Electrophoresis

Protein Normal MW MW with dye Color ovalbumin 45,000 51,000 yellow carbonic anhydrase 29,000 30,000 orange trypsin inhibitor 20,100 23,000 green -lactalbumin 14,200 16,500 purple aprotinin 6,500 10,500 blue

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

Electrophoresis

• Protein detection using dyes– Coomassie blue– Sypro– Cybergreen– Silver staining

coomassie brilliant blue A595

Staining with dyes AFTER electrophoresis - no change in MWnon-covalent interaction

Western blotting

• Separate proteins by electrophoresis

• Transfer to membrane (e.g. nitrocellulose)

• Bind primary antibody• Bind secondary

antibody• Detection

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

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

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 0

Salt ppt. 165 2,800 1190 0.425 48 3.9

IEC 65 100 720 7.2 29 66

SEC 40 14.5 555 38.3 23 355

Affinity 6 1.8 275 152 11 1407

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.

Protein purification simuation

• http://www.tlsu.leeds.ac.uk/courses/bioc2060/proteinlab102/proteinlab.html

Enzymes

BL4010 10.12.05

• 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

Objectives

What is an enzyme?

Macromolecular biological catalyst

Can be protein or RNA

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

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)

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 !

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

How do enzymes work?• How do catalysts in general work?

catalysts lower the activationenergy of a reaction

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!

How do enzymes work?

• Enzymes accelerate reactions by lowering the free energy of activation

HOW?

Four contributing factors to enzyme catalysisNO 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

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