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Enzyme purification
Why purify proteins ?? -

(chapter 1)

to understand what is going on in a cell and between cells: reconstruction of metabolic and regulatory pathways. Pure enzymes/proteins required to study reactions, kinetics, regulation, etc. to understand deviations in normal metabolism or regulation processes, etc., due to abnormal enzymes/proteins (e.g. mutants of globins --> thalassemia's and sickle cell disease). to make a rational design of drugs possible, based on the 3D-structure of a protein. many proteins/enzymes have themselves an added value, as biocatalysts (proteases, lipases, glucose isomerase), therapeutics (insulin, interleukins), etc. any many other reasons.

Degree of purity required depends on application

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Enzyme purification
What is purification ??

(chapter 1)

Extraction of a single enzyme/protein from e.g. cells, tissue, etc. which may contain more than 1000 different proteins and lots of other biomolecules Keep structure and activity intact Physical boundaries of protein stability: pH temperature salt concentration oxygen sensitivity storage mechanical forces Each enzyme requires a specific strategy for purification 5 main types of purification methods described in this manual How is purification measured ?? Determination of specific activity Purification table Physical methods: SDS-PAGE; gelfiltration

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Preparing extracts for purification

Sources animal tissues plants microorganisms culture media subcellular fractions (e.g. mitochondria, membranes) etc.

Recombinant DNA technology is a very useful tool for protein purification for 'overproduction' of proteins using expression vectors for application of 'tags' to proteins (chapters 6,7) for excretion of proteins into the culture medium

Disruption and homogenization of cells, tissue, etc. Different techniques: shearing, grinding, sonication, French pressure cell disruption, depending of cell types Isolation of organelles; solubilization of membranes Excreted proteins

Clearing of extracts

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Cleared extracts should be used for column steps; clearing by centrifugation or filtration; solubilization of membranes

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Protein stability
Temperature pH

(chapter 2)
often 0 4o C (ice, cold room) determine pH where enzyme is most stable before attempting purification; work in buffers (chapter 2) chapter 3 anaerobic conditions for oxygen-labile enzymes

Salt Oxygen

Proteolytic enzymes add inhibitors: EDTA, PMSF, benzamidine, etc. Storage Mechanical forces frozen, suspension, addition of glycerol avoid frothing, foaming

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Buffers buffering capacity pKa pKa /T

(chapter 2)

Henderson-Hasselbalch equation: pH = pKa + log (base / conjugated acid) Examples Tris buffer: Acetate buffer: Tris NaAc + + HCl HAc KH2PO4 pKa 8.0

pKa 5.0 pKa 7.0

Phosphate buffer: K2HPO4 +

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buffering capacity of Tris-buffer

1,2

moles of H+ added/mole of Tris

0,8

0,6

pKa

0,4

0,2

0 4,5 5,5 6,5 7,5 8,5 9,5 10,5

-0,2

pH

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Main types of purification methods

(chapter 1)

The following main types of purification methods for proteins can be distinguished: 1. 2. 3. 4. 5. Precipitation methods Separation based on molecular size Separation based on charge Separation based on specific interaction with other biomolecules Separation based on other principles

Examples: 1. 2. 3. 4. 5. Ammonium sulfate precipitation Gel filtration Ion-exchange chromatography Bio-affinity chromatography Hydrophobic interaction chromatography; hydroxyapatite chromatography

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Enzyme activity
-

(chapter 1)

stoichiometry of participating reagents requirement for cofactors pH-optimum temperature optimum concentration of substrates [S] >> KM v [E]

continuous vs. discontinuous assay

Example Alcohol dehydrogenase: NAD+ + EtOH ----> NADH + aceetaldehyde

enzyme activity
1 0,8 0,6 A340 0,4 0,2 0 0 20 40 60 80 100 time (s) 140 120

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Calculation enzyme activity (see also page 5)

Assay mix: 0.99 ml 0.1 M Tris/HCl, pH 9.0, 100 mM EtOH, 0.5 mM NAD+,

Assay started by addition of 0.01 ml ADH, 50x diluted from stock solution Increase of absorbance at 340 nm followed: A340 = 0.6/min. v = [ A340/min ]/NADH, 340 = 0.6/6.22 = 0.096 mM.min-1 Amount of enzyme in assay in Units: 0.096 mM.min-1 = 0.096 mol.ml-1. min-1 = 0.096 U.ml-1. Amount of enzyme in ADH stock solution: 0.096 x 1000/10 x 50 = 480 U.ml-1.
(dilution in cuvet: 0.01 ml enzyme + 0.99 ml buffer) (dilution prior to addition to cuvet)

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Purification table
Measure for each purification step: 1. 2.

(chapter 1)

The volume of the enzyme solution (ml) The protein content of the solution (mg.ml-1) (chapter 13: Protein quantitation) The activity of the enzyme solution (U.ml-1)

3.

Total amount of enzyme (U): Activity (U.ml-1) x volume (ml) Specific activity (U.mg-1): Activity (U.ml-1) / protein content (mg.ml-1) Yield (%): Total amount of enzyme after a purification step / total amount of enzyme before that step Purification factor: Specific activity of enzyme after a purification step / specific activity before that step

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Purification table (example)

Step Volume (ml) Total activity (U) Total protein (mg) Specific Yield Purification activity factor (U/mg) (%)_____________

CE (1) 500 3,000 15,000 0.2 100 -AS (2) 100 2,400 4,000 0.6 80 3.0 IEC (3) 45 1,440 500 2.9 48 14.5 GF (4) 50 1,000 125 8.0 33 40.0 _________ __________________________________________________ Steps: (1) Crude cell extract; (2) ammonium sulfate fractionation; (3) ion exchange chromatography; (4) gel filtration.

Purity-check:

SDS-PAGE (chapter 10)

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Enzymes/proteins to be purified (ch. 12)

chymosin (rennin) calf veal rennet Leeuwarder kaasstremsel -glucosidase Pyrococcus furiosus E. coli overproducing clone lipoamide dehydrogenase Azotobacter vinelandii E. coli overproducing clone riboflavin binding protein eggs lipase Candida rugosa cell powder (Sigma)

p. 104

p. 96

p. 100 p. 112

p. 109

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Assays/activity measurements (ch. 12)

Chymosin (discontinuous assay) Measure clotting time of milk after addition of specified vol. of enzyme solution to milk; compare with standard Activitysample = (t2.v1/ t1.v2) Activitystandard t1, t 2 v1, v2 : : clotting time sample and standard dilution of sample and standard

-glucosidase (discontinuous assay) pH 5.0 p-nitrophenyl-glucopyranoside (NPG) p-nitrophenol + glucose pH 9.0 p-nitrophenol
NO2 p-nitrophenol OH H+ pKa = 7.0 p-nitrophenolate (yellow)

p-nitrophenolate (yellow)
NO2 O-

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Riboflavin binding protein No activity Riboflavin absorption measured (455 nm)

Lipase (continuous) pH = 7.5 p-nitrophenylacetate p-nitrophenolate + acetate Reaction at pH 7.5 (> pKa p-nitrophenol/p-nitrophenolate)

lipoamide dehydrogenase (continuous) NADH + oxidized lipoamide NAD+ + reduced lipoamide

NH2 S oxidized lipoamide lipoamide reduced NH2 S SH SH

Measure oxidation NADH (340 nm)