Announcements Columbus Day 10/8 – No class, 10/9 BU Monday!

Make up section for Tuesday discussion: Wednesday 10/10, 5-6 pm, SAR 102

Monday Section: Tuesday 10/9, 10-11 am KCB 106

Wednesday Section: Wed. 10/10, 10-11 am SAR 300

No quiz in any section next week

Chapter 3 – Week 2 Parts D,E

Purification of Lactate Dehydrogenase (LDH)

Purpose:

Understand the effects of ionic strength on proteins and precipitate proteins via ammonium sulfate

Use dialysis to de-salt a protein sample

Protein Solubility ● Depends on number of hydrophilic and

hydrophobic residues on protein surface

● Majority of hydrophobic residues on inner globular core of protein

● Hydrophilic residues tend to stay on outer surface to interact with aqueous solution

● Charged and polar groups more likely to form salt bridges and hydrogen bonds

● Also depends on properties of the solution in which the protein is dissolved

Blue = hydrophillic White = hydrophobic

Major Factors Influencing Protein Stability

● Ionic Strength

● Many ions effect stability of other ions in solution

● pH

● Extremes tend to have poor protein stability

● Temperature

● Extremes tend to have poor protein stability

● Presence of Denaturants – SDS, Urea, Guanidinium chloride, [SCN-], etc.

● Necessary in some complex mixtures

● Dielectric Constant of the Solvent

● Relative polarity of solvent

Ionic Strength Effects

● Salts have different effects on proteins depending on ionic strength

● Protein solubility increases with neutral salts at low ionic strength – “Salting-in”

● Protein solubility decreases with neutral salts at high ionic strength – “Salting-out”

● Salting-in stabilizes charged groups of proteins

● Salting-out is competition between protein and salt for waters of hydration

● As salt concentration increases protein molecules aggregate and some fall out of solution

Ammonium Sulfate Precipitation

● Method that allows us to use the relative ionic strength of different proteins to purify individual proteins

● Different proteins precipitate at different levels of ionic strength due to different secondary and tertiary structure

● Ammonium sulfate used in protein purification and crystallography to help “salt-out” proteins

● Need to watch pH

● % Saturation is unit used to denote ionic strength

● “Salted-out” proteins are separated after salt addition by centrifugation

Dialysis

Selectively permeable membrane

Large Enzyme Molecules

Small salt/solvent molecules

● Method to separate solvents from the rest of the protein

● Semi-permeable membrane allows salt and solvent molecules out

● Protein molecules remain inside membrane

● Water diffuses in as salt diffuses out

● [Salt] inside and outside diffuse to equilibrium

● Buffer switched 2-3 times

● [Salt] from ~4 M to 4 μM

1S Supernatant – CRUDE EXTRACT

2P Pellet – Extraneous proteins

2S Supernatant – LDH + Other proteins

3P Pellet – LDH + Other proteins

Dialysis – 2 Buffer exchanges by TF’s –

3P–Dialyzed

Affinity Purification

Ultrafiltration

PURE LDH!

3S Supernatant – Extraneous proteins

Flow Chart for LDH Purification

Add (NH4)2SO4 (aq) to 40% Sat.

Centrifuge 12krpm, 10 min

Add (NH4)2SO4 (s) to 75% Sat. Centrifuge

12krpm, 10 min

See flow chart p. 70

Week 2: Procedure ● Ammonium Sulfate Precipitation

● Activity Assays

● Protein Concentration via Dye Binding

● Dialysis

Keep everything on ice! Especially LDH extract!

Week 2: Procedure

SAVE ALIQUOTS OF EVERYTHING!

● Ammonium Sulfate Precipitation

● Thaw protein from week 1, save 1 mL aliquot

● Set up buret with saturated (NH4)2SO4 solution

● Bring to 40% Saturation

Add 33 ml (NH4)2SO4 (aq) for 67 ml of crude extract

Use appropriate proportion

● Add dropwise in beaker, and let equilibrate 5 min after adding

● Centrifuge 10 min at 12,000 rpm

● After Spin: 2S and 2P Where is LDH?

● Resuspend 2P in 15 ml of buffer

● Ammonium Sulfate Precipitation

● Measure volume of 2S

● Add solid (NH4)2SO4 to bring final solution to 75%

– See table p. 65 (21.2 g/100 mL)

– Add while stirring over ~15 min

– Check pH midway through, ~7-8, adjust with NH3(aq)

● Equilibrate 5 min after addition

● Centrifuge 10 min at 12,000 rpm

● After Spin: 3S and 3P Where is LDH?

● Resuspend 3P in 15 ml of buffer

Week 2: Procedure

SAVE ALIQUOTS OF EVERYTHING!

● Activity Assays

● Do LDH Activity Assays on:

– 1S – Compare to week 1 activity

– 2S

– 2P – No dilution

– 3S – No dilution

– 3P

● Use 1 ml aliquots for assays

● All assays need to be in range of ΔA340/min of 0.05-0.25

What do you use to blank your spectrophotometer?

Week 2: Procedure

● Protein Concentration – Dye Binding Assay

● Make new standard curve if necessary

● Find protein concentration for:

– 1S

– 2S

– 2P

– 3S

– 3P

● Use 1 ml aliquots for protein concentration

● A595 should be within linear region of your standard curve

– Dilute protein when necessary

What do you use to blank your spectrophotometer?

Week 2: Procedure

Week 2: Procedure

● Dialysis

● TF’s will show to how prepare bag

● Put all but 1 ml aliquot of 3P sample in dialysis bag

● Leave some head space at top of bag for expansion during dialysis

● Label tape in clamp with initials and leave same labeled tube with TF’s

● TF’s will switch dialysis buffer at least twice before your next lab and then will freeze your protein sample – 3P Dialyzed Fraction

[Activity] = Units/ml = μmol of Substrate Consumed or Product Formed

min * ml

[Activity] = ΔC = ΔA340/min / εapp in mM-1 = (0.05/min)/(6.21 mM-1) =

0.0081 units/ml in the assay

You must account for the dilutions of your protein!

[ActivityUndiluted] = (ΔC)(Total Volume of Assay)(Dilution Factor)

(Volume of enzyme used in assay)

[ActivityUndiluted]= (0.0081 units/ml)(3.0 ml)(400) = 193 units/mL

(0.05 ml)

Activity Calculation

● Total Activity = (Activity)(Total Volume) = Units/ml* ml = Units

● Protein = Mass Protein/Volume Extract = mg/ml

● Total Protein = (Protein)(Total Volume) = mg/ml* ml = mg

● Specific Activity = Total Activity/Total Protein = Units/mg

● % Yield = Total Activity in Given Step

Remember to account for the dilutions of your protein!

For needed calculations, see purification table, p. 86

More Enzyme Calculations

Total Activity in Crude Extract x 100

Purification Table Calculations

Fraction Volume

(mL)

Corrected Volume

(mL)

Activity (units/

mL) Protein

(mg/mL)

Total Activity (units)

Total Protein

(mg)

Specific Activity (units/

mg) Yield (%) ΔA340/

min Dilution Factor

Homogenate 160 160

1S 80 160.0 272.5 125 43594 20000 2.2 100.0 0.071 400

2S 30 60.0 456.0 30 27362 1800 15.2 62.8 0.118 400

2P 15 30.0 10.0 44 301 1320 0.2 0.7 0.208 5

3S 35 70.0 12.0 17 839 1190 0.7 1.9 0.248 5

3P 17 34.0 560.4 26 19053 884 21.6 43.7 0.145 400

3P-D 18 36.0 510.1 22 18365 792 23.2 42.1 0.132 400

Pooled 8 32.0 499.7 4 15991 128 124.9 36.7 0.129 400

Conc. 2 8.0 1959.4 15 15675 120 130.6 36.0 0.169 1200

Only used 80 ml of original homogenate, therefore everything is multiplied by 2

Only loaded 9 ml for 5000 units for affinity column, therefore volume is multiplied by 4 (8 ml*4 = 32 ml)

Fraction

Corrected Volume

(mL) Activity

(units/mL) Protein

(mg/mL)

Total Activity (units)

Total Protein

(mg)

Specific Activity

(units/mg) Yield (%)

Homogenate 160

1S 160.0 272.5 125 43594 20000 2.2 100.0

2S 60.0 456.0 30 27362 1800 15.2 62.8

2P 30.0 10.0 44 301 1320 0.2 0.7

3S 70.0 12.0 17 839 1190 0.7 1.9

3P 34.0 560.4 26 19053 884 21.6 43.7

3P-D 36.0 510.1 22 18365 792 23.2 42.1

Pooled 32.0 499.7 4 15991 128 124.9 36.7

Conc. 8.0 1959.4 15 15675 120 130.6 36.0

Final Example Purification Table