Chapter 3 – Parts A,B,CChapter 3 – Week 2 Parts D,E Purification of Lactate Dehydrogenase (LDH)...
Transcript of Chapter 3 – Parts A,B,CChapter 3 – Week 2 Parts D,E Purification of Lactate Dehydrogenase (LDH)...
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