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
●
●
pH
●
●
Extremes tend to have poor protein stability
Presence of Denaturants – SDS, Urea, Guanidinium
chloride, [SCN-], etc.
●
●
Extremes tend to have poor protein stability
Temperature
●
●
Many ions effect stability of other ions in solution
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
●
●
●
●
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
Dialysis
Selectively
permeable
membrane
Large
Enzyme
Molecules
Small
salt/solvent
molecules
Add (NH4)2SO4 (aq)
to 40% Sat.
Centrifuge 12krpm,
10 min
1S Supernatant –
CRUDE EXTRACT
2P Pellet – Extraneous
proteins
Add (NH4)2SO4 (s)
to 75% Sat.
Centrifuge
12krpm, 10 min
Flow Chart for LDH
Purification
2S Supernatant – LDH +
Other proteins
3P Pellet – LDH + Other
proteins
3S Supernatant –
Extraneous proteins
Dialysis – 2 Buffer
exchanges by TF’s –
3P–Dialyzed
Affinity Purification
Ultrafiltration
PURE LDH!
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
●
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
SAVE ALIQUOTS OF EVERYTHING!
Week 2: Procedure
●
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
SAVE ALIQUOTS OF EVERYTHING!
Week 2: Procedure
●
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
●
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 Calculation
[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)
More Enzyme Calculations
●
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
Total Activity in Crude Extract
x 100
Remember to account for the dilutions of your protein!
For needed calculations, see purification table, p. 86
Purification Table Calculations
Specific
Activity
(units/
ΔA340/ Dilution
mg)
Yield (%) min
Factor
Corrected Activity
Total
Total
Volume Volume (units/ Protein Activity Protein
Fraction (mL)
(mL)
mL) (mg/mL) (units)
(mg)
160
160
Homogenate
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)
Final Example Purification Table
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