C2-3. Page 144
Table C.2-3. Lactate Dehydrogenase Reaction Time Courses
Reading
number
time
(seconds)
1
0
2
15
3
30
4
45
5
60
6
75
7
90
8
105
9
120
A340 readings
50 µl
sample
100 µl
sample
200 µl
sample
300 µl
sample
400 µl
sample
Protein Purification Lab C2
Pages 115 to 168
Lab C.2
Four Periods
Protocol Page 135-160
Benchtop Protocols begin on page
398
Be sure to read theory starting
page 120
Exam
• Exam October 19,20
• Includes Carbohydrates, Enzyme kinetics,
and all protein labs and material related
there to.
• Pay attention to the powerpoints
– Read theory sections in the lab manual
• Will be about one hour in length
• Example of exam with answers is posted
on web
This Lab
•
•
•
•
4 lab periods
Prelab= 12 points
Lab Report= 55 points
First exam in period 4
You Have:
• Become skilled at using micro pipetters
• Have learned to use the spectrophotometer
– To determine concentration of an unknown
• Beers Law
– To measure activity of an enzyme
• Have learned how to organize experimental
protocols
• Have learned how to prepare a report.
In the next days
• You will use all of these skills to perform a
fundamental exercise in
Biochemistry/Molecular Biology
• Will learn basic protocols in protein
purification and analysis
Protein Purification
• A black art (proteins have personality)
• Requires knowledge of protein
– What kind of cell is it coming from
– What part of cell
– What does it do
• Particularly helpful
– Size
– Composition
Strategy
• Move from organism to pure protein in as
few steps as possible with as little loss of
activity (assayable quality) as possible
– Time and temperature are factors
Next 4 sessions
• Day one: Protein fractionation by
centrifugation, salt precipitation and
dialysis
• Day 2: Purification by affinity
Chromatography
• Day 3: Determination of concentration by
BCA assay
• Day 4: Determine purity by PAGE
Will fill out this critical table as we
proceed page 162 (day 4)
Table C.2-4. Enzyme Purification Table
Step
Net volume
(ml)
V0 units per
ml
V0 units
Total
(an “amount”)
Protein
content
(% of total)
Protein
concentration
(mg/ml)
Net
amount
of protein
(mg)
A
B
C
D
E
F
1.
Cleared
2.
(NH4)2SO
4
Supernata
nt
3. diluted
dialyzed
sample/
solution
placed on
column
4. pooled
peak tubes
from
column
Column C = (Column A)(Column B)
Column F = (Column A)(Column E)
Column G = Column C/Column F = Column B / Column E
Column D = Column C/first value in Column C
Specific
Activity
(V0/mg
protein)
G
Protocols for Protein Purification
• Highly individualized
• Use a common approach
– Fractionate crude extract in a way that protein
of interest always goes into the pellet or the
supernatant.
– Follow progress with functional assay
Lactate Dehydrogenase
• NADH + H+ + Pyruvate =NAD+ + Lactate
• Enzyme clears lactic acid from working muscles
• The obvious source of enzyme is muscle tissue
(heart & skeletal muscle, H&M, isomers)
• We will assay for the enzymes ability to convert
Pyruvate to Lactate
Begin with intact tissue
• Disrupt (step4&5)
– Blender, homoginizer
• Remove debris (step7)
– Centrifugation
• Precipitate/concentrate (step 14-16)
– Ammonium sulfate
• Remove salt (step 22)
– dialysis
• Purify (next Lab)
– Chromatography
• Analyze (Part B and week 3 & 4)
– Activity, molecular weight
Ammonium Sulfate ppt
page 124
• Has a wide range of application
• Relies on fact that proteins loose solubility as
concentration of salt is increased
– Is characteristic of particular protein
– Results in a partial purification of all proteins with
similar solubility characteristics
– Must determine [amm sulf] to precipitate your protein
empirically.
• Produces “salt cuts”
Salting in / Salting out
• Salting IN
• At low concentrations,
added salt usually
increases the solubility of
charged macromolecules
because the salt screens
out charge-charge
interactions.
• So low [salt] prevents
aggregation and
therefore precipitation or
“crashing.”
• Salting OUT
• At high concentrations
added salt lowers the
solubility of
macromolecules because
it competes for the
solvent (H2O) needed to
solvate the
macromolecules.
• So high [salt] removes
the solvation sphere from
the protein molecules and
they come out of solution.
Kosmotrope vs. Chaotrope
Page 125
• Ammonium Sulfate
• Increasing conc
causes proteins to
precipitate stably.
• Kosmotropic ion =
stabilizing ion.
• Urea
• Increasing conc
denatures proteins;
when they finally do
precipitate, it is
random and
aggregated.
• Chaotropic ion =
denaturing ion.
Dialysis Page 180
• Passage of solutes through a semi-permeable
membrane.
• Pores in the dialysis membrane are of a certain
size.
• Protein stays in; water, salts, protein fragments,
and other molecules smaller than the pore size
pass through.
Column Chromatography
2nd Day Page 126
Available in any volume
Gel Filtration
Ion Exchange
Affinity Chromatography
We will use bound Adenosine
-5’-monophosphate
(page 128 & 152). This is
part Of NAD+. LDH will
Bind. Release LDH by adding
NADH
NAD+
AMP
Affinity chromatography
• Remember: NADH is a co-substrate for lactate
dehydrogenase.
• We use AMP-Sepharose: AMP is covalently
bound to the affinity gel, which will not pass
through the filter.
• LDH binds to the AMP b/c it looks like half an
NADH.
• Thus LDH remains immobilized in the column
until we add NADH which binds tighter to the
LDH.
Protein Purification
page 130
Activity
A280
NADH
Protein Concentration
• Lowry ( most cited reference in biology)
– Color assay
• A280
– Intrinsic absorbance Page 132
– Relies on aromatic amino acids
• BCA page 133
– Modification of Lowry: increased sensitivity and
consistency
• Bradford
– Shifts Amax of dye from 465nm to 595nm
A280 Page 132
• Uses intrinsic absorbance
• Detects aromatic residues
– Resonating bonds
• Depends on protein structure, native state
and AA composition
• Retains protein function
Protein separation using SDS-PAGE
Page 158
(Laemmli system)
1. Apply protein/dye samples
into polyacrylamide gel wells
Stacking
gel
Resolving
gel
3. Remove the gel from the
apparatus and stain for
proteins
2. Run the electrophoresis until dye
reaches the end of the gel
SDS PAGE of Purification
Process
1.
2.
3.
4.
5.
Complete mix of proteins
High Salt
Ion exchange
Gel-filtratio
Affinity
10micrograms loaded in each lane
IMPORTANT
• Do not throw away anything until you are
certain you no longer need it
– Biggest source of problem in this lab
• Label everything clearly copy labels into
lab book
• Throwing out wrong fraction results in
starting over
– 3 days into experiment huge problem
Will follow Flow sheet: Page 138
Ground sirloin
(or alternative LDH source)
Place in blender, add buffer,
homogenize
Initial meat suspension
Centrifuge
Discard
precipitate
Cleared meat
extract
(save 1 ml)
Step 1
Ammonium sulfate
precipitation, Centrifuge
We will do only one NH4SO4 cut
Supernate
Precipitate
(save 1 ml)
Resuspend in
buffer
(save 1 ml)
Step 2a
Discard
remainder
Add PMSF,
Dialyze
Remove dialysate,
Store at -20oC
Step 2b
Save 3 samples
Will determine protein concentration
activity and purity
Will fill out this critical table as we
proceed page 162
Table C.2-4. Enzyme Purification Table
Step
Net volume
(ml)
V0 units per
ml
V0 units
Total
(an “amount”)
Protein
content
(% of total)
Protein
concentration
(mg/ml)
Net
amount
of protein
(mg)
A
B
C
D
E
F
1.
Cleared
2.
(NH4)2SO
4
Supernata
nt
3. diluted
dialyzed
sample/
solution
placed on
column
4. pooled
peak tubes
from
column
Column C = (Column A)(Column B)
Column F = (Column A)(Column E)
Column G = Column C/Column F = Column B / Column E
Column D = Column C/first value in Column C
Specific
Activity
(V0/mg
protein)
G
Today. Page 138 (part of group)
• Steps 1-5: Weigh muscle sample place in
blender with 50ml ice cold buffer homogenize for
2 minutes.
• Steps 6&7: remove large debris by centrifugation
Save Supernatant (remove 1ml (Microfuge tube)
for later analysis).
• Steps 9-13: Measure the volume of the
supernatant determine amount of ammonium
sulfate required for precipitation, weigh out 0.4
grams per/ml (NH4)2SO4
Today group 1 continued
• Step14-16: Slowly add salt to gently stirred
supernatant . Keep Cold!!See step 12
• Step 17: Centrifuge precipitate to a pellet
• Step 18-21: Save supernatant (1ml in microfuge
tube). Suspend pellets in 5ml cold buffer
• Step 22, 23: Add PMSF and place suspended
pellet in dialysis tubing and give to TA
Today group 2
• Set up standard assay as on page 142
– Measure loss of absorbance as NADH is converted to
NAD+
• Step 4 is similar to Kinetic curve you did for ADH
(page 124) only reversed as measure loss of
absorbance
• Steps 8-12: You will determine the velocity of
LDH catalyzed reaction by varying the
concentration of LDH with constant substrate
and cofactor. Be sure to adjust the amount of
reaction buffer to give 3.2 ml final volume in
each assay
Very Important: Page 145
Blank without NADH
Blank with NADH
Today group 2 continued
• You are establishing the assay conditions
you will use next week to follow the
purification of LDH. You must become
proficient at this assay.
Flow chart 1B (page 142)
Prepare the reaction mixtures
Each reaction will contain 3.200 ml:
Zero the spectrophotometer:
3.00 ml 50 mM buffer, pH 7.5
Add buffer and pyruvate
to the cuvette then set
the zero.
50 l NADH
50 l pyruvate
100 l Enzyme solution,
column fraction
or diluted Step 1, 2, 3 or 4
Add NADH and check
the A340 value.
Determine A340 at 15 sec and 45 sec
after adding the enzyme sample.
Note: You may have to adjust the
time frame of the rate measurement
or the amount of added enzyme to
achieve a non-spurious V0 value.
Calculate V0.
Divide the raw answer by the
product of 340 (for NADH)
times the cuvette path length to
convert the units to mole/liter
per sec units.
Spurious Vo Measurements
Same as with ADH
(this is similar to your [ADH] exp)
B) Increasing [E]
A) Small [E]
0.6
0.6
more
enzyme
0.4
A340
0.4
A340
0.2
0.2
0
0
0
15
30
45
time (sec)
60
75
0
15
30
45
time (sec)
60
Procedure (Page 143)
• 1 Step 1-6. Will create a kinetic curve for
LDH (adjust volume of buffer to make
3.2ml)
– Similar to ADH
• 2. Repeat kinetic curve with different
concentrations of enzyme
– This is protocol you will use as you purify LDH
• Do this assay on the unknown samples
from step one and 2a from group 1.
C2-3. Page 144
Table C.2-3. Lactate Dehydrogenase Reaction Time Courses
Reading
number
time
(seconds)
1
0
2
15
3
30
4
45
5
60
6
75
7
90
8
105
9
120
A340 readings
50 µl
sample
100 µl
sample
200 µl
sample
300 µl
sample
400 µl
sample
Today 200 microliter
200 micro
900
800
700
600
500
400
300
200
100
0
0
20
40
60
80
100
120
140
Next Week Column
Chromatography
• Due next time: Prelab assignment for
period 2 of ‘LDH Purification’
• You really should write up or otherwise
arrange what you did today as soon as
possible. Do Not Trust Your Memory
Next lab
• Need member of group to be here at 1:30
to begin washing column
• Will need to measure absorbance at 280
to determine that contaminating protein is
lost from column. Wash and measure until
A280 is constant.
Strategy
• For samples generated determine amount
of protein (A280 ) and activity
• Activity per microgram of protein =s
specific activity
• You strive for maximal activity per unit of
protein. (table C2-4 Column G, Page 162)
Will generate this elution profile
Page 153
contaminant protein
LDH
A280
V0
NADH
added
0
0
10
20
30
40
50
60
fraction (tube) number
(approximate only)
70
80
Will fill out this critical table as we
proceed page 162 (day 4)
Table C.2-4. Enzyme Purification Table
Step
Net volume
(ml)
V0 units per
ml
V0 units
Total
(an “amount”)
Protein
content
(% of total)
Protein
concentration
(mg/ml)
Net
amount
of protein
(mg)
A
B
C
D
E
F
1.
Cleared
2.
(NH4)2SO
4
Supernata
nt
3. diluted
dialyzed
sample/
solution
placed on
column
4. pooled
peak tubes
from
column
Column C = (Column A)(Column B)
Column F = (Column A)(Column E)
Column G = Column C/Column F = Column B / Column E
Column D = Column C/first value in Column C
Specific
Activity
(V0/mg
protein)
G