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INQUIRY:
What are some reasons that could contribute to failure of kinetic activity during the
enzyme measurement assay?
Purification of an enzyme into an in vitro setting requires that the enzyme be isolated
from the tissue that contains it. The main thrust of this course will be to isolate and
purify LDH from beef heart, and to express and purify barracuda LDH. To this point you
have done several procedures to prepare for your work with the LDH preparation. Here
we will proceed with the homogenization, centrifugation and ammonium sulfate
precipitation of the beef heart LDH. These are the initial purification steps before we do
chromatographic separations.
Enzymes lower the activation energy of a chemical reaction. This is shown below.
reaction (pyr and NADH) can either be substrates or products, depending on the
direction of the reaction. In order to follow the purification of the LDH protein, you will
follow its activity. We will assay the activity of LDH in the lactate to pyruvate direction,
in other words, the oxidation of lactate and reduction of NAD+. We need to determine
the number of moles (review the definition of moles) of pyruvate and NADH formed per
unit time. This is the rate or kinetics of the reaction.
We need to know how much protein in the extract. Go from a gemish to single protein
(LDH). Go from LDH being 1/10,000th of the total protein to being the only protein.
Protein determination
You have already seen a standard curve for protein determination in Week 2, so you
have seen how protein determinations are set up. Your values for mg protein/ml will go
into your purification table. We use the Bradford method.
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BioC 4025W Fall 2017 Lab 3
There are many other protein determination methods that have various limitations. A
comprehensive (more than you ever wanted to know) is at the following website:
http://wolfson.huji.ac.il/purification/Protocols/Comparision_of_Methods.htm
The father of protein determination is Oliver Lowry, who invented the Lowry Method.
For many years this was the most cited paper in the biochemical literature:
(Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) Protein
measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275)
In lecture, you see a graphic presentation of the first steps in the purification of LDH
from beef heart. Animal is slaughtered, heart removed (weighed), and cut into cubes.
Tissue is then homogenized, aliquoted and given to you to commence purification.
You need to follow the purification by constructing a purification table, as illustrated for
the first purification steps. Your table will grow as you add more purification steps.
Crude 100
homogenate
20,000Xg
supernatant
40%
(NH4)2SO4
supernatant
65%
(NH4)2SO4
pellet
65%
(NH4)2SO4
supernatant
Next step
here
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BioC 4025W Fall 2017 Lab 3
You can see that in order to fill in the table you must keep track of all volumes, dilutions,
proteins, and activities. It is sort of like keeping an accountant’s ledger. It doesn’t hurt
to keep too much data, if you are not sure what you will need to fill in the table. A note
of caution, when you back calculate, don’t forget to figure in the aliquots that were
removed for assay, they need to be added back to get an accurate determination.
You will need to convert the change in absorbance (at 340 nm for NADH) per unit time.
Just for thought: Look at the equation and consider what each part is responsible
for and how it relates to our assay.
What is the 6220? What does the -1 coefficient do? Why do we multiply by 106?
Why do we multiply by 0.001L?
How does specific activity differ from the first step in purification to the next?
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BioC 4025W Fall 2017 Lab 3
Centrifugation
Dialysis
The high salt (NH4)2SO4 from the salting out needs to be removed by dialyzing the
protein preparation. The dialysis membrane is has a pore size which retains the protein
but allows the salt to
be removed. A dialysis
membrane is a semi-
permeable film
(usually a sheet of
regenerated cellulose)
containing various
sized pores.
Molecules larger than
the pores cannot pass
through the membrane
but small molecules
can do so freely. In
this manner, dialysis
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BioC 4025W Fall 2017 Lab 3
Remember this week you will not be doing anything on Expressed Barracuda LDH
(Tract II)
Next week you will begin purification by column chromatography. This week you will
pour your first column (manual) for ion exchange chromatography. The theory for ion
exchange chromatography, for both manual and using the Duoflow (BioRad’s Brand)
Fast Protein Liquid Purification (FPLC), will discussed in the intro to next week’s lab.
2. What must be done before putting centrifuge tubes into the rotor?
3. Why do you add ground (NH4)2SO4, and why do you add it slowly?
Procedure 1
ENZYME PURIFICATION
1. Beef heart was cut up into small pieces. Fatty and connective tissue was
removed.
2. 75g of beef heart was put into a blender with 225 mls of cold 0.05M sodium
phosphate, pH 7.0 and in a Waring blender at high the highest speed for 2
minutes.
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BioC 4025W Fall 2017 Lab 3
4. Tubes with homogenate were allowed to thaw. For each homogenate tube, a
0.5 ml aliquots were dispensed into 1.5 microfuge tube and labeled CRUDE. This
tube is in your ice bucket. Please save this sample. You will do an assay on it
later.
5. The homogenates were put into white centrifuge tubes then spun at 16,500 RPM
for 25 minutes at 4ºC and put into your ice buckets. You will use the supernatant
and NOT the PELLET.
Please note: since 0.5 ml was removed from your centrifuge tube, this leaves a
starting volume of 29.5 ml.
You have in your ice bucket the white centrifuge tube from step 5. Save a
0.5 ml aliquot of the supernatant from that tube for assays. Label the tube
20,000 x g and put the tube in your ice bucket.
6. Pour the supernatant into a graduated cylinder to measure the volume then into a
beaker. Put the beaker with the supernatant into a “sandwich” box, add ice
around the beaker, add a stir bar and turn on. Do not turn on too high. SLOWLY
add ground ammonium sulfate to the supernatant so that is becomes a 40%
saturated solution. This requires 0.242 g of ammonium sulfate for every
milliliter of supernatant you have.
7. Make sure the ammonium sulfate is mixed in SLOWLY and completely. Remove
the stirrer and pour the contents of the beaker into one of the 50 ml white
centrifuge tubes at your station. Put the tube into your ice bucket and let the
sample sit in ice for 15 minutes. PUT A VERY TINY PIECE OF TAPE ON THE
LID. LOOK AT THE ORIGINAL TUBE THAT WAS IN YOUR ICE BUCKET.
9. Save a 0.5 ml aliquot of the supernatant and label the tube 40% SN
(supernatant). Pour the rest of the supernatant into a graduated cylinder to
measure the volume then into a beaker.
10. To the supernatant, add ammonium sulfate until the final concentration makes a
65% saturated solution. Use 0.166g of ammonium sulfate per milliliter of
supernatant and process like you did in step 6. Add the ammonium sulfate
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BioC 4025W Fall 2017 Lab 3
SLOWLY, mix well. Let stand for 15 minutes on ice then spin at 16,000 RPM for
20 minutes.
11. Save a 0.5 ml aliquot of the supernatant and label the tube 65% SN. Pour the
supernatant into a graduated cylinder and measure the volume. Note that this
fraction (65% SN) should not have significant activity when you do your assay
but save it temporarily, until you know where your activity is (see next step).
12. To the pellet left in the centrifuge tube, add 8 ml of 0.03 M Bicine, pH 8.5 buffer,
and dissolve the pellet well. An easy way to dissolve the pellet is to gently
pipette up and down using the liquid in the tube to “squirt” against the
pellet. Save a 0.2 ml aliquot of the solution and label the tube 65% pellet.
13. Place the remainder of the re-dissolved pellet into a dialysis bag and suspend in
a jar of 0.03 M bicine, pH 8.5 buffer which will be dialyzed at 4ºC overnight. You
should have about 10 ml that will go into the dialysis bag.
Everyone’s samples will be taken out of the dialysis jar the next day and put into
50 ml conical tubes that will be placed in a -80° freezer.
Each fraction you collected needs to be assayed today. Enzymes are notoriously
unstable in crude form, and we need to find out how much activity is there before LDH is
hydrolyzed by endogenous proteases.
LDH will be assayed by monitoring the formation of NADH, which absorbs at 340 nm.
Each assay has a reaction volume of 1 ml. The assays will for run 2 minutes.
The reaction is initiated by adding a sample composed of enzyme plus water totaling
100 µl. For your cruder samples, start with10 µl of sample and add 90 µl of water. Even
10µl might be too much.
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BioC 4025W Fall 2017 Lab 3
You should do at least 2 assays for each aliquot from Part 1. In a beaker, make up
enough cocktail to do 30 assays. Let the beaker sit on your table until it reaches RT
(room temp):
10 x 0.5 ml 6 mM NAD+ = 5 ml
Put 900 μl of cocktail into a 1ml cuvette. Decide how much enzyme sample you will use,
such as 10 µl. The difference between that and 100 µl is 90 µl (remember, the final
volume is 1 ml), so put 90 µl water into the cuvette with the cocktail. Have at least 6-7 of
these set up and ready to have the 10 µl of your sample added. It will save you time.
Put the cuvette with cocktail and water into the NanoPhotometer, add 10 µl of the
enzyme sample and pipette up and down three times then hit start,
or mix quickly using the vortex mixer and hit start, (cover cuvette with parafilm if mixing
with vortex and you do not need to remove parafilm).
Do not mix the enzyme and the water together to put into the cuvette.
Before you are ready to do your first reaction, make sure you have blanked the
NanoPhotometer using dH2O. If you forget and mix up your sample and it is not
blanked, you cannot use that sample.
You want to do the assays with the cocktail buffer at room temp.
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BioC 4025W Fall 2017 Lab 3
When you are finished with all your assays, put your samples into your freezer
box. Be sure that you have labeled them properly and don’t forget the Crude
sample you were given in your ice bucket at the beginning of your purification.
Practice Prep: This is a great place to stop and think about how to calculate
Units, relative activity and specific activity!
Procedure 2
There will be a demo on how to pour your columns. Practice pouring, note how
the slurry moves, and be sure NOT to let the column dry out.
1. Use the long Pasteur pipette provided to fill the column with matrix. Make sure
the stop cock is in the off position.
a. First slowly draw up as much as you can of the Q-Sepharose with the
pipette. Put the tip against the inside wall of the column and release
gently. You do not want air bubbles so do not “squirt” it into the column.
b. Repeat step above two more times being careful not to overflow the
column.
2. Open stopcock and let it run for 30 seconds then TURN OFF the stopcock. Let it
settle. If it doesn’t settle to the black line that is right under the Crown Mark, add
more Q-Sepharose to fill the column to that mark. You may have to remove some
of the liquid from the top of the column so you can add more mixed matrix. You
can put that liquid into the 50 ml conical of Bicine.
Do not let the matrix dry – make sure there is plenty of buffer above the Q-
Sepharose! A rule of thumb is one inch above the matrix.
3. Put parafilm on top of the column and around the stop cock for storage. Make
sure the stop cock is in the off position.
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BioC 4025W Fall 2017 Lab 3
4. Label your column with tape and do not write on the columns or use color
dots. Put the tape on the parafilm.
Lab Notebook:
Cleanup: Yep, this was a long lab and lots of work. But that’s no reason not to clean up.
The handout is there to help you. Just think, you are on the way to purifying LDH from
smelly beef heart homogenates. Be happy you didn’t have to make them.
INQUIRY:
What type of column chromatography method is this DNA clean up? Hint: What is the charge
of DNA and how does it bind and elute?
Prep LLR steps: Now is a great time to think about the LLR and how you would write a
materials and methods (experimental procedure for the Bradford assay you did in lab 2,
and the kinetic assay you did in lab 3). Look at the Journal of Biological Chemistry for
an example of how to write methods providing enough information to reproduce the
experiment. It’s not a protocol, so summarize. Type them up single spaced and bring
them to lab for review. It’s a soft deadline, but don’t dally too long. Then write out the
results as you would for JBC using the results of your kinetic assay. Note how the
methods do not provide results, nor do the results provide methods.
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