ldh purification
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Biochemistry 331
Spring 2014
Experiment II: Purification of Lactate Dehydrogenase (LDH)
The Experiment II write-up will be due Monday, 3/24, in lecture.
Table of Contents
Page(s) Content
2!5 Overview
6!8 Week 1:Theory, Prelab Instructions & Detailed Protocols
9!14 Week 2:Theory, Prelab Instructions & Detailed Protocols
15!17 Week 3:Theory, Prelab Instructions & Detailed Protocols
18!19 Week 4:Theory, Prelab Instructions & Detailed Protocols
20!22 Weeks 1 3:Write-up Instructions
23 Week 4:Write-up Instructions
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Experiment II - Purification of Lactate Dehydrogenase (LDH)
OVERVIEW
Mastery of a protein purification scheme is a rite of passage for all biochemists. Thetechniques employed in protein purification utilize important biochemical ideas about thestructure, catalytic activity, and the optical and electronic properties of proteins. The commonthemes that emerge in purification strategy are outlined below.
1. Devise an assay for your protein that is simple, quick and specific . The presence of anenzyme is typically assayed by observation of the formation of a specific product or thedepletion of a specific reactant. This method, which gives both qualitative andquantitative information, is the one that you will employ in this protein isolation. If theprotein being purified is not an enzyme, it sometimes can be detected by measurement ofa unique spectral characteristic, such as the 410 nm absorption of a heme protein or 615
nm absorption of a blue copper protein.
2. Production of a clear solution from something alive. This one step has been thenemesis of many novice biochemists. From bacterial cell paste, human blood serum,bovine brain, or chicken muscle, you must obtain a solution that will flow throughcolumns, dialyze without precipitating and, in general, be well behaved. Extra time spenthere to make your solution as pristine as possible will always save you time andheartache later.
3. Isolate the protein you want from those you don't want. In order to do this, it helps toknow something about the protein you're isolating, such as its molecular weight,
isoelectric point, solubility, color, or functional activity. As you're precipitating,dialyzing, and running your solution through columns, it's crucial to monitor yourprogress by using your assay from #1 above.
4. Evaluation of the purity and percent yield. Usually this involves the quantification ofyour data from #1 aboveperhaps a translation of an absorption reading into the activityof the protein per mg. This is usually charted for each step in the purification scheme.
* An unequivocal standard of purity is a single band on a polyacrylamide gel. We willsave a sample of your purified protein from this lab to run later in the semester.
We will carry out all of these steps for the enzyme lactate dehydrogenase (LDH) fromchicken. LDH is a tetramer of 36 kD subunits. Two different types of subunit, known as M (formuscle) and H (for heart), exist. (In general, enzymes that catalyze the same reaction, but differin structure, are referred to as isozymes.) Overall, five different tetrameric assemblies of LDHare possible: M4, M3H1, M2H2, M1H3, and H4. These differences in subunit composition lead tofunctionally important differences in pyruvate binding affinities and inhibition by pyruvate.
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The muscle enzyme (M4) plays a critical role in anaerobic glycolysis by coupling thereduction of pyruvate to lactate with the oxidation of NADH to NAD
+. The product NAD
+can
then again participate in glycolysis. The product lactate is a dead end in muscle metabolism andmust be first transported to the liver where it is converted by liver LDH (also M4) back into
pyruvate, and ultimately into glucose through the gluconeogenic pathway. In a sense, LDH buystime by sustaining glycolysis during periods of stress and shifting the metabolic burden from themuscle to the liver. The heart isozyme (H4) functions in vivoby oxidizing lactate to pyruvate,which can then be funneled into the citric acid cycle.
The particulars to our application of the steps outlined above are as follows:
1. Assay: In this prep, we will use two complementary assays; first, an enzyme assayspecific to LDH and, second, a so-called Bradford assay that quantitates by visibleabsorption the total amount of protein present, and is notspecific for LDH. Bycalculating the ratio of the results from these two assays (i.e.the amount of LDH to theamount of total protein), we will be able to quantitate the purification of LDH fromchicken heart and muscle.
a. Enzyme Assay: We will monitor the catalysis of the following reaction by ourLDH enzyme.
Even after hours of incubation, no products are formed when just pyruvate andNADH are mixed in buffer. Add just a small amount of LDH, and the reactiontakes place immediately. Since the NADH reactant (not the NAD
+product)
absorbs light at 340 nm, the course of the reaction can be monitored by thedecrease in the 340 nm absorption as reactant is converted to product. Our assaywill monitor the drop in OD340as a function of time when an aliquot containingan unknown amount of LDH is added to a mixture of pyruvate and NADH. Theslope of a plot of OD340vs. time can be related to the amount of enzyme present.We will use this assay to monitor the purification procedure and to measure the
kinetics during Week 3.
b. Bradford Assay: The enzyme assay will be complemented by an assay for totalprotein which makes use of the reaction between proteins and Coomassie blue, a
coumarin dye. By monitoring the blue absorption and comparing it to standards,you can determine the total amount of protein present. Again, this assay is not
specific for LDH, but rather measures total protein.
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2. Purification: The enzyme will be isolated from chicken muscle.
a. The chicken muscle will homogenized with a blender to break the myofibrils andrelease LDH and the rest of the soluble proteins into the buffer. Centrifugation of
this murky extract at moderately high speeds sediments the nuclei, mitochondria,
membranes and other insoluble material. The remainder of the purification is anattempt to separate the LDH contained in the clear supernatant from the othersoluble proteins.
b. The remainder of the purification scheme used here will consist of only two steps.The first is a classical differential precipitation by ammonium sulfate to partiallypurify the LDH and reduce the load of protein that enters the second step. This
latter step consists of specific binding to and selective elution from an affinitycolumn. The column material is constructed of insoluble polysaccharide beadscalled sepharose. In this lab we will be using Blue Sepharose, which consists of
sepharose particles covalently attached to a blue dye, Cibacron Blue, that shows
good affinity for NAD
+
/NADH-binding proteins such as LDH. Cibacron Blueacts as a (loose) structural analogue for NAD+/NADH. The structures of both are
shown below for comparison.
Cibacron Blue 3GA Sepharose NAD+LDH, like most dehydrogenases, possesses a strong binding affinity for NAD+and this dye analogue. Dehydrogenases are thus selectively retained on the
column when the extract is poured over the polysaccharide beads. This process byitself is not sufficient to completely purify the LDH since it is still contaminated
by other dehydrogenases that are also bound to the column. The final purificationis effected by eluting just the LDH from the column with a solution of a special
adduct composed of NADH bound to pyruvate. The adduct binds to the LDHselectively, displacing it from the Cibacron Blue and allowing the LDH adduct
complex to be eluted from the column.
c. We will be able to chart the progress of our purification by severalcomplementary methods. First, we will calculate the unit activity per mg protein
by combining our enzyme assay results (NADH absorption vs. time) with ourBradford assay results (Coomassie dye). A combination of these assays is used to
determine the specific activity of our LDH.
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Specific Activity is defined as units of activity per mg of protein where 1.0
unit of activity is that amount causing transformation of 1 mol of substrate
per minute at 25
Cunder conditions where the enzyme is saturated with both
pyruvate and NADH.
d. Now you will have purified LDH suitable for experimentation. Kinetic analysesand calculation of kcatandKMwill be performed for the enzyme.
In the last wet lab of the semester we will run a denaturing polyacrylamide gelelectrophoresis (SDS-PAGE) on sample aliquots that have been collected along the wayand stored in the -20C freezer. Under the influence of an electric field, a SDS-denatured
protein will migrate in the gel according to its molecular weight. A protein mixture willbe resolved on the gel into numerous bands, which can be stained with a protein specific
dye. We will compare two samples, before and after the purification column, to assess the
efficiency of the protein purification technique we have used.We will save two samples:one from the first dialysis step (week 2) and another from the second dialysis of the
pooled samples (week 3).
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Experiment II, Week I
Pre-lab Instructions
Use Notebook A for this experiment!
A. Table of contents
Use the first page of your lab notebook B (front and back side) for the table of contents.Neatly copy the following items for Experiment II. Dont forget to come backand record the corresponding pages in the table of contents as you complete each section.
Table of Contents PagesTitle and goals (and list lab partners)Week 1
Prelab Questions & NotesProcedure, Notes & Observations (dated)Week 2
Prelab Questions & NotesProcedure, Notes & Observations (dated)Data and calculations
Analysis and conclusionsWeek 3
Prelab Questions & NotesProcedure, Notes & Observations (dated)Data and calculationsAnalysis and conclusionsWeek 4
Prelab Questions & CalculationsProcedure, Notes & Observations (dated)Data and calculationsAnalysis and conclusions
B. Prelab Questionsand Notes(must be completed in your lab notebook before coming to lab-may be written or typed and pasted in)
1. Based on the reaction shown on p. 3, it looks like the enzyme should be named pyruvate
hydrogenase not lactate dehydrogenase. Is LDH a misnomer?
2. Why is monitoring the production of NAD+an appropriate way to measure the activity of
lactate dehydrogenase?
3. Copy and paste the detailed procedure for the first weeks activities so you may use it as a
reference during lab.
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Experiment II, Week 1:Initial Extraction
DETAILED PROCEDURES FOR WEEK 1
NOTE: ALL LDH solutions should be kept ON ICE to protect your proteinfrom denaturation and to inhibit endogenous proteases!
SECOND NOTE: You MUST record the total volume of all solutions from
which an aliquot is taken!
Preparation of the Extract. Each group should weigh a plastic beaker containing 50 mL
of 50 mM potassium phosphate buffer (PB). After chilling the beaker and contents to 0
C, place approximately 25 cm3of tissueeither heart, liver, or musclefrom the
chicken into the beaker to bring the total volume to about 75 mL. Weigh the
beaker/buffer/tissue and determine the weight of tissue taken. From this weight, you will
eventually calculate the weight-percent of LDH in your tissue.
Homogenize it in 50 mL of buffer with the hand blender. Blend for four 30-second
bursts, separated by 1-minute intervals in which the blender jar is chilled on ice to keepthe whole extract cold. Split the homogenate exactly equally between two chilled
centrifuge tubes. (You can increase your yield by rinsing the residual goop off of theblender with a little cold PB, but dont use more volume than will fit into the two
centrifuge tubes.) Thoroughly rinse the blender with distilled water when you are finishedso as not to contaminate the next group's preparation. When everyone has completed this
step, the tubes will be centrifuged at 17,000 x g for 20 minutes.
Ammonium Sulfate [(NH4)2SO4] Precipitation. During the centrifugation, each groupshould prepare for the ammonium sulfate precipitation by rinsing a 100 mL beaker withPB and adding a clean stirring bar. The supernatant from the centrifugation should be
decanted away from the pellet in one smooth motioninto this beaker. Remove an aliquotof approximately 0.5 mL of this homogenate (Homogenate)remember: record the
total volumeof the supernatant solution!for later assay. Weigh out 0.35 g ofammonium sulfate per mL of supernatant, and add this salt gradually to the supernatant
with continuous stirring (on ice use a weigh boat with ice under the beaker), make surethe salt has dissolved before adding more. When all of the ammonium sulfate has
dissolved, continue stirring for 10 more minutes. Be sure to make sure all of the salt hasdissolved before starting the timer for 10 minutes. Divide the protein suspension exactly
between the two centrifuge tubes, and centrifuge at 17,000 x g for 20 minutes. This stepsalts out the LDH and some other proteins from solution. The pellet will contain the
LDH, while the supernatant will contain other proteins not currently of interest.
Dialysis. While the tubes are spinning, each group should prepare dialysis tubing. Takeone section of tubing from the beaker provided. Rinse the tubing thoroughly inside and
out with distilled water. Place a dialysis clip as near as possible to one end of the tubing.
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Store the tubing in a beaker of water until ready to use so as to prevent it from drying out.
When the centrifugation is complete, remove a 0.5 mL aliquot (Supernatant)of the
supernatantdid you record the total volumeof the supernatant solution?for lateranalysis, and then pour off the supernatant as before, this time retaining the pellets. The
pellets should be resuspended in 10 mL of cold PB. A Pasteur pipette can be used toencourage the pellet to dissolve. Remove a 0.5 mL aliquot (Pellet)surely, you have
alreadyrecorded the total volume of the solution!and transfer the remainder of thesolution to your dialysis tube. Don't allow the contents to warm up. Place a dialysis clip
at the free end of the tubing as before. Attach a piece of thread to one of the clips, and tothe thread attach a piece of tape with your group number, group initials, and the date.
Submerge the bag in the large beaker of PB provided for dialysis.
Dialysis tubing keeps the large protein molecules inside the bag and allows solvent andsmall molecules to travel through the small pores in the tubing. This step effectively
removes salt and other small molecules. Your T.A. will change the external PB at leasttwo times over the next week so as to thoroughly dialyze away the residual ammonium
sulfate. When you return next week, your protein will be happily dissolved in pure PB.
Before you leave,make sure you have:
1. Recorded in your notebook the weight of the tissue used.
2. Recorded in your notebook the weight of (NH4)2SO4employed for precipitation.
3. Recorded in your notebook the volume of the solutions from which theHomogenate, Supernatant and Pellet aliquots were taken.
3. Collected three labeled aliquots. Store these in the freezer until next week. You
will then perform enzyme assays and Bradford assays on the (thawed) samples.Write down how you labeled them and where to find them.
4. A sample of crude LDH dialyzing in the cold room. Write down how you labeled
this and what it looks like. This sample is precious and should be treated withrespect, tenderness, and affection.
5. Disinfected all glassware and tools that came in contact with the raw chicken
tissue.
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Experiment II - Purification of Lactate Dehydrogenase (LDH)
Week 2: AFFINITY CHROMATOGRAPHY
Prelab Questionsand Notes
1. Below is a sample table for setting up dilutions for your Bradford assay standard curve, pleasecomplete the table, and use this as a reference while performing assays in lab.
Bradford Standard curve
g BSAl of stock BSA(1 mg/ml) l of Phosphate Buffer ml Bradford reagent
0 0 50 1
2.5 2.5 47.5 1
5
7.5
15
17.5
20
25
2. You will be making a lot of dilutions in both weeks 2-4 of the LDH lab, so it will be helpful tothink a bit about the best way(s) to make dilutions before you come to lab. One handy rule of
thumb: never pipet less than 2 L. Why? Whats wrong with making a 1000-fold dilution bypiptetting 0.5 L of your sample into 499.5 L of buffer?
3. Another handy rule of thumb: dont generate ridiculously large dilution volumes if you need
only small amounts for analysis. But this rule seems to be in tension with the rule in the previousquestion. For example, lets say you want to make a 10,000-fold dilution of your sample, but we
just told you not to pipet less than 2 L. Does that mean that you have to make at least 20 mL of
your dilution (2 L in 19,998 L buffer)? Theres got to be a better way, right? What is the
better way? Please be specific, noting the steps that you would carry out to prepare a reasonableamount (say, 500 L) of the 10,000-fold dilution.
4. Copy and paste the detailed procedure for the weeks activities so you may use it as a
reference during lab.
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Week 2: Affinity Chromatography
In this second week of the laboratory, you must complete two separate tasks. (Division of labor
among the various members of the group is essential.) During today's lab, you must:
1. Set up and run the Blue-Sepharose affinity columna description of this step is given inthe LDH Lab Overview. (Note: Some years agarose is the resin, as opposed to sepharose.
This change has no effect; the resinsepharose or agarose, which are bothpolysaccharidesmerely serves as an inert matrix to which the Cibacron Blue is
attached.)
2. Assay for the total protein content in the various fractions and batches.
DETAILED PROCEDURES FOR WEEK 2
A. Purification of LDH on a Blue-Sepharose Affinity Column (requires
preternatural dexterity and extraordinary poise)
Since this portion of the experiment must be run before the other two sections can becompleted, start it immediately.
1. Carefully remove the top clip from your dialysis bag containing your protein from
last week and transfer your LDH solution into a chilled centrifuge tube. It requiressome dexterity to avoid spilling the contents of the bag and thereby running out of
things to do. Centrifuge your sample at 34,000 x g for 15 min and CAREFULLY
transfer the supernatant to a plastic conical tubedo not let ANYof the pellet, whichcontains column-clogging gunk, contaminate the supernatant. The supernatant, ofcourse, contains the LDH and will be loaded onto the column. To make sure that this
solution is absolutely clear, filter the supernatant through a 0.45 M syringe filter into
a clean conical tube. Take two 0.5 mL aliquots of the dialyzed and filtered LDH
solution (Dialysis)and set it aside (recording, as always, in your notebook the totalvolumeof the solution whence came this aliquot). One aliquot will be used for later
analysis of protein content and LDH activity, the other will be frozen until the end ofthe semester and run on an SDS-PAGE gel.
2. The basic setup of the affinity column and its operation will be demonstrated to you
in lab. Follow the instructions in parts a.-f. below. DO NOTlet your column run dry.NOTE: you should record the volume of every batch of eluent you collect.
a. Your column will be pre-equilibrated with PB. When you are ready to start your
column, remove the clip and let the excess PB flow out by gravity. Remember:DO NOTlet your column run dry. You will want to carefully watch every step so
that you can stop the flow when the liquid level is just above the column bed.
b. When the PB level is just above the beads, stop the flow and carefully load the
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LDH (your LDH-containing supernatant from week 1) onto your column.Immediately place a conical tube labeled load and wash under your column and
start to collect the eluent. When your LDH has been entirely loaded (again, DONOTlet your column run dry), carefully rinse down the sides of the column with
3 mL of PB and allow this buffer to pass through the column. This rinse willensure that all of your protein has been loaded onto the column.
c. When the rinse has been drawn into the column, carefully load 10 mL of
additional PB, and continue to collect the eluent into the load and wash conicaltube. Repeat this process with another 10 mL of PB, again collected in the same
load and wash conical tube. These washes should remove any proteins from thecolumn that haven't bound to the Blue-Sepharose.
d. Now, you will elute the LDH. Make sure the level of PB is within 1 cm of the top
of the column bed and add 10 mL of NAD-pyruvate adduct, collecting the eluentin a fresh tube. Repeat this process two more times. Collect the eluent in three
"10 mL batches labeled adduct 1, adduct 2, and adduct 3.
e. Clean your column of residual protein by running high salt regeneration buffersthrough it: 10 mL of pH 8.5 salt wash, followed by 10 mL of pH 5.0 salt wash.
Collect the eluent into a tube labeled salt wash. Other people will use yourcolumn material in the future, so although your precious LDH should be eluted at
this point, pleaseDO NOTlet your column run dry.
f. Continue with washes according to the checklist (attached as Appendix I, p. 14).When done, securely clamp the outlet tube at the bottom of the column and hand
it over to your TA to return to the cold room.
g. Be sure to take 0.5 mL aliquots of each of your column batches (Load & wash,adducts 1-3 and Salt wash), after, of course having measured the actual volumes.
REMEMBER: you should record the volume of every batch of eluent you collect
that is, the volume of the load and wash, the three adduct-eluted batches, and the
salt wash.
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B. Bradford Protein Assays (requires unusually compulsive analytical technique)We will use a dye-binding assay known as the Bradford Assay to determine the concentration
of protein in our various samples. A protein is added to a solution containing the dye,Coomassie Brilliant Blue. When the dye binds to proteins, its visible spectrum changes and
an absorption maximum appears at 595 nm. We will estimate the total amount of protein ineach aliquot by measuring the absorbance at this wavelength (also referred to optical density
at 595 nm, or OD595) and comparing it that of standards.
Basic Procedure: The volume of protein will vary for different samples as described below.Accurately pipette y mL of a protein sample into a plastic cuvette containing (50 y) L of
PB. Add 1000 L (1.0 mL) of dye reagent. (The total volume in every sample should thus
always be 1050 L.) After about 2!5 minutes, read the OD595.Make sure that all of your
experimental absorbance readings are well within the absorbance range of your standard
curve (see point 1, below). Outside of this range, useful data are not obtained. You mayhave to dilute your protein in PB to get a reading within this range. For suggestions about
preparing samples for this assay, see this weeks pre-lab questions.
Set up a table called Bradford Assay with the following columns: Dilution; !L of dilutionto add; !L of PB; !L of Bradford reagent; OD595. Leave enough rows for each of your
samples (and a few more).
1. In the first series of experiments, run a standard curve containing at least six pointsranging from roughly 2.5 g to 25 g of the BSA standard. (Your lab instructor will
inform you of the concentration, in mg/mL, of the BSA standard.) Also, measure theabsorbance of a 0 mg/mL sample (i.e., just PB, no protein). From the resulting data,make a graph of OD595vs. amount of BSA. (Your plot may show some curvature.) Rerun
any suspicious points. The total amount of protein in each of the samples below can bedirectly determined by reference to this standard curve. Plot your data in a spreadsheet
and calculate the r2value for the best-fit line; you want this correlation to be very good.
Orientation of cuvette: to take a reading, make sure you have the cuvette orientedproperly with respect to the path of light in the spectrophotometer: there should be an
arrow on the spec that indicates the direction of the light path, and this should align withthe V-shaped mark on the cuvette (this will mean that the light shines through the 1 cm
long column of liquid, not the "cm long side).
2. In the second experiment, you will be measuring the protein concentrations of your fouraliquots from purification (homogenate, supernatant, pellet, dialysis). Again, the volume
of protein sample (y) should be chosen to give OD595
readings well within the bounds ofyour standard curve. Start by assaying 5 L of each aliquot, if your aliquots contain a
large amount of protein, you may need to dilute these samples to get an accurate readingin the Bradford assaythis is best done in a microcentrifuge tube. (Remember: the P20
pipettors will not accurately pipette less than 2 L, so use serial dilutions as needed).
3. In the third series of experiments, you will measure the protein concentration of thecolumn batches: the load and wash batch, the three adduct-elution batches, and the final
salt wash batch. If you pool multiple adduct-elution batches with high LDH activity,
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perform a final Bradford assay on the pooled sample.
Look over your results and make sure they make sense and are complete. Do not
discard any of your tubes as these dilutions may be useful for the LDH activity
assays you will complete next week.
Before you leave, make sure you have performed the following:
1. Bradford assay: a) standard curve
b) homog., super., pellet and dialysis aliquotsc) column batches (5), plus pooled LDH
2. Saved well-labeled aliquots and fractions in the freezer
a) homog., super., and pellet aliquotsb) dialysis aliquot
c) column batches (5)
3. Cleaned up your lab bench
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Appendix I: Column regeneration for Biochem 33ChecklistD o t h e f o l l o w i n g
Check off as you go
10mL TrisHCL (pH 8.5) Wash _________
10 mL Sodium Acetate (pH 5) Wash _________
The two above washes are combined and collected for assaying,
Now- Continue to wash, and from now on all the flow through can be discarded
10mL TrisHCL (pH 8.5) Wash _________
10 mL Sodium Acetate (pH 5) Wash _________
10mL TrisHCL (pH 8.5) Wash _________
10 mL Sodium Acetate (pH 5) Wash _________
10mL TrisHCL (pH 8.5) Wash _________
10 mL Sodium Acetate (pH 5) Wash _________
Fill column with PB, let run through _________
Fill once more with PB, _________
Let run until about 4-5 inches above resin
Clamp, and give to your TA
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Experiment II - Purification of Lactate Dehydrogenase (LDH)
Week 3: LDH Activity Assay and Selecting Active Column Elutions
Prelab Questionsand Notes
This week aliquots from your purification steps and column batches will be assayed both forLDH activity:
a. Protein from the purification steps taken in week 1 will be assayed to confirm thelocation of LDH in the steps prior to the affinity column.
b. Batches from the NAD-pyruvate elutions containing high LDH activity will beidentified. If more than one batch has high activity, these should be pooled for
dialysis against PB to rid the solution of NAD-pyruvate adduct. Record the totalvolume of the pooled sample, and make sure this pooled sample is again analyzed
for LDH activity and total protein before leaving it to dialyze.b. Protein from the initial load & wash (Week 2 step A.2.c.) and the salt wash (Week
2 step A.2.e.) will also be assayed using the LDH Activity Assay.
1. Predicting the location of LDH activity throughout the purification.a. Based on the premise of affinity purification predict the presence and level of LDH
activity for each of the following samples you will test.b. Use your results from the Bradford assays to fill in the relative total protein
concentrations of each sample.
Assay
Sample
a) LDH Activity Prediction(High, Medium, Low, None)
b) Total [Protein] Prediction(High, Medium, Low, None)
Homogenate
Supernatant
Pellet
Dialysis
Load + Wash
Adduct Elution 1
Adduct Elution 2
Adduct Elution 3
Salt Wash
Pooled Adduct
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DETAILED PROCEDURES FOR WEEK 3
LDH Assays (requires a facile mind and great witand an intimate familiarity with the
proper use of a micropipettor)
LDH activity will be assayed by adding protein samples to 50 mM PB buffer, pH 7.5,
containing 0.12 mM NADH and 0.33 mM pyruvatesaturating concentrations for eachof these substrates. The absorbance change at 340 nm is then monitored. Note: the change
in OD340can be related to the amount of NADH converted to NAD+using the conversion
factor 6.22 OD340/(mM NADH#NAD+)
Prepare a table called LDH Assay with the following columns: Dilution;!l of dilution
to add; ml of LDH assay buffer; Linear initial rate?(Y/N); File name; file opens in excel.Leave enough rows for each of your samples (and a few more).
1. To determine the background rate of NADH oxidation, run one control with 1.5 mLof assay buffer containing no enzyme. This rate should be essentially zero. Be sureyou are orienting the cuvette properly in the spectrophotometer (see Basic
Procedure: Orientation of Cuvette for description).
2. Activity Assay:a. Dialysis Aliquot. Measure the OD340using 1.5 mL of assay buffer, and then add
10 L of enzyme directly to that cuvette. Mix by inverting 5 times (cover withparafilm) and reinsert into the spectrophotometer. Record the OD vs. time. If the
change in OD occurs too quickly to measure (i.e., if you dont see a nice, lineardrop in OD), make a note of that and prepare a diluted sample in a
microcentrifuge tube: dilute 10 L of enzyme into 90 L of PB. Add 10 L of thisdiluted enzyme to 1.5 mL of assay buffer. Try various dilutions until linear
kinetics are obtained. Once you have a dilution that works to give you linearkinetics in the assay, do another dilution to show that the rate of change of OD
with time is proportional to the amount of enzyme added.
b. Homogenate, Supernatant and PelletAliquots. With the information from yourdialysis aliquot as a guide, assay the amount of enzyme in the remaining three
aliquots taken during the purification.
c. Column Batches. Assay the LDH activity of the several column batches: the loadand wash batch, the three adduct-elution batches, and the final salt wash batch. As
noted above, you should pool the adduct-elution batches with the highest activity.
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3. Use the known protein concentrations and volumes of all pooled adduct-elution
batches determine the final protein concentration of the pooled sample.
4. You should also measure the LDH activity in this final pooled solution.
5. Dialyze your pooled sample in preparation for week 4.
Before you leave, make sure you have performed the following:
1. LDH assay: a) homog., super., and pellet aliquots
b) dialysis aliquot (two different concentrations)c) column batches (5), plus pooled LDH
2. Dialysis: adduct (or pooled sample) with highest LDH activity
3. Saved aliquots in the freezera) homog., super., and pellet aliquots
b) dialysis aliquotc) column batches (5), plus pooled LDH
4. Cleaned up your lab bench
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Experiment II - Purification of Lactate Dehydrogenase (LDH)
Week 4:Today, you will analyze the kinetic properties of your purified LDH.
As you are well aware, LDH catalyzes the following reaction:
CH3-C-COO!+ NADH + H
+ CH3-C-COO!+ NAD
+
pyruvate L-lactate
Below is a simplified reaction scheme that permits the kinetics to be expressed in the Michaelis-Menten form. The NADH concentration will be held constant (and saturating), while that of
pyruvate will be varied. As suggested by the scheme, NADH does in fact rapidly bind to LDH
prior to pyruvate.NADH k1
NADH kcat
E - H++ PYR E - H
+ E + LAC + NAD
+
k-1 PYR
Analysis of this kinetic scheme yields the familiar equation:
kcat[Eo] [PYR]
initial velocity (vi) = -----------------KM
pyr+ [PYR]
Experiment II, week 4
Prelab Questionsand Notes(must be completed in your lab notebook before coming to lab-may be written or typed and pasted in).
1. Why did you bother dialyzing your sample between Weeks 3 and 4? In other words, whatwere you trying to get rid of and why were you trying to get rid of it?
2. What will be the final concentration of NADH in your Week 4 kinetics reactions? Showyour calculation. (You will, of course, need to consult Detailed Procedures for Week 4,
below.)
3. For reference, what were the pyruvate and NADH concentrations you used last week inthe LDH activity assays.
4. Copy and paste the detailed procedure for this weeks activities so you may use it as areference during lab.
OHO
H
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DETAILED PROCEDURES FOR WEEK 4
The following reagents should be prepared first in a cuvette (one lab partner can prepare all of
the reactions (without enzyme) mixed in a cuvette:
1400 L 50 mM phosphate buffer, pH 7.5
50 L NADH Stock Solution (4 mM)
y L Pyruvate solution. The initial pyruvate concentration in the cuvette
should vary between 0.5 mM and 0.025 mM; you will be providedwith a 30 mM stock solution. Calculate how much of this stock
solution you will need to add the cuvette to achieve the desiredpyruvate concentrations (run about 8 different concentrations).
The most useful data will be collected below about 0.2 mM, sodon't run too many points above this concentration.
50 - y L 50 mM phosphate buffer. That is, add enough buffer so that the
total volume in the cuvette is now exactly 1.5 mL.
To initiate the reaction, add 10 !L of enzyme solution (your dialyzed LDH sample,
which of course should be removed from the dialysis tubing and kept on ice throughoutthe afternoon). Cover the cuvette with a square of parafilm, mix the solution thoroughly,
and place the cuvette into the spectrophotometer and record the change in absorbance at340nm (#OD340)
Notes:
1. Try the 0.5 mM pyruvate reaction first. You may need to experiment with enzymedilutions until reasonable kinetics are obtained with 0.5 mM pyruvate (but on the fast
side of reasonabledo you see why?). In all subsequent reactions, use the samedilution of enzyme. Note that the total volume of solution in the assay mix is 1510
L.2. Your kinetic runs may well show some curvature due to depletion of the pyruvate
substrate. In such cases, determine initial velocities by using only the linear data atthe beginning of the runs.
3. If you have data points that do not make sense (e.g., did you add a lower substrateconcentration and get a higher initial velocity?!?), you probably made a pipetting or
diluting error somewhere. Make new samples for these data points.4. You may need to re-run particular substrate concentrations once you plot the data for
initial velocity vs. substrate concentration.
5. You will need to determine, together with your lab partners, whether you are satisfiedwith the quality of your data and will be able to extract kcatandKmvalues in whichyou are confident (it is unwise to rely on one person alone to determine this, or to
conclude that getting out of lab is more important than having reliable data).6. Once you are satisfied that your data are complete and of good quality, dont forget to
clean up your lab station; discard all waste and leave it ready for the next days lab(check with the TA before you go to make sure this is complete).
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Before you leave, make sure you have performed the following:
1. LDH assay:
Collected quality data with linear portions of the kinetics.
2. SDS-PAGE sample:Saved a 0.5 ml aliquot of your pooled, purified LDH to use later in the semester
when we run an SDS-PAGE gel.
3. Freezer storage of SDS-PAG samples:Make sure you transfer your two samples for future SDS-PAGE analysis (Dialysis
1 and pooled samples) into the provided freezer box and noted theirlocation in you lab notebook.
4. Cleaned out the freezer:
All of your samples in the freezer can now be disposed of properly.
5. Cleaned up your lab bench
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The write-up for experiment II is due on Monday, March 24th
. It is a longand
complicatedwrite-up. To make the process less daunting, the write-up is
broken into two parts that can be completed independently (weeks 13 and 4).It is highly recommended that you complete the sections as you goand not
wait until the end of the four weeks to write everything up.
LDH Weeks 1-3 Write-up Instructions and Hints
Note:When using data gathered by your lab partner(s), you must include your own calculations.Please see the courses Organization Details handout under Intellectual Responsibility, andif you are unclear on these intellectual-responsibility requirements, ask.
Preparation of the tables described below and answers to questions 1-3:
Your Weeks 1!3 write-up should include tables with data for each of the solutions from which
you took aliquots and each of your batches from the affinity column. The tabular data should
contain:I) the total units of LDH (units = mol/min)
II) the total milligrams of proteinIII) the specific activity of LDH. (units/mg)
I. Total Units of LDH:
Note:If you are so inclined, feel free to save some number-crunching time and set up anExcel spreadsheet to carry out these calculations. You must, however, show one sample
calculation with all of the relevant units in your notebook.
Step 1: First, calculate the total units of LDH in the cuvette. A unit of LDH will turn over onemol of substrate per minute. MAKE SURE THAT YOUR UNITS WORK OUT!
Helpful hints and reminders:
OD is unitless
the conversion factor is 6.22 OD340/(mM NADH#NAD+)
1 mM = 1 mol/mL
your total volume of solution in the cuvette was 1510 L
Step 2: Calculate the total units of LDH in the entire sample.
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Your table of data (or spreadsheet) will look something like this:dilution factor
(if any)
OD340/min cuvette
volume
units
assayed
volume of
sample
assayed
total sample
volume
total units
in sample
Aliquot
Homog.
Supernat.
Pellet
Dialysis
Column
L&W
A1
A2
A3
Salt washPOOL
This procedure needs to be adjusted if you had to dilute the aliquot in order to get a reasonable
OD340/min. In that case, you need to include a dilution factor (10, 50, 100) if the activity of the
neat aliquot was too high. Of course, if no dilution was made, the dilution factor in the above
table would be 1.
II) Total milligrams protein from Bradford assays
Remember that this assay is designed to measure total protein concentration and is notspecific for LDH. One compares the OD595of the aliquots to the standard curve todetermine the micrograms protein in the cuvette. Dividing the micrograms of protein in
the cuvette by the small volume of aliquot assayed yields the concentration of protein inthat aliquot. To calculate the total micrograms protein in the entire biological sample,
one needs to multiply this concentration by the volume of the sample. Again, rememberto take into account any dilution factors that were made.
MAKE SURE THAT YOUR UNITS WORK OUT!
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Your table of Bradford data should look something like this. Again, please show at least onesample calculation with all of the relevant units:
dilution factor
(if any)
volume
of
sample
assayed
OD595 total
protein
assayed
conc. of
protein in
sample
sample
volume
total
protein in
sample
Aliquot
Homog.
Supernat.
Pellet
Dialysis
Column
L&W
A1
A2
A3Salt wash
POOL
III. Calculate the specific activity of LDH
For each aliquot and column fraction, divide the Total Units of activity by the totalmilligrams of protein.
Question 1:How does the specific activity of your column-purified enzyme compare with that
given in the Handy LDH information below? Answer:It is likely lower. Why?
Question 2:Do the data make sensefor example, is the enzymatic activity where you expect itto be?
Question 3:How well did your purification work? Discuss the efficacy of each step.
Handy LDH Information(From the 2000 Sigma Chemical Company Catalog)
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LDH Week 4 Write-up Instructions and Hints
Generate a Lineweaver-Burk plot by plotting 1/Vinitialvs. 1/[S]. For the latter number, rememberthat a change of 6.22 OD340/min corresponds to 1 mM/min. Your plot will provideKM
pyrand
Vmax. For this plot, please use a graphing program (Excel will do). Report the equation for the
line and the square of the correlation coefficient (r
2
). If you are a graphing-program whiz, youmay, of course, fit your Vinitialvs. [S] data directly to the Michaelis-Menten equation (equation 26on page 232 of Stryer); you will get the same answers, just without a linear fit.
Question 4: How does your KMpyr compare with the literature value (0.32 mM for the M4
isozyme, 0.08 mM for the H4isozyme)? Suggest an explanation for any discrepancy.
Using your Bradford data on the pooled, purified LDH and the molecular weight persubunit from your gel (see below), and assuming that your enzyme is completely pure,
determine the kcatfor a LDH subunit from the equation Vmax= kcat[ET]. Also determinethe kcatusing the known subunit molecular weight of 35,500. Note:[ET] is, of course, the
enzyme concentration in the cuvettefor each of your kinetic runs in week 3, not in yourstock solution (again, you may assume that the stock is pure LDH). Remember, the
enzyme concentration was the same in every assayonly pyruvate concentration varied.
Question 5: How does your value for kcatcompare with the literature value (approximately 500s
-1per subunitfor both the M4and H4isozymes)? Suggest an explanation for any discrepancy.
Also, provide a recalculation of the specific activity of your LDH from your kcat value
(just to be sure that you have become a kinetics expert). How is this done? kcat =(molecules substrate turned over/s)/molecule LDH = mol substrate turned over/s)/ mol
LDH %(mol substrate turned over/s)/mg LDH %(mol substrate turned over/min)/mg
LDH = units/mg LDH = specific activity. For the purposes of the above calculation youmay assume each subunit of LDH is catalytically identical and hence may simply use the
subunit molecular weight to convert from moles of LDH into mass of LDH.
Question 6:How does this recalculated value compare with the value that you determineddirectly from your Bradford and kinetic assays? Propose a possible reason for a substantial
discrepancy (>10%).
Question 7: How would your values ofKMpyrand kcatchange if you assumed a protein purity of
only 80%? Calculate what the new values would be if you expect them to change.
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