ldh purification

5/28/2018 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




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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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.


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







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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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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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.


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



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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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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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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.



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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.

ldh purification

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