LDH Purification

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


15!17


18!19


20!22

Weeks 1 – 3: Write-up Instructions

23

Week 4: Write-up Instructions

1

Experiment II - Purification of Lactate Dehydrogenase (LDH)

OVERVIEW Mastery of a protein purification scheme is a rite o f passage for all biochemists. The techniques employed in protein purification utilize important biochemical ideas about the structure, catalytic activity, and the optical and electronic properties of proteins. The common themes that emerge in purification strategy are outlined be low.

1.

Devise an assay for your protein that is simple, quick and specific . The presence of an enzyme is typically assayed by observation of the formation of a specific product or the depletion of a specific reactant. This method, which gives both qualitative and quantitative information, is the one that you will employ in this protein isolation. If the protein being purified is not an enzyme, it sometimes can be detected by measurement of a 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 the nemesis of many novice biochemists. From bacterial bac terial cell paste, human blood serum, bovine brain, or chicken muscle, you must obtain a solution that will flow through columns, dialyze without precipitating and, in general, be well behaved. Extra time spent here to make your solution as a s pristine as possible will always save you time and heartache later.

3.

Isolate the protein you want from those you don't want . In order to do this, it helps to know 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 your progress by using your assay from #1 above.

4.

Evaluation of the purity and percent yield. Usually this involves the quantification of your data from #1 above—perhaps above —perhaps a translation of an absorption reading into the activity of the protein per mg. This is usually charted for each step in the purification scheme.

* An unequivocal standard standard of purity purity is a single band on a polyacrylamide gel. We will save 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) from chicken. LDH is a tetramer of 36 kD subunits. Two different types of subunit, known as M (for muscle) and H (for heart), exist. (In general, enz ymes that catalyze the same reaction, but differ in structure, are referred to as “isozymes.”) Overall, five different tetrameric assemblies of LDH are possible: M4, M3H1, M2H2, M1H3, and H4. These differences in subunit composition lead to functionally important differences in pyruvate binding affinities and inhibition by pyruvate.

2

The muscle enzyme (M4) plays a critical role in anaerobic glycolysis by coupling the + + reduction 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 and must 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 buys time by sustaining glycolysis during periods of stress and shifting the metabolic b urden from the muscle to the liver. The heart isozyme (H4) functions in vivo by 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 assay specific to LDH and, second, a so-called Bradford assay that quantitates by visible absorption the total amount of protein present, and is not specific for LDH. By calculating the ratio of the results from these two assays (i.e. the amount of LDH to the amount of total protein), we will be able to quantitate the purification of LDH from chicken heart and muscle.

a.

Enzyme Assay: We will monitor the catalysis of the following reaction by our LDH enzyme.

Even after hours of incubation, no products are formed when just pyruvate and NADH are mixed in buffer. Add just a small amount of LDH, and the reaction + takes place immediately. Since the NADH reactant (not the NAD product) absorbs light at 340 nm, the course of the reaction can be monitored by the decrease in the 340 nm absorption as reactant is converted to product. Our assay will monitor the drop in OD340 as a function of time when an aliquot containing an unknown amount of LDH is added to a mixture of pyruvate and NADH. The slope of a plot of OD340 vs. time can be related to the amount of enzyme present. We will use this assay to monitor the purification procedure an d to measure the kinetics during Week 3. b.

Bradford Assay : The enzyme assay will be complemented by an assay for total protein 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. 3

2.

Purification : The enzyme will be isolated from chicken muscle.

a.

The chicken muscle will homogenized with a blender to break the myofibrils and release 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 an attempt to separate the LDH contained in the clear supernatant from the other soluble 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 partially purify 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 affinity column. The column material is constructed of insoluble polysaccharide beads called 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 Blue + acts as a (loose) structural analogue for NAD /NADH. The structures of both are shown below for comparison.

+

NAD

LDH, like most dehydrogenases, possesses a strong binding a ffinity 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 by itself 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 purification is 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 LDH selectively, 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 several complementary methods. First, we will calculate the unit activity per mg protein by combining our enzyme assay results (NADH absorption vs. time) with our Bradford assay results (Coomassie dye). A combination of these assays is used to determine the specific activity of our LDH. 4

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 µmol of substrate per minute at 25 C under conditions where the enzyme is saturated with both pyruvate and NADH.

d.

•

Now you will have purified LDH suitable for experimentation. Kinetic analyses and calculation of k cat and K M will be performed for the enzyme.

In the last wet lab of the semester we will run a denaturing polyacrylamide ge l electrophoresis (SDS-PAGE) on sample aliquots that have been collected along the way and stored in the -20°C 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 will be 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. Don’t forget to come back and record the corresponding pages in the table of contents as you complete each section. Table of Contents Title and goals (and list lab partners)

Pages

Week 1

Prelab Questions & Notes Procedure, Notes & Observations (dated) Week 2

Prelab Questions & Notes Procedure, Notes & Observations (dated) Data and calculations Analysis and conclusions Week 3

Prelab Questions & Notes Procedure, Notes & Observations (dated) Data and calculations Analysis and conclusions Week 4

Prelab Questions & Calculations Procedure, Notes & Observations (dated) Data and calculations Analysis and conclusions

B. Prelab Questions and Notes (must be completed in your lab notebook before coming to labmay 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 week’s activities so you may use it as a reference during lab.

6

Experiment II, Week 1: Initial Extraction DETAILED PROCEDURES FOR WEEK 1 NOTE: ALL LDH solutions should be kept ON ICE to protect your protein from 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 3 °C, place approximately 25 cm of tissue—either heart, liver, or muscle—from 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 keep the 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 the blender with a little cold PB, but don’t use more volume than will fit into the two centrifuge tubes.) Thoroughly rinse the blender with distilled water when you are finished so 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 [(NH 4)2SO4] Precipitation. During the centrifugation, each group should prepare for the ammonium sulfate precipitation by rinsing a 100 mL beaker with PB and adding a clean stirring bar. The supernatant from the centrifugation should be decanted away from the pellet in one smooth motion into this beaker. Remove an aliquot of approximately 0.5 mL of this homogenate (“Homogenate”) —remember: record the total volume of the supernatant solution!—for later assay. Weigh out 0.35 g of ammonium 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 sure the 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 has dissolved 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 step “salts 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. Take one 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. 7

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 supernatant—did you record the total volume of the supernatant solution?—for later analysis, 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 to encourage the pellet to dissolve. Remove a 0.5 mL aliquot (“Pellet”) —surely, you have already recorded the total volume of the solution!—and transfer the remainder of the solution 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 to the 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 and small 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 least two 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)2SO4 employed for precipitation.

3.

Recorded in your notebook the volume of the solutions from which the Homogenate, 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 with respect, 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 Questions and Notes

1. Below is a sample table for setting up dilutions for your Bradford assay standard curve, please complete the table, and use this as a reference while performing assays in lab. Bradford Standard curve

µl of stock BSA (1 mg/ml)

µg BSA

µl 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 to think 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 µL. Why? What’s wrong with making a 1000-fold dilution by piptetting 0.5 µL of your sample into 499.5 µL of buffer? 3. Another handy rule of thumb: don’t 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 previous question. For example, let’s say you want to make a 10,000-fold dilution of your sample, but we just told you not to pipet less than 2 µL. Does that mean that you have to make at least 20 mL of your dilution (2 µL in 19,998 µL buffer)? There’s 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 reasonable amount (say, 500 µL) of the 10,000-fold dilution. 4. Copy and paste the detailed procedure for the week’s 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 column—a description of this step is given in the LDH Lab Overview. (Note: Some years agarose is the resin, as opposed to sepharose. This change has no effect; the resin—sepharose or agarose, which are both polysaccharides—merely 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 be completed, 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 requires some 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 tube—do not let ANY of the pellet, which contains column-clogging gunk, contaminate the supernatant. The supernatant, of course, contains the LDH and will be loaded onto the column. To make sure that this solution is absolutely clear, filter the supernatant through a 0.4 5 µM 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 total volume of 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 e nd of the 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 NOT let 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 NOT let 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 10

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, DO NOT let 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 will ensure 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” conical tube. 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 the column 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 eluent in 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 buffers through 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 your column material in the future, so although your precious LDH should be eluted at this point, please DO NOT let 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 conc entration of protein in our various samples. A protein is ad ded 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 in each 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) µL of PB. Add 1000 µL (1.0 mL) of dye reagent. (The total volume in every sample should thus always be 1050 µL.) 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 may have to dilute your protein in PB to get a reading within this range. For suggestions about preparing samples for this assay, see this week’s pre-lab questions.

Set up a table called ‘Bradford Assay” with the following columns: Dilution; !L of dilution to 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 points ranging from roughly 2.5 µg to 25 µg of the BSA standard. (Your lab instructor will inform you of the concentration, in mg/mL, of the BSA standard.) Also, measure the absorbance of a “0 mg/mL” sample (i.e., just PB, no protein). From the resulting data, make a graph of OD595 vs. 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 be directly determined by reference to this standard c urve. Plot your data in a spreadsheet 2 and calculate the r value for the best-fit line; you want this co rrelation to be very good. Orientation of cuvette: to take a reading, make sure you have the cuvette oriented properly 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 with the 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 four aliquots from purification (homogenate, supernatant, pellet, dialysis). Again, the volume of protein sample (y) should be chosen to give OD595 readings well within the bounds of your standard curve. Start by assaying 5 µL of each aliquot, if your aliquots contain a large amount of protein, you may need to dilute these samples to get an accurate reading in the Bradford assay—this is best done in a microcentrifuge tube. (Remember: the P20 pipettors will not accurately pipette less than 2 µL, so use serial dilutions as needed). 3. In the third series of experiments, you will measure the protein concentration of the column 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, 12

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 aliquots c) column batches (5), plus pooled LDH

2. Saved well-labeled aliquots and fractions in the freezer a) homog., super., and pellet aliquots b) dialysis aliquot c) column batches (5) 3. Cleaned up your lab bench

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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

_________


_________

10mL TrisHCL (pH 8.5) Wash

_________


_________

10mL TrisHCL (pH 8.5) 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 Questions and Notes

This week aliquots from your purification steps and column batches will be assayed both for LDH activity: a. Protein from the purification steps taken in week 1 will be assayed to confirm the location of LDH in the steps prior to the affinity column. b. Batches from the NAD-pyruvate elutions containing high LDH activity will be identified. If more than one batch has high activity, these should be pooled for dialysis against PB to rid the solution of NAD-pyruvate addu ct. Record the total volume 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 Homogenate Supernatant Pellet Dialysis Load + Wash Adduct Elution 1 Adduct Elution 2 Adduct Elution 3 Salt Wash Pooled Adduct

a) LDH Activity Prediction

b) Total [Protein] Prediction

(High, Medium, Low, None)

(High, Medium, Low, None)

15

DETAILED PROCEDURES FOR WEEK 3 LDH Assays (requires a facile mind and great wit—and 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 pyruvate—saturating concentrations for each of these substrates. The absorbance change at 340 nm is then monitored. Note: the change + in OD340 can 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 mL of assay buffer containing no enzyme. This rate should be essentially zero. Be sure you are orienting the cuvette properly in the spectrophotometer (see “Basic Procedure: Orientation of Cuvette” for description). 2. Activity Assay: a. Dialysis Aliquot . Measure the OD340 using 1.5 mL of assay buffer, and then add 10 µL of enzyme directly to that cuvette. Mix by inverting 5 times (cover with parafilm) and reinsert into the spectrophotometer. Record the OD vs. time. If the change in OD occurs too quickly to measure (i.e., if you don’t see a nice, linear drop in OD), make a note of that and prepare a diluted sample in a microcentrifuge tube: dilute 10 µL of enzyme into 90 µL of PB. Add 10 µL of this diluted 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 linear kinetics 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 Pellet Aliquots . With the information from your dialysis 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 load and 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 freezer a) homog., super., and pellet aliquots b) dialysis aliquot c) 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: O

OH +

CH3-C-COO + NADH + H !

+

CH3-C-COO + NAD !

H L-lactate

pyruvate

Below is a simplified reaction scheme that permits the kinetics to be expressed in the MichaelisMenten 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 +

k 1

E - H + PYR

NADH +

E-H k -1

k cat

+

E + LAC + NAD

PYR

Analysis of this kinetic scheme yields the familiar equation: k cat [Eo] [PYR] initial velocity (vi) = ---------------- pyr K M + [PYR] Experiment II, week 4 Prelab Questions and Notes (must be completed in your lab notebook before coming to labmay be written or typed and pasted in).

1. Why did you bother dialyzing your sample between Weeks 3 and 4? In other words, what were 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? Show your 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 in the LDH activity assays. 4. Copy and paste the detailed procedure for this week’s activities so you may use it as a reference during lab.

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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 µL

50 mM phosphate buffer, pH 7.5

50 µL

NADH Stock Solution (4 mM)

y µL

Pyruvate solution. The initial pyruvate concentration in the cuvette should vary between 0.5 mM and 0.025 mM; you will be provided with a 30 mM stock solution. Calculate how much of this stock solution you will need to add the cuvette to achieve the desired pyruvate concentrations (run about 8 different concentrations). The most useful data will be collected below about 0.2 mM, so don't run too many points above this concentration.

50 - y µL

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 throughout the 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 at 340nm (#OD340) Notes: 1. Try the 0.5 mM pyruvate reaction first. You may need to experiment with enzyme dilutions until reasonable kinetics are obtained with 0.5 mM pyruvate (but on the fast side of reasonable—do you see why?). In all subsequent reactions, use the same dilution of enzyme. Note that the total volume of solution in the assay mix is 1510 µL. 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 at the beginning of the runs. 3. If you have data points that do not make sense (e.g., did you add a lower substrate concentration 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 satisfied with the quality of your data and will be able to extract k cat and K m values in which you 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, don’t forget to clean up your lab station; discard all waste and leave it ready for the next day’s 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 their location 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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th

The write-up for experiment II is due on Monday, March 24 . It is a long and complicated write-up. To make the process less daunting, the write-up is broken into two parts that can be completed independently (weeks 1 3 and 4). It is highly recommended that you complete the sections as you go—and 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 course’s “Organization Details” handout under “Intellectual Responsibility,” and if 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 = µmol/min) II) the total milligrams of protein III) 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 an Excel 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 one µmol 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 µmol/mL your total volume of solution in the cuvette was 1510 µL

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 wash POOL

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 not specific for LDH. One compares the OD595 of the aliquots to the standard curve to determine the micrograms protein in the cuvette. Dividing the micrograms of protein in the cuvette by the small volume of aliquot assayed yields the conc entration of protein in that 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, remember to 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 one sample 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 A3 Salt wash POOL

III. Calculate the specific activity of LDH For each aliquot and column fraction, divide the Total Units of activity by the total milligrams 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 sense—for example, is the enzymatic activity where you expect it to 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/V initial vs. 1/[S]. For the latter number, remember pyr that a change of 6.22 OD340/min corresponds to 1 mM/min. Your plot will provide K M and V max. For this plot, please use a graphing program (Excel will do). Report the equation for the 2 line and the square of the correlation coefficient (r ). If you are a graphing-program whiz, you may, of course, fit your V initial vs. [S] data directly to the Michaelis-Menten equation (equation 26 on page 232 of Stryer); you will get the same answers, just without a linear fit. pyr Question 4: How does your K M compare with the literature value (0.32 mM for the M4 isozyme, 0.08 mM for the H4 isozyme)? Suggest an explanation for any discrepancy.

Using your Bradford data on the pooled, purified LDH and the molecular weight per subunit from your gel (see below), and assuming that your enzyme is completely pure, determine the k cat for a LDH subunit from the equation Vmax = k cat[ET]. Also determine the k cat using the known subunit molecular weight of 35,500. Note: [ET] is, of course, the enzyme concentration in the cuvette for each of your kinetic runs in week 3, not in your stock solution (again, you may assume that the stock is pure LDH). Remember, the enzyme concentration was the same in every assay—only pyruvate concentration varied. Question 5: How does your value for k cat compare with the literature value (approximately 500 -1 s per subunit for both the M4 and H4 isozymes)? Suggest an explanation for any discrepancy.

Also, provide a recalculation of the specific activity of your LDH from your k cat value (just to be sure that you have become a kinetics expert). How is this done? k cat = (molecules substrate turned over/s)/molecule LDH = µmol substrate turned over/s)/ µmol LDH % (µmol substrate turned over/s)/mg LDH % (µmol substrate turned over/min)/mg LDH = units/mg LDH = specific activity. For the purposes of the above calculation you may 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 determined directly from your Bradford and kinetic assays? Propose a possible reason for a substantial discrepancy (>10%). pyr Question 7: How would your values of K M and k cat change 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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LDH Purification

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