Michelle Grau Lactate Dehydrogenase Protein Purification and Analysis Laboratory September 17 2009-October 16 2009-Notebook Pages: 58-96

Michelle Grau Lactate Dehydrogenase Protein Purification and Analysis Laboratory September 17 2009-October 16 2009- Notebook Pages: 58-96

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Introduction: In this lab investigation, the aim was to purify and analyze Lactate Dehydrogenase (LDH) enzyme from chicken breast. Protein purification is essential when studying the function, structure, and interactions of a particular protein. It’s important to determine the concentration of protein if  using it a variety of physical analytic methods such as steady-state kinetics or ligand binding that rely on molar measurements. LDH is an enzyme found in most plants and animals and plays a role in glycolysis for the formation of ATP.1 This enzyme catalyzes the reaction of pyruvate and NADH to form lactate and  NAD+, while at the same time it can catalyze the reverse reaction if there’s a large concentration of  lactate. If you wanted to purify LDH, or any protein, you must start with a tissue sample containing the protein. Most proteins are typically found within a cell, so the tissue must be subjected to a homogenizing process in order to break cell walls and release protein. If the protein is in solution, you expose it to a selective precipitation. Selective precipitation of proteins can be used as a rough method to recover a desired protein in a purification. This process depends on the physical or  chemical interaction between the protein and the precipitating agent. In this lab, (NH4)2SO4 is used to precipitate out LDH. Dialysis is a process of separating molecules in solution by differences in rates of diffusion across a semi-permeable membrane. Dialysis can most often remove a large amount of small impurities in a heterogeneous solution containing your protein. We used dialysis in this lab to remove the excess, unwanted (NH4)2SO4 and other small impurities while simultaneously exchanging the extraction buffer with dialysis buffer. Affinity chromatography is used to obtain a specific substance if it’s mixed in a heterogeneous solution. Columns used for affinity chromatography are typically composed if inert, chemically stable polymers, that have specific binding proteins or molecules. Yo u would use a column that was composed of a specific molecule to which the protein of o f interest would bind to with a high affinity. Once the protein solution is app lied to the column, all substances and proteins that do not bind or that bind loosely to the column are removed with buffer washes. Then the column is washed with a solution to which the desired protein binds strongly to and is isolated. In this lab we used a Cibacron blue affinity column to purify LDH. This molecule mimics the shape and charge characteristics of pyridine nucleotides to which dehydrogenase proteins frequently bind to. To obtain pure LDH from the column, it was washed with an NADH solution because of the high affinity LDH has for NADH. Once your protein is purified, there are many techniques to determine the purity and concentration of your protein. First you typically run an activity assay if the protein has enzymatic  properties, to determine which fractions from the chromatography contain the protein. SDS-PAGE gel electrophoresis is a good method to use for determining the purity of the protein. This method separates proteins according their molecular weight and length of polypeptide chain. Proteins all exhibit the same charge per unit mass due to the binding of o f SDS resulting in fractionation by size and mass. Using the gel from SDS-PAGE, you can detect the protein using a specific and visible antibody against the protein. Bradford Assays are a routine method for determining protein con centration. A standard curve made with known protein concentrations is constructed using Coomassie blue dye which

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Aims of Experiment: The purpose of this experiment was to extract and purify LDH enzyme from chicken breast muscle using a variety of techniques including centrifugation, selective p rotein precipitation, dialysis and affinity chromatography. Many different analytical methods were employed to determine the presence, purity and concentration of LDH such as activity assays, SDS-PAGE, Western Blot, Bradford Assay, Edelhoch, and QAAA. Results: Figure 1.  Flowchart Depiction of LDH Protein Purification Procedure and Analysis Step 1: 50g of Chicken Breast in 75mL of Extraction Buffer  Extraction Buffer: 10mM TrisHCl pH 8.6, 1mL 2mercaptoethanol, mercaptoethanol, 100mM PMSF, 1mM ethylene diamine Chicken tissue and extraction buffer homogenized using 4x 30sec  bursts allowing 10 seconds between bursts Step 2: Centrifugation 27,000 x g, 4°C for 20 min in 250mL conical vials Supernatant Collected – 52mL (Crude Homogenate) Step 3: Ammonium Sulfate Used 20.28g (NH4)2SO4 (0.39g of (NH4)2SO4 per mL of  supernatant) Added in cold room slowly over 15 minutes and then stirred for an additional 15 minutes. Step 4: Centrifugation Centrifugation Same conditions as above. Supernatant Collected- 52mL (Ammonium Sulfate Supernatant) Step 5: Resuspended Pellet in 5mL of Extraction Buffer  Same buffer as described above. Suspension contained protein and (NH 4)2SO4 Volume of pellet in extraction buffer- 7.2mL

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Used a Cibacron Blue Affinity Column Absorbance of all fractions was measured with a UV-Vis Spectrophotometer Spectrophotometer at 280nm. Blank was measured with milli-Q water. Absorbance of PMSF PMSF buffer was about 0.004. 0.004. Absorbance of each fraction was below 0.1 Abs before moving on to each consecutive wash. Flow Through

Tris PMSF Wash #1:

10mL of NAD+ wash: Tris PMSF Wash #2:

10mM TrisHCl pH 8.6, 0.5mM 2-mercapto ethanol, 10mM TrisHCl pH 1mM Lithium Lactate, 8.6, 0.5mM 2+ mercaptoethanol, 1mM 1mM NAD PMSF

Fracti Fractions ons:: 1-3

Fract Fraction ions: s: 4-9

10mL of NADH wash: 10mM TrisHCl pH 8.6, 0.5mM 2-mercapto ethanol, 1mM NADH

Same solution as wash #1

Fract Fraction ions: s: 11,12 11,12

Fracti Fractions ons:: 13-16 13-16

Tris PMSF Wash #3: Same solution as wash #1

Fract Fraction ions: s: 17,18 17,18

Fracti Fractions ons 19,20 19,20

Step 8: Analysis of LDH Purification

Activity Assays of  LDH Samples

SDS-PAGE Gel Electrophoresis

Analysis parameters located in Table 1

Bradford Assay for  determination of  LDH concentration

Analysis parameters located in Figure 2

Edelhoch Analysis Analysis parameters located in Table 2 and Figure 5

Analysis parameters located in Table 1 Western Blot analysis Analysis parameters Analysis parameters located in Figure 4

LDH mass determination via BenchMark TM Protein Ladder Standard Curve Analysis parameters located in Figure 3

Bradford Assay for  determination of  LDH concentration in ooled fractions Analysis parameters located in Table 2

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A crude homogenate of chicken breast was obtained and to this, ammonium sulfate was added to precipitate LDH. This precipitate was treated as described in the flowchart in Figure 1. Following dialysis, the LDH sample was subjected to a Cibacron blue affinity column. LDH activity assays were performed to determine which samples and fractions contained a significant amount of LDH. The results of the LDH activity assays are located in Table 1. The highest enzyme activity was observed in the crude homogenate. Fraction 17 produced the highest observed activity from the chromatography fractions. A higher enzyme activity was observed in the (NH4)2SO4 supernatant than the (NH4)2SO4 dialyzed protein solution.  Bradford Assay Analysis:

Table 1: LDH Concentration, Purity, and Yield Determination via Bradford Assay Sample

Crude Homogenate

(NH4)2SO4 Supernatant

(NH4)2SO4 Dialyzed

Fraction #11

Fraction #12

Fraction #17

Fraction #18

Fraction #17 + #186

Dilution

12,500

12,500

125

125

125

125

125

200

Abs

0.141

0.065

0.45

0.347

0.359

0.613

0.396

0.221

75.19

13.11

18.95

33.99

38.57

45.86

8.51

27.19

0.0028

0.0003

0.0130

0.0096

0.0100

0.0184

0.0112

0.0066

34.810

3.353

1.627

1.201

1.250

2.302

1.404

1.362

52.0

52.0

7.2

5.0

5.5

6 .0

6.0

12.5

1810.099

174.338

11.715

6.004

6.877

13.810

8.421

17.025

3909.88

681.72

136.44

169.95

212.135

275.16

51.06

339.875

2.160

3.910

11.647

28.307

30.846

19.924

6.063

19.963

Fold Purification4

1.00

1.81

5.39

13.11

14.28

9.22

2.81

9.24

Yield (%)5

100.00

17.44

3.49

4.35

5.43

7.04

1.31

8.69

LDH Activity (μmol NADH min 1 mL-1)1

Diluted Protein Concentration (mg/mL)

Undiluted Protein Concentration (mg/mL)

Volume (mL)2 Total Protein (mg)

Total LDH Activity (μmol -1

NADH min )

Specific LDH Activity (μmol NADH min -1 mg-1)3

Table 1: Bradford Assay standard curve was constructed using IgG protein concentrations between 1.25 μg/mL-25.0μg/mL. BioRad Assay Reagent was added to standards, samples, and fractions that were diluted in 50mM KH 2PO4 buffer at pH 8.0. The final volume was 1mL. The blank was measured with 50mM KH2PO4 buffer at pH 8.0. Absorbance was measured at 595nm using a UV-Vis Spectrophotometer. Spectrophotometer. Path length was 1cm. The equation of the standard curve determined through the Bradford Assay was: y = 0.0302x + 0.056. Dilutions were made for samples to obtain an absorbance between 0-1. 1LDH ActivitiesActivity of 10μL LDH was determined via time course measurements using the UV-Vis Spectrophotometer, monitoring the NADH production by following the absorbance at 340nm. Duplicates were measured for samples with significant enzyme activity. 2 Volumes- See flowchart. 3 Specific LDH Activity = LDH Activity/ Undiluted LDH Concentration. 4 Fold Purification = Specific LDH Activity/ Crude Homogenate Specific LDH Activity. 5 % Yield = (Total LDH Activity/ Crude Homogenate LDH Activity) *100. 6 The absorbance and both diluted and undiluted concentrations for this fraction was determined by the Bradford Assay as described in Table 2. Absorbance value is an average of two measurements. The enzyme activity was estimated based on the average of the two fractions. All the subsequent calculations are therefore also estimations.

A Bradford analysis was used to determine the co ncentration of LDH in a selection of  o f 

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largest observed amount of LDH as well as the greatest total LDH activity and % yield while  producing the lowest specific LDH activity and fold p urification. Of the column fractions (not combined), 17 had the largest observed LDH concentration, mass and total LDH activity, while 12 had the largest specific LDH activity, fold purification and % yield. The (NH4)2SO4 supernatant had a larger protein concentration, con centration, mass, total LDH activity, and % yield as compared to the (NH4)2SO4 dialyzed protein solution which had a larger specific LDH activity and fold purification. After the SDS-PAGE analysis (Figure 2), fractions 17 and 18 were combined because they had the largest observed specific LDH activity of the fractions that resulted from the NADH wash. An enzyme activity was estimated based on the average activity of both fractions. The absorbance and concentration was determined through a different Bradford Assay as described in Table 2. The same calculations were made for the combined fractions that were made for all the samples and individual fractions. This fraction had the largest mass, total LDH activity and % yield than all the other fractions. The overall yield of the pooled fractions was 17.025mg LDH/50g chicken breast.  SDS-PAGE Gel Analysis:

Figure 2. SDS-PAGE Gel of LDH Samples and Fractions

An SDS-PAGE gel was run to determine the purity of  1 2 3 4 5 6 7 8 9 10 11 12 12 13 14 15 LDH in select samples and fractions that had observed enzyme activity. An image of  the gel is in Figure 2. An LDH control was used as a reference for determining which band corresponded to LDH. The lanes with crude homogenate, dialyzed (NH4)2SO4 protein, F11, F12, F13, F17, and F18 all appeared to have a band that corresponds to the LDH control band. Fractions 11, 12, SDS-PAGE-Each sample was made in SDS-sample buffer: 0.0583M TrisHCl, TrisHCl, 5% (v/v) Figure 2: SDS-PAGE-Each and 17 have the thickest glycerol, 1.713% (w/v) SDS, 10mM DTT, 0.0017% (w/v) Bromophenol blue. 15μL of each sample and fraction was added to each well. 5μL of BenchMark  Protein standard was added to wells 1 an 14, and  bands. There were a lot of  2μL was added to well 15. Lane 1: Standard, Lane 2: Crude Homogenate, Homogenate, Lane 3: Na2SO4 observed bands in the crude Supernatant, Lane 4: Na2SO4 Dialyzed Protein, Lane 5: Fraction (F)11, Lane 6: F12, Lane 7: F13, Lane 8 F14, Lane 9: F17, Lane 10: F18, Lane 11: F19, Lane 12: F20, Lane 13: Control LDH protein, homogenate lane that didn’t Lane 14: Standard, Lane 15: Standard. SDS-PAGE was run in running buffer: 0.025M TrisHCl, correspond to LDH. In all the 0.192M glycine, 0.1% (w/v) SDS. 12% ac rylamide gel was used. Proteins were visualized with Coomassie blue staining. fractions, there were other   bands that didn’t correspond to LDH, however, there were a significantly less number of bands in the fractions as compared to the crude homogenate as well as the (NH4)2SO4 supernatant and (NH4)2SO4 dialyzed protein. TM

15 14 13 12

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Figure 3. Standard Curve of BenchMark TM  Protein Ladder from The distance was measured SDS-PAGE Gel For LDH Mass Determination  between the LDH band in lane 17 was measured to and the  baseline. This distance, 3.05cm, was used in the equation of the standard curve to calculate for an experimental molecular weight of LDH. The distance measured was 3.05cm. The experimental molecular mass was determined to be 36,791g/mol. The percent difference was