Meat Juice Lab (Anna Peskin, C5)

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  • 8/14/2019 Meat Juice Lab (Anna Peskin, C5)

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    Anna Peskin (1020425) Biochemistry 426

    Lab Partner: Anthony Cho (Group C5) November 13, 2013

    ISOLATION AND CHARACTERIZATION OF LACTATE DEHYDROGENASE

    OBJECTIVE :

    The objective of this experiment was to purify and characterize a protein of interest. In this lab, lactate

    dehydrogenase (LDH) was the protein of interest. LDH has various isozymes made of heart or muscle LDH (H4, M3H,

    H2M2, HM3, and M4). This lab looked specifically at porcine muscle, but both heart and muscle LDH are expressed in

    muscle. The protein was first purified on an affinity column. Afterwards, a BCA assay was used to determine the

    amount of protein that was purified. The reaction that is catalyzed by LDH (lactate + NAD+pyruvate + NADH + H+)was run while observed in a spectrophotometer and LDH activity was measured by observing NADH formation. SDS-

    PAGE was then used to measure to observe the purity of the purifiedprotein. Finally, a spectrophotometer was used to

    run kinetic analysis of the reaction catalyzed by LDH in order to determine kinetic constants of the reaction.

    PROCDURE :

    The first step of this experiment was to prepare a lipid-free sample of porcine muscle (prepared by the TA).

    This homogenized sample was run through an Affigel Blue affinity column, washed with various buffers, and collected

    into several fractions (F1-10). The expected contents of these fractions are in table 1, below. The fractions were then

    dialyzed to remove NaCl, NAD, and NADH. These molecules would otherwise interfere with assays performed later on

    in this experiment.

    Native gel electrophoresis was then run on each fraction. The H and M subunits differ in net chargethe heart

    subunits are negative while the muscle subunits are slightly positive. Native gel electrophoresis uses this to its advantage

    as it separates the subunits based on charge and allows experimenters to see the relative amount of each isozyme in each

    fraction.

    Afterwards, a BCA Assay was run to determine the total amount of protein in each fraction. BCA works

    because peptide bonds (and thus proteins) reduce Cu+2to Cu+1. Cu+1binds to BCA and forms a colored complex. This

    color can be measured with absorbance and thus the amount of protein in each sample can be quantified.The amount of LDH activity in each fraction was determined by adding the LDH containing fraction to lactate

    and NAD and catalyzing the reaction: lactate + NAD+pyruvate + NADH + H+(this reaction is catalyzed by LDH).

    A spectrophotometer measured absorbance at 340nm, where NADH absorbs while NAD does not absorb. The formation

    of NADH was representative of the activity of LDH in each sample.

    SDS-PAGE was used to separate proteins based their size. The SDS-PAGE of our fractions was compared to

    known contaminants as well as the known size for LDH. The SDS-PAGE allowed for experimenters to visually

    observe purity in each fraction as well as see if LDH was present in each fraction.

    Finally, kinetic constants for LDH were calculated by running the reaction catalyzed by LDH while observing

    NADH formation in a spectrophotometer. This was done while varying NAD or lactate concentrations one at a time.

    From here, kinetic constants could be calculated.

    F1: crude homogenate F6: column washed with NAD, expect to see heart LDH

    F2: homogenate ran through column, no LDH F7: column washed with NAD, expect to see heart LDH

    F3: homogenate ran through column, no LDH F8: column washed with NADH, expect to see muscle LDH

    F4: homogenate ran through column, no LDH F9: column washed with NADH, expect to see muscle LDH

    F5: column washed with NAD, expect to see heart LDH F10: column washed with NADH, expect to see muscle LDH

    Table 1, expected contents of various fractions.

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    Anna Peskin (1020425) Biochemistry 426

    Lab Partner: Anthony Cho (Group C5) November 13, 2013

    RESULTS AND CONCLUSION :

    ACTIVITY AND RECOVERY OF LACTATE DEHYDROGENASE :

    Fraction Volume (mL) Dilution? Volume

    Assayed (L)

    Absorbance

    / minute

    [NADH] in the cuvette

    after 1 minute (mol/mL)

    units LDH

    in 10 mL

    Activity

    (units)

    F1 2.7 1:5 10 0.38 0.061 31 84

    F2 2.7 n/a 10 0.018 0.0030 0.30 0.81

    F3 2.9 n/a 10 0.0060 0.00097 0.096 0.28

    F4 3.3 n/a 10 0.0014 0.00022 0.023 0.075

    F5 4.2 n/a 10 0.0028 0.00045 0.045 0.19

    F6 3.2 n/a 10 0.0096 0.0015 0.15 0.49

    F7 4.1 n/a 10 -0.00080 -0.00013 -0.013 -0.052

    F8 3.1 n/a 10 0.60 0.096 9.6 30.

    F9 3.0 n/a 10 0.64 0.10 10. 31

    F10 3.8 n/a 10 0.11 0.018 1.8 6.7

    Table 2, activity of all of the fractions. Note: volume amounts were measured with a pipette after dialysis. Negative units are

    indicative of so little LDH that it was barely measureable.

    Sample Activity Calculation for F1:

    Absorbance at 0sec: .11

    Absorbance at 30sec: .30

    abs / 30 sec = (Absorbance30sec - Absorbance0sec) / 30 sec

    = (.30 - .11) / 30 sec

    = .19 / 30 sec

    abs / 1 min = (abs / 30 sec) * (60 sec / 1 min)

    = (.19 / 30 sec) * (60 sec / 1 min)

    = .38 / minute

    [NADH] in the cuvette after 1 minute (mol/L)use A1/ C1= A2/ C2 (the molar extinction coefficient which is

    6220M-1cm-1 for NADH)

    (6220M-1cm-1)/ 1M = (abs / 1 min) / x

    x = .38 / (6220M-1cm-1 / 1M)

    x= 6.1*10-5 mol/L

    Convert units to mol/mL:

    6.1*10-5 mol 106 mol 1 L = .061 mol/mL

    1 L 1 mol 103 mL

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    Anna Peskin (1020425) Biochemistry 426

    Lab Partner: Anthony Cho (Group C5) November 13, 2013

    Account for dilution : .061 mol/mL * 5 (dilution factor) = .31 mol/mL

    Units LDH in 10 L: .31 units LDH 1.0*103L = 31 units / mL

    10. L 1 mL

    Activity = (units / mL) * (mL fraction)

    = 31 units / mL * 2.7 mL

    = 84 units

    Percent Recovery :

    Fraction Percent Recovery

    F2 0.96 %

    F3 0.33 %

    F4 0.089 %

    F5 0.22 %

    F6 0.58 %

    F7 -0.062 %

    F8 36 %

    F9 37 %

    F10 7.9 %

    Total Recovery 82 %

    Table 3, percent recovery of the various fractions. Negative percent recovery is indicative of negative LDH units (explained

    in table 2).

    Sample Percent Recovery Calculation for F8:

    Percent Recovery F8 = (LDH Activity in F8 / LDH Activity in F1 (crude homogenate)) * 100%

    = (30 / 84) * 100%

    = 36 %

    It is expected that the NAD and NADH buffers will elute LDH from the column. NAD has a high affinity for

    heart isozyme and NADH has a high affinity for muscle isozyme. Because of this, it is expected that NAD will elute

    heart isozyme and NADH will elute muscle isozyme. This assay was run on porcine muscle and thus it is expected the

    majority of the LDH isozyme to be muscle isozyme. Because of this, the NADH is eluting most of the product off of the

    column.

    These results showed that the LDH that was applied to the column did indeed bind to the resin. The fraction

    that did not go through the column (F1) had a high activity (84 activity units) which is indicative of a lot of LDH present

    in the fraction. The fractions that went through the column but were not washed with an elution buffer (F2, F3, and F4)

    had a very low activity (.81, .28, and .075respectively) which indicates that little to no LDH was present in these

    fractions. The fractions that were washed with NAD (in which it was expected to see heart isozyme (F5, F6, and F7) also

    had low activitynone of these activities surpassed 1 unit. This is not surprising because porcine muscle does not have

    a high amount of heart LDH isozyme. Thus, there was not a large amount of LDH in the fractions and the activity was

    low. It is expected to see the most LDH recovered in F8, F9, and F10 because those were washed with NADH and

    NADH has a high affinity for muscle LDH isozyme. This turned out to be truethe activity in F8F10 was much

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    Anna Peskin (1020425) Biochemistry 426

    Lab Partner: Anthony Cho (Group C5) November 13, 2013

    higher (reaching up to 31 activity units) than all of the other fractions that passed through the column. Out of these 3

    fractions, F10 had the lowest activity. This is due to the fact that most of the LDH likely washed off into F8 and F9. This

    data supports the conclusion that LDH was bound to the resin and was later eluted with NADH.

    Overall, 82% of the LDH was recovered. A majority of the LDH was recovered when the column was washed

    with NADH. This supports the conclusion that a majority of the LDH present was muscle LDH (because muscle LDH

    has a high binding affinity for NADH). 18% of the LDH was not recovered. It may have stuck to the column and not

    washed off with the elution buffers. Alternatively, since this experiment was done over the course of a few weeks it

    might have denatured over time and thus would not be active.

    PROTIEN DETERMINATION:

    Figure 1, protein determination by BCA. The lighter lines represent 1 standard deviation from the mean.

    Fraction Concentration

    (Sample 1, mg/mL)

    Concentration

    (Sample 2, mg/mL)

    Concentration

    (Average, mg/mL)

    Specific

    Activity

    F1 7.8 8.3 8.1 1.4

    F2 0.27 0.46 0.36 0.30

    F3 1.7 2.2 1.9 0.017

    F4 1.6 1.6 1.6 0.0042

    F5 0.45 0.45 0.45 0.024

    F6 0.17 0.21 0.19 0.26

    F7 0.14 0.10 0.12 -0.026

    F8 0.20 0.17 0.19 16

    y = 0.43x + 0.080

    R = 0.98

    y = 0.43x + 0.040

    y = 0.43x + 0.12

    0

    0.2

    0.4

    0.6

    0.8

    1

    1.2

    0 0.5 1 1.5 2 2.5A

    bsorbance(absorbanceunits)

    Protein Concentration (mg/mL)

    PROTEIN DETERMINATION BYBCA

    : Protein Determination: Standard Curve : 1 Standard Deviation

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    Anna Peskin (1020425) Biochemistry 426

    Lab Partner: Anthony Cho (Group C5) November 13, 2013

    F9 0.32 0.28 0.30 12

    F10 0.13 0.15 0.14 3.4

    Table 4, total protein concentration as determined by BCA-Assay and specific activity of fractions.

    Based on the specific activity of LDH in each fraction, the recovered LDH is more pure then the crudehomogenate (F1). In F8, F9, and F10, each mg/mL of protein has 16, 12, and 3.4 (respectively) units of LDH activity.

    This can be compared to F1 where there was only 1.4 units of LDH activity for each mg/mL of protein. From this, it can

    be concluded that the recovered LDH was much more pure then the starting material.

    BCA-Assay Absorbance Results

    Table 5, BCA-Assay absorbance results. S1-S11 were standards of known concentrations made with BSA.

    Sample Concentration Calculation for F1:

    Equation from linear regression: y= .43x + .080 ; y= absorbance, x=concentration

    Concentration = (absorbance - .080)/.43

    = (.75 - .080)/.43

    = 1.6 mg/mL

    S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11

    F1 F1, 1:5 F1, 1:25 F2 F2, 1:5 F2, 1:25 F3 F3, 1:5 F3, 1:25

    F4 F5 F6 F7 F8 F9 F10

    S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11

    F1 F1, 1:5 F1, 1:25 F2 F2, 1:5 F2, 1:25 F3 F3, 1:5 F3, 1:25

    F4 F5 F6 F7 F8 F9 F10

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    Anna Peskin (1020425) Biochemistry 426

    Lab Partner: Anthony Cho (Group C5) November 13, 2013

    Account for dilution 1.6 * 5.0 (dilution factor)

    = 7.8 mg/mL

    Sample Specific Activity Calculation for F1:

    mg total protein = volume fraction (from dialysis) * concentration

    = 2.7 mL * 8.1 mg/mL

    = 22 mg

    Specific Activity = Units LDH (from table 2) / mg total protien

    = 31 / 22 mg

    = 1.4 activity units

    Standard Deviation Calculations:

    xi yi yl*

    (yi-yl) (yi-yl)

    2

    0.00 0.12 0.080 0.039 0.0015

    0.00 0.12 0.080 0.038 0.0014

    0.20 0.20 0.17 0.028 0.00081

    0.20 0.17 0.17 -0.051 0.0026

    0.40 0.25 0.25 0.0010 1.1*10-6

    0.40 0.26 0.25 0.0110 0.00012

    0.60 0.33 0.34 -0.014 0.00021

    0.60 0.39 0.34 0.046 0.0021

    0.80 0.43 0.43 0.0073 5.3*10-5

    0.80 0.31 0.43 -0.12 0.0131.0 0.52 0.51 0.0089 7.9*10-6

    1.0 0.50 0.51 -0.017 0.00029

    1.2 0.60 0.60 0.00052 2.7*10-7

    1.2 0.58 0.60 -0.015 0.00024

    1.4 0.66 0.68 -0.021 0.00044

    1.4 0.68 0.68 -0.0019 3.5*10-6

    1.6 0.77 0.77 -0.0042 1.8*10-5

    1.6 0.74 0.77 -0.027 0.00074

    1.8 0.88 0.86 0.025 0.00064

    1.8 0.83 0.86 -0.030 0.00088

    2.0 1.0 0.94 0.088 0.0077

    2.0 0.95 0.94 0.0030 9.0*10-6

    SUM = 0.033

    SD

    *21 = (number of samples -1)

    = (0.033/ 21)

    = 0.040

    Table 6, standard deviation calculations for protein concentration as determined by a BCA-Assay.

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    Anna Peskin (1020425) Biochemistry 426

    Lab Partner: Anthony Cho (Group C5) November 13, 2013

    *ylis the absorbance that is expected based on the linear regression. To calculate, the x values were plugged into the

    equation y=.43x+.080 and y was solved for.

    Example calculation for xi = 0:

    yl = .43x+.080

    = .43(0)+.080

    = .080

    Expected absorbance (yl) for 0 protein concentration = .080

    Actual absorbance (yi) for 0 protein concentration: 0.12

    (yi-yl) = 0.12 - .080

    = 0.039

    (yi-yl)2 = 0.0392

    =0.0015

    NATIVE GEL

    Figure 2, native gel showing amounts of LDH in various fractions.

    The heart LDH seemed to be more prominent in the fractions. Heart LDH was prominent in every fraction

    other than F2 and F10. Muscle LDH was only present in F1, F8, F9, and F10. As expected, F8 (which was washed with

    a NADH buffer to elute M4LDH) had the highest abundance of muscle LDH. F9 and F10 were also washed with NADH

    F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F1

    H4

    M4

    M3H

    M2H2

    MH3

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    Anna Peskin (1020425) Biochemistry 426

    Lab Partner: Anthony Cho (Group C5) November 13, 2013

    so it is expected to see muscle isozyme in F9 and F10. The native gel shows dimmer smears in the F9 column and by

    F10 they are virtually nonexistent other than a light marking that represents the muscle isozyme. This is because most of

    the muscle LDH got eluted off into fractions 8 and 9.

    The native gel confirms that heart isozyme has a higher affinity for NAD while muscle isozyme has a higher

    affinity for NADH. Fractions 5, 6, and 7 were washed with NAD buffer. These bands are dark and appear where it is

    expected to see H4and maybe MH3isozymes. On the other hand, fractions 8, 9, and 10 were washed with NADH buffer.

    F8 and F9 contain darker smears where it is expected to see M4isozyme which confirms the affinity of M4for NADH.

    Also, light M4bands are seen in the F10 column (which was also washed with NADH), but it is reasonable to conclude

    that these bans are light because by the 10thfraction almost all of the LDH has been washed out.

    SDS-PAGE

    Figure 3, SDS-PAGE gel showing presence of LDH as well as the purity of various fractions.

    From the SDS-PAGE, it can be concluded that the final preparation was pure. Bands for other proteins (such as

    ovalbumin or lysozyme) clearly showed up in the crude homogenate F1 and the wash fraction (F3). Bands for other

    proteins were faint, but nevertheless present in F5, F6, and F7. This lab was concerned with muscle isozyme (which was

    expected in F8-F10), and these bands were absent by the time that F8 was eluted. This is indicative of purity in F8 and

    F9.

    The gel pattern agreed with the LDH standard. It was expected that LDH was seen in F5-F7 (heart LDH) and

    F8-F9 (muscle isozyme). There is a clear, thick band in the LDH standard column which also shows up in the F8

    column. This band is also present in F5-F7 and F9, although it is much fainter. This faint band suggests that a majority

    of the LDH that was present was indeed muscle LDH that had mostly eluded off into F8.

    F1 F2 F3 F5 F6 F7 F8 F9 MW std LDH std

    Carbonic anhydrase (31 kDa)

    Phos hor lase B 97 kDa Serum albumin (66 kDa)

    Ovalbumin (45 kDa)

    T...

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