Meat Juice Lab (Anna Peskin, C5)

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

1/12

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.


2/12



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


3/12



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


4/12



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


5/12



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


6/12



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.


7/12



*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


8/12



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)

Trypsin inhibitor (21 kDa)

Lysozyme (14 kDa)

LDH


9/12


10/12



Figure 5, a graph of the y-intercepts found in the Lineweaver-Burke plot (figure 4).

Note that 25mM lactate data was excluded because there was likely a problem with dilution due to pipetting accuracy. The intercept

of the 25mM lactate data was inconsistent with the rest of the data and thus excluded.

Figure 6, a graph of the slopes found in the Lineweaver-Burke plot (figure 4).

Note that 25mM lactate data was excluded because there was likely a problem with dilution due to pipetting accuracy. The slope of

the 25mM lactate data was inconsistent with the rest of the data and thus excluded.

y = 26x + 2.2

R = 0.97

0

0.5

1

1.5

2

2.5

3

3.5

0 0 .005 0 .01 0 . 015 0 .02 0 .025 0 .03

Intercept

1/Lactate (mM-1)

INTERCEPT REPLOT (EXCLUDING 25MM

LACTATE)

y = 15x + 0.20

R = 0.98

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 0 . 0 0 5 0 . 0 1 0 . 0 1 5 0 . 0 2 0 . 0 2 5 0 . 0 3

Slope

1/Lactate (mM-1

)

SLOPE REPLOT (EXCLUDING 25MM

LACTATE)


11/12



CALCULATIONS EXCLUDING 25MMLACTATE

Kiafor NAD = -(1/x value of interception) = -(1/-1.7) = 0.59 mM

Intercept Replot Equation: y = 26x+2.2

Y-intercept (Vmax-1) = 2.2

NAD Vmax(max rate of product formation)

= 2.2-1

= 0.45 units/30 sec

X-intercept (-Kb-1) = -0.083

Kbfor lactate

(Michaelis constant for lactate)

= (-1.0)(-0.083-1)

= 12 mM

Slope Replot Equation: y = 15x + 0.20

NAD Ka(Michaelis constant for NAD)

= y-intercept of slope replot * Vmax

= 0.20 * .45

= 0.093 mM

DISCUSSION :

The column seemed to work well in general. The activity of LDH was significantly higher in the fractions

which were washed with NADH to elute muscle LDH (when compared to all of the other fractions that were washed

through the column). A high activity in this fraction when compared to other fractions shows that the column efficiently

allowed for separating of muscle LDH. Also, the SDS-PAGE showed no bands that represented contaminate proteins in

F8 and F9. It did, however, show lines that indicated the presence of LDH. From these results, it can be concluded that

the column worked well to purify muscle LDH.

The LDH is purer at the end that in the pure homogenate. Comparing F8F10 to the crude homogenate shows

how many fold the increase in purity was after the homogenate was applied to the column.

F8 16 / 1.4 = 11 fold

F9 12 / 1.4 = 8.6 fold

F10 3.4 / 1.4 = 2.4 fold

It is important to note that throughout this lab F2 showed low activity, low LDH concentrations, and even low

contamination on the SDS-PAGE. It is suspected that F2 was mostly wash buffer left over in the column from when the

column was created rather than actual fraction.

There were some inconsistencies in the data. For example, one of the lines (the line representing 25mM lactate)

on the Lineweaver-Burk plot did not intersect and gave an inconsistent slope and y-intercept. That line was excluded

from the calculations. It is likely that error occurred with that sample because the pipets that were used had less than

perfect accuracy. This caused error to occur when working with very small, diluted samples. It would be ideal to redo

the kinetics experiment for 25mM lactate with a more accurate pipet and see if results differ.

Also, all of the assays showed a low LDH activity in F3-F7. However, the native gel showed dark bands for

heart LDH in F3-F7. This was unexpected for two reasons. First of all, F3-F7 had otherwise consistently showed low


12/12

Documents

Meat Juice Lab (Anna Peskin, C5)