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Investigate The Mitochondrial Purity

Abstract

The main goal of this lab report was to investigate the mitochondrial purity of samples derived from a liver homogenate. This was done by the separation of mitochondria from the rest of the cellular material. The experiments used to achieve this were, centrifugation to separate out the liver homogenate and by enzyme and protein assays. The enzyme assay was done to measure the absorption of various samples of the homogenate of liver as time progresses. This was done using DCPIP, succinate, water and phosphate in buffer. The protein assay was done to determine the protein concentrations of the samples, this was done using Bio-Rad dye which binds to the protein in the samples. The results of which both assays were used to calculate the specific activity of the samples and showed P2 to have the greatest specific activity. Exemplifying that, as a result of multiple centrifugations, P2 has a greater ratio of mitochondria to the rest of the cellular material than the other samples. This concludes that the hypothesis that P2 will have the greatest specific activity and as a result, it will also be most pure was correct as proven by this lab exercise. Introduction

Centrifugation separates the inner components of cells, due to the different sizes and other sedimentation properties causing the components to sediment at different times during the centrifugation, giving us a pellet and a supernatant. (Fishersci.co.uk, 2018)

In these set of experiments we will be using centrifugation to separate a liver homogenate to be able to purify mitochondria. Mitochondria are membrane bound organelles, located in the cytoplasm of eukaryotic cells. They are made up of: a smooth outer membrane, an inner membrane which folds to form cristae, the matrix and the intermembrane space.

Mitochondria convert food we eat into ATP by the process of oxidative-phosphorylation; they also have other functions, such as the break down of harmful chemicals in the body and play a key role in apoptosis. (Mrc-mbu.cam.ac.uk, 2018)

The mitochondria had to be purified, as the liver homogenate sample has other cellular material. In order to achieve more accurate results, we needed to remove the other cellular material. At each centrifugation, more of the other cellular material will be separated from the mitochondria; therefore, I hypothesise that P2 will have the greatest specific activity and as a result, it will also be most pure as the specific activities are directly proportional to the amount of mitochondria in each sample relative to the total cellular material.

To achieve the specific activity of succinate dehydrogenase in each sample, both a protein and enzyme assay must be carried out. The enzyme assay was carried out to show the change of absorption of light over time, to show the initial rate of reactions of each sample.

The protein assay was carried out to find the unknown concentrations of 5 samples. This was done by recording the absorbance of proteins with known concentrations and plotting them on a concentration vs absorbance graph and then plotting the absorbance of the 5 samples.

Method

Cell Fractionation : purifying mitochondria

500µl of the liver homogenate was removed and placed into an eppendorf tube, which was consequently put in ice. Following this, 10 ml of the homogenate was taken and centrifuged in a 15ml centrifuge tube at 3000 rpm, which is a g force of 1509.3 g

Once the centrifugation was complete, the supernatant was removed and placed into a 25 ml beaker in ice. 5 ml of tris homogenisation buffer was then added to the pellet and resuspended, making up 7.49 ml of homogenous suspension.

500µl of both the supernatant and the resuspended pellet were placed in individual eppendorf tubes and put in ice. After, 6.2 ml of the supernatant was centrifuged for 5 minutes at 30,000g in an ultracentrifuge. Once finished, the new pellet and supernatant were separated. The pellet was placed in 1 ml of homogenisation buffer making 1.4 ml of homogeneous suspension, and 5.9 ml of supernatant.

Once all 5 samples were obtained, an assay was carried out, containing 7 different cuvettes.

Table 1

Reagents /cuvette

1

2

3

4

5

6

7

DCIP in buffer (0,0165 mg/ml)

1.5ml

1.5ml

1.5ml

1.5ml

1.5ml

1.5ml

Phosphate buffer (0.275M, pH 7.4)

1.5ml

Succinate (0.5M, pH 7.4)

30µl

30µl

30µl

30µl

30µl

Water

1.2ml

1.2ml

1.2ml

1.2ml

1.2ml

1.2ml

1.2ml

Tissue sample 60µl

H

H

H

S1

N

S2

P2

When the tissue samples were added, the contents were mixed via inversion and immediately placed into the spectrophotometer which was set to A600 and H was added to cuvette 1 and used to calibrate it.

Measurements were taken every 5 seconds for the first 60 seconds and then every 10 seconds after for another 2 minutes.Once all results were achieved, a graph was plotted on graphpad to show absorbance against time and linear regression was used for the first 30 seconds to find the initial rate of reaction.

Purifying mitochondria part 2 : Protein assay

Known concentrations of protein were prepared using Bio-Rad dye. 8 standard solutions of BSA were made up, ranging from 0 – 1.4mg/ml at intervals of 0.2mg/ml. Next 1.5ml of Bio-Rad dye and 25µl of each BSA solution were added to different cuvettes; two of each were made and 1:20 dilutions were carried out. Once mixed, the absorbance for each concentration using the spectrophotometer at 595nm was recorded. Cuvette 8 was used to calibrate the spectrophotometer and the average of the two readings per each concentration were plotted on a graph. The protein concentrations of the tissue samples were then determined by recording the concentrations which were plotted on the graph of known concentrations.

Table 2

Standard Solution number

BSA concentration required (mg/ml)

Volume of stock 1.4mg/ml

Volume of water

1

1.40

2.00

0.00

2

1.20

1.71

0.29

3

1.00

1.43

0.57

4

0.80

1.14

0.86

5

0.60

0.86

1.14

6

0.40

0.57

1.43

7

0.20

0.29

1.71

8

0.00

0.00

2.00

Results

Enzyme assay

This graph shows the change in absorbance over time of the 6 cuvettes, all of which show a decrease in absorbance over time. Cuvette 7 has the greatest absorbance and cuvette 4 has the lowest; cuvette 3 shows the greatest overall decrease in absorbance.

Table 3

Time (s)

Absorbance

Cuvette 1

Cuvette 2

Cuvette 3

Cuvette 4

Cuvette 5

Cuvette 6

Cuvette 6 – re run

Cuvette 7

0

0.000

0.214

0.205

-0.194

0.274

-0.250

0.247

0.522

5

0.205

0.195

-0.196

0.261

-0.255

0.239

0.514

10

0.192

0.185

-0.198

0.248

-0.260

0.228

0.505

15

0.182

0.171

-0.204

0.239

-0.265

0.227

0.499

20

0.172

0.156

-0.205

0.229

-0.270

0.222

0.492

25

0.163

0.146

-0.21

0.224

-0.276

0.217

0.485

30

0.157

0.136

-0.216

0.217

-0.281

0.211

0.48

35

0.15

0.129

-0.222

0.209

-0.290

0.206

0.475

40

0.145

0.122

-0.227

0.201

-0.295

0.201

0.471

45

0.14

0.112

-0.233

0.192

-0.296

0.2

0.467

50

0.131

0.105

-0.234

0.187

-1

0.195

0.464

55

0.127

0.099

-0.239

0.18

0.19

0.46

60

0.122

0.092

-0.244

0.175

0.19

0.455

70

0.114

0.082

-0.251

0.163

0.185

0.451

80

0.108

0.072

-0.257

0.152

0.179

0.443

90

0.099

0.062

-0.263

0.143

0.173

0.436

100

0.092

0.055

-0.269

0.135

0.168

0.431

110

0.08

0.048

-0.275

0.128

0.166

0.427

120

0.077

0.04

-0.277

0.118

0.161

0.421

130

0.072

0.035

-0.282

0.112

0.159

0.418

140

0.067

0.028

-0.284

0.106

0.157

0.414

150

0.062

0.024

-0.29

0.1

0.152

0.41

160

0.058

0.02

-0.292

0.091

0.15

0.405

170

0.054

0.015

-0.297

0.085

0.147

0.401

180

0.051

0.012

-0.299

0.082

0.146

0.398

Protein assay

Table 4

BSA concentration (mg/ml)

Absorption

A

B

average

1.4

0.955

1.043

0.999

1.2

0.944

0.787

0.866

1.0

0.688

0.669

0.679

0.8

0.569

0.549

0.559

0.6

0.475

0.442

0.459

0.4

0.320

0.345

0.333

0.2

0.145

0.158

0.152

0.0

0.000

-0.013

-0.007

Table to represent change in absorbance of two sets of known BSA concentrations, and their averages. This shows that as the concentration of BSA(mg/ml) decreases (from 1.4mg/ml to 1.2mg/ml), the absorption also decreases.

Table 5

Sample

Absorption

(Entered)

BSA concentration (mg/ml)

(Interpolated)

H

0.824

1.15610212765957

S1

0.633

0.883012765957447

N

0.863

1.21186382978723

S2

0.59

0.821531914893617

P2

0.624

0.870144680851064

Table to show the absorption of the samples obtained from the spectrophotometer and BSA concentrations which were interpolated from the graph below. Showing sample H has the greatest concentration of 1.156mg/ml.

Table 6

Sample

Absorbance (595 nm)

Concentration (mg/ml)

H

0.824

23.12

S1

0.633

17.66

N

0.863

24.24

S2

0.590

16.44

P2

0.624

16.40

Table to show the concentration of protein in each sample prior to the dilution. This is calculated by multiplying the concentration given from the interpolation of X values in the protein assay by 20 to give the overall concentration of protein in the sample; this is done as a 1:20 dilution was carried out on each sample.

Table 7

Sample

Amount of protein in each sample (mg)

H

231.20

S1

132.27

N

150.29

S2

97.00

P2

22.96

This table represents the total amount of protein in mg in each fraction, the results show a decrease in the amount of protein in each sample excluding N.

Table 8

Sample

As a yield of H (%)

H

100

S1

57.2

N

65

S2

41.95

P2

9.65

Table to show the percentage yields of each sample as a yield of H. The table shows that the percentage yield shows an overall decrease from H to P2.

Table 9

Sample

As a yield of S1 (%)

S2

73.33

P2

17.36

Table to show the percentage yields of S2 and P2 as a yield of S1. This shows that S2 has a greater percentage yield than P2.

Table 10

Sample

Protein (mg) used in each succinate dehydrogenase assay

H

1.387

S1

1.06

N

1.455

S2

0.986

P2

0.984

Table to shows the amount of protein (mg) used in each succinate dehydrogenase assay, the table shows that N has the greatest amount of protein used, and that S2 has more protein used than P2.

Table 11

Sample

Gradient

Specific Activity

H

0.004529

0.00327

S1

0.0007214

0.00068

N

0.001886

0.00130

S2

0.001129

0.00115

P2

0.001407

0.00143

This table shows the specific activities of each sample, which tells us the relative ratio of mitochondria to the total cellular material. The table shows that H has the greatest specific activity out of all samples and P2 has the greatest specific activity out of all samples that are a result of centrifugation.

Discussion

Enzyme assay

The enzyme assay shows that over time, the absorbance in Au decreases. This means that as time progresses, the rate of respiration decreases, suggesting that the activity of the enzyme is decreasing. This could be due to the substrate being used up over time – over time the substrate concentration decreases causing the number of collisions between the enzymes and the substrates to decrease.

In the enzyme assay, cuvette 6 was ruled out as an outlier as the machine read -1 at 50 seconds. So the spectrometer was re-calibrated by cuvette 1, but instead of adding H, S2 was added. Then cuvette 6 was recorded again.

When recording the enzyme assay, measurements were taken every 5 seconds for the first minute and then every 10 seconds thereafter. Towards the start of the experiment measurements are recorded more frequently, to increase the accuracy of the initial rate of reaction. However, as the experiment progresses, the change in rate of reaction slows down and consequently, measurements could be more spaced out.

A problem that could have occurred during the experiment is when separating the supernatant from the pellet, some of the supernatant may have been missed or taken up some pellet by accident with the pipette, this would have an effect on the final specific activities of each sample. Another error that could have occurred is recording the volumes of the samples, which could have been inaccurate due to human error.

Protein assay

As the concentration of BSA (mg/ml) decreased, the absorption of light also decreased. This is due to there being less protein for the Bio-Rad dye to bind to; therefore, the degree of pigmentation decreases allowing more light to pass through the cuvette and be detected.

A 1:20 dilution was carried out, with 20µl of sample and 380µl of water. This was done because the recorded absorbance of H was too high, so it was no longer on a linear area of the graph. Therefore, a 1:20 dilution was made so that the samples would lie on an area of the graph which was more linear, increasing the accuracy of the results.

P2 has the greatest specific activity, demonstrating that P2 has the greatest ratio of mitochondria to the rest of the cellular material. Therefore my hypothesis is correct.

An improvement to increase the accuracy of the data could be to make 3 sets of samples, as if an error is made in making one of the samples, that error could affect the other samples results. Thus, running the test 3 times will allow any anomalies to be identified and excluded, and then an average can be calculated.

Another way in which the experiment could be improved would be to take all class data and carry out a statistical test.

Reference list

Fishersci.co.uk. (2018). Centrifugation Theory. [online] Available at: https://www.fishersci.co.uk/gb/en/scientific-products/centrifuge-guide/centrifugation-theory.html [Accessed 13 Dec. 2018].

Mrc-mbu.cam.ac.uk. (2018). What are Mitochondria? | MRC Mitochondrial Biology Unit. [online] Available at: http://www.mrc-mbu.cam.ac.uk/what-are-mitochondria [Accessed 13 Dec. 2018].

Appendix

Calculations to show the protein concentrations in each sample:

Sample

calculation

H

1.156 x 20 = 23.12

S1

0.883 x 20 =17.66

N

1.212 x 20 = 24.24

S2

0.822 x 20 = 16.44

P2

0.820 x 20 = 16.40

To calculate the total amount of protein in each fraction, multiply the concentration of the protein by the volume of the sample used.

Calculations :

Sample

calculation

H

23.12 x 10 = 231.20

S1

17.66 x 7.49 = 132.27

N

24.24 x 6.2 = 150.29

S2

16.44 x 5.9 = 97.00

P2

16.40 x 1.4 = 22.96

Calculations to show the amount of protein (mg) used in each succinate dehydrogenase assay

Amount of protein = concentration of protein x 0.06

Sample

concentration of protein x 0.06

H

23.12 x 0.06 = 1.387

S1

17.66 x 0.06 = 1.06

N

24.24 x 0.06 = 1.455

S2

16.44 x 0.06 = 0.986

P2

16.40 x 0.06 = 0.984

To calculate the specific activity, the amount of protein in each dehydrogenase assay must be divided by the amount of protein for each sample.