Cystochrome C Oxidase Lab Report

Introduction:
Cytochrome c oxidase plays a vital role in cellular respiration by accepting e- from cytochrome c and transferring them to an acceptor oxygen molecule in the final step of electron transfer chain. Carbon monoxide and cyanide are few of the inhibitor of this enzyme. 4 Fe2+ -cyt c + 8H+ + O2 4 Fe3+ -cyt c + 2H2O + 4H+ [out]
Cytochrome c oxidase locates to the inner membrane which separates the mitochondrial matrix from the intermembrane space. However, Potato tubes can be used as the source of mitochondria, where this enzyme is located and active. We used potato in this lab instead of using other things because we can easily abstracts cytochrome c from it. Cytochrome c oxidize is very important in human organisms.
To assay the activity of the redox enzyme cytochrome c oxidase. Cytochrome c will be the substrate in the experiment. The cyt c oxidation can be absorbed through spectrophotometer by oxidation of heme iron in cytochrome c. In addition, the ferrocytochrome contains an oxidase ferros iron and the ferricytochrome contains oxidase feeri iron and they both have different structure. Spectrophotometer helps us to measure the intensity light of the substance. The colorimetric assay is based on observation of the decrease in absorbance at 550 nm of ferrocytochrome c caused by its oxidation to ferricytochrome c by cytochrome c oxidase. We are trying to assay the activity of the enzyme cytochrome c oxidase, which is in charge of O2 consumption in cell respiration. Absorbance level of substance should decrease over time because the substrate is getting eaten up overtime. This experiment is to assay the activity, which is change of O2 consumption in cellular respiration.
Methods:
Isolate mitochondria from potato; start by washing potatoes, peeling potatoes and cutting them in small cubes of 1cm. pour 50 g into blender and add 100 ml of mitochondria extraction buffer; Blend for 15 seconds until the tissue is in a creamy consistency. Filter it through four layers of cheesecloth and the two layers of sterile muslin sported by a funnel. Collect the tubes and centrifuge at 2o C for 5 minutes at 1000 Xg (1000 rpm). Discard the pellet and centrifuge tubes and spring at 2o C for 20 minutes at 14,000Xg (8,000 rpm). In this case discard the supernatant and gently resuspend the pullet in 1.5-2.5 ml of mitochondria extraction buffer. Warm up tungsten lamp and set the wavelength to 550nm. Zero the spectrophotometer with 950 ml of 1 X assay buffer. Prepare 5 samples according to the table below.
Sample
Assay Buffer
(μL)
Enzyme Dilution
Buffer (μL)
Sample (μL)
Ferro cytochrome c
Substrate Solution (μL)
Blank
950
100
---
50
Unknown Sample 1
950
100 – x1 =75
25= x1
50
Unknown Sample 2
950
100 – x2 =50
50= x2
50
Unknown Sample 3
950
100 – x3 =25
75= x3
50
Unknown Sample 4
950
0
100
50
Table 1:
Results:

Time (sec)
Unknown 1
( A550)
Unknown 2
(A550)
Unknown 3
(A550)
Unknown 4
(A550)
Blank
(A550)

0
0
0
0
0
0.18
15
0.312
0.564
0.549
0.692
0.184
30
0.308
0.559
0.537
0.685
0.18
45
0.304
0.549
0.52
0.675
0.18
60
0.301
0.541
0.513
0.666
0.181
75
0.297
0.534
0.509
0.659
0.182
90
0.293
0.528
0.506
0.652
0.18
105
0.29
0.521
0.5
0.648
0.179
120
0.287
0.515
0.495
0.641
0.18
135
0.284
0.511
0.491
0.639
0.179
150
0.281
0.506
0.487
0.634
0.179
165
0.278
0.501
0.484
0.63
0.179
180
0.276
0.496
0.481
0.627
0.179

Table 2:

Figure 1:
The graph above shows the absorbance at 550nm.
Wavelength of blank and the form unknown samples prepared by mixing solution according to the table in procedure.

Activity of the sample:
Units/mL = (ΔA550/min) (dilution) (1.1) (21.84)(Vol. of enzyme)

Unknown 1: (0.00875) (25/1100) (1.1) = 4.01*10-4 Units/ml (21.84)(0.025)

Unknown 2: (0.01675) (50/1100) (1.1) = 7.67*10-4 Units/ml (21.84)(0.05)
Unknown 3: (0.01675) (75/1100) (1.1) = 7.67*10-4 Units/ml (21.84)(0.075)
Unknown 4: (0.016) (100/1100) (1.1) = 7.33*10-4 Units/ml (21.84)(0.1)
Blank (0.00025) (0/1100) (1.1) = 0 Units/ml (21.84)(0)
Discussion: The blank which contained only 950 ml assay buffer, enzyme dilution buffer and ferrocytochomre c substrate but not the sample had a relatively consistent absorbance reading throughout the 3-minutes duration.
Compared to other samples, sample 4 had the highest absorbance readings. There wasn’t any enzymes in the blank sample because there wasn’t any enzyme samples in that tube. Hence, the oxidation process did not occur and the products weren’t formed, not resulting in significant change of absorbance reading. On the other hand, all other sample showed enzyme activity through decreased in their absorbance. Samples 2 and 3 had the greatest activity compared to all samples. As we expected the absorbance level of the enzymes decreasing over time. If we didn’t measure time corrected it then it might have affected out result because time is a key in
oxidation-reduction.
Conclusion:
Cytochrome c oxidase is important because subcellular structure where an essential enzyme are stays active and that helps us to regulated energy production in mitochondria. Sometimes, it effect several of our body parts. Moreover, by doing this experiment we can synthesis that the absorbance of unknown sample is more related with spectrophotometer. We can see through the graph and tables that the results of the experiment were very consistent because blank had no activity and sample 1,2,3 and 4 had enzyme in it so it shows the absorbance decreasing. So we can conclude that our experiment was consist with the oxidation of cytochrome c oxidase.

References:

http://scholar.google.com/scholar?q=cytochrome+c+oxidase+activity&hl=en&as_sdt=0&as_vis=1&oi=scholart&sa=X&ei=wtJbVJ6XDs6cyASxiAE&ved=0CBsQgQMwAA
http://www.sigmaaldrich.com/life-science/metabolomics/enzyme-explorer/learning-center/cytochrome-c.html