lab report 6

Topics: Cellular respiration, Mitochondrion, Adenosine triphosphate Pages: 6 (961 words) Published: June 30, 2015
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...


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
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