Experiment A
Rate DCPIP reduction calculations:
Part 2 = 0.96-0.96 / 5 =
0 / 263 x 1000 =
0 nmol-1 min-1
Part 3 = 0.95-0.935 / 5 =
0.003 / 263 x 1000 =
0.011406844 nmol-1 min-1
Part 4 = 0.91-0.72 / 5 =
0.038 / 263 x 1000 =
0.144486692 nmol-1 min-1
Part 5 = 0.54-0.55 / 5 = -0.002
-0.002 / 263 x 1000 = -0.007604562 nmol-1 min-1
The negative value here is due the experiment changing from a reduction reaction to an oxidation reaction.
Experiment B
Maximum rate of DCPIP reduction calculations:
Part 1 = 0.91-0.40 / 263 =
0.51 / 263 x 1000 =
1.939163498 nmol min-1
Part 2 = 0.84-0.75 / 263 =
0.09 / 263 x 1000 =
0.342205323 nmol min-1
Part 3 = 0.75-0.495 / 263 =
0.255 / 263 x 1000 =
0.969581749 nmol min-1
Discussion
The chain complex which donates electrons to DCPIP is Chain III. This is because when cyanide is added, the DCPIP becomes gradually colourless as can be seen by the spectrometer readings (dropping from 0.19 to 0.46) over the 15 minute run. Because DCPIP loses its colour, this means that it is still being reduced and if complex 4 was the donator then this would have stopped when the cyanide was added. When the Antimycin A was added the reduction stopped which meant that electrons were no longer being donated. Because the reduction stopped when Complex III was inhibited, it can be concluded that Complex III is the electron donor for DCPIP.
Cyanide increases the rate of DCPIP reduction because it inhibits Complex IV. Complex IV accepts electrons from DCPIP. Therefore, if it is inhibited it can no longer accept electrons and so DCPIP no longer donates electrons. Because it is accepting electrons from Complex III and not donating any, DCPIP becomes decolourised quickly because it is being reduced at a much faster rate than before. Therefore because cyanide inhibition stops the donation of electrons, the rate of DCPIP reduction is increased dramatically.
To be able to accept electrons, DCPIP must be slightly positively charged, as any electron acceptor needs to be able to have a slight pull on the electron to attract it. Because Cytochrome C is a redox agent between Complex III and Complex IV and will also attract electrons, to be able to attract electrons itself, DCPIP must have a higher positive charge and therefore redox potential than Cytochrome C. As Cytochrome C has a redox potential of 0.22 E0’ (volts) and DCPIP must have a higher redox potential than this then the estimated redox potential for DCPIP would be around 0.33 (0.23-0.35).
The most efficient of the respiratory chain complexes, for donating electrons to the respiratory chain is Complex III. This is because when there was only cyanide in the mitochondrial assay and therefore, Complex IV was inhibited, the reduction of DCPIP was at a fairly constant rate and also reached it highest rate of reduction over 15 minutes as shown by the gradient of the line on the plot.(part 1 line on plot). When compound x was added the reduction of DCPIP was reduced.
The mode of action of compound x could either be a cyanide inhibitor, which would slow the inhibition of Complex IV also and so would allow DCPIP to donate electrons again, slowing the reduction of DCPIP. It could also be another redox agent, which by also accepting electrons, would lower the reduction of DCPIP.
I have a high confidence in the reliability of the results. From what we know respiratory inhibitors, it is clear that the experiment acted as it was expected to. However, the exception is in the results from the assay which contained compound x. This is because as an unknown compound, it can not be said that this experiment acted as it should, as what effect compound x has is unknown.
References
Lecture Notes
Practical Assessment Notes
Biochemistry – Stryer, Lubert
