The concentration of equine cytochrome c is calculated using the extinction coefficient at a given wavelength.

Reduced cytochrome c at 550 nm using e29.5 x 103 xM-1 cm-1

Reduced minus oxidised using e21.1 x 103 xM-1 cm-1

Oxidised cyto c at 550 nm using e8.4 x 103 xM-1 cm-1

Complex 4 activity was analysed using cytochrome c (reduced using ascorbate and purified via Sephadex column) in phosphate buffer (10 mm pH 7.4) and mitochondria at 50ug/ml. The spectrum of reduced cytochrome c was read from 400-600 nm before the addition of mitochondria (to confirm it was reduced) and repeated following the assay to confirm that oxidation had taken place. Absorbance at 550 nm was usually ~ 0.7

30ul of Reduced cytochrome c had an absorbance of 0.702 at 550 nm = 0.0332M (before adding mitochondria) and an absorbance of 0.135 at 550 nm =0.0063M (following the assay). By calculating the slope of the reaction and the extinction of reduced minus oxidised cytochrome c I can estimate how much oxidation of reduced cytochrome c occurred.

My question is this: Why would I need to calculate the isosbestic point of cytochrome c? My reduced cytochrome c was made in batches and stored at -80 for ~2 months at a time. I reduced the same cytochrome c for all assays and calculated from the absorbance at 550 nm what volume I required to get 100um in a 600ul cuvette (30ul was the usual vol. used).

My understanding of the Beer-Lambert law where A=e l c says that I know the concentration of both oxidised and reduced cytochrome c. However I am advised that this is not total cytochrome c.

Are my missing some unknown extinction coefficient for total (as opposed to oxidised and reduced)?

I have attempted to reduce cytochrome c by starting with oxidised and adding ascorbate at different concentrations however my isosbestic points are not in agreement with the literature possible because my reduced cytochrome c now contains ascorbate, and is thus not a suitable method for determining some unknown in cytochrome c. It is not comparable to the cytochrome c I used for assays as this was purified via Sephadex column.

Despite a trawl through the journals beginning from 1959 I am none the wiser.

Any advice/suggestions /references/job offers are gratefully received.