[hugez]

I'm feeling very lost about a small part of a question from my biochemistry lab.

We were doing cation exchange chromatography and we were separating ferritin from cytochrome c. The question is:

"After you added the phosphate buffer you should have observed a second band. What was the colour of this band? What do you think this protein is? Explain why in relation to the buffer and pH. WHy does this protein have this colour"

-> Now I know that it is cytochrome c because we had already eluted the ferritin from the mixture and the pH was 11 (greater than its pI of 10.5). It's the colours that confuse me. When I did the experiment, the colour of the band was exactly the same as the gel we were running it through (a yellow-cream). Yet this question seems to imply that cytochrome c is a coloured protein, and that there is a 'reason' for this, yet I can't seem to figure it out, and I can't find any research that suggest cytochrome c is coloured.

Any suggestions? Do any of you have any personal experience with cytochrome c having colour?

[Babcock_Hall]

Yes, I can verify that cytochrome c has an intense color at neutral pH.  Do you know the identity of the prosthetic group of this protein?  Can you double-check the value of the pH?  Although I can see the logic of going to high pH, it might have an effect on the protein that would be difficult to predict without more information.

[hugez]

I know that cytochrome c contains a heme group, though I'm not sure how this would be affected by a change in pH. The pH was definitely 11.5 (so the protein was negatively charged overall at this pH). I read in my observations that I noted that the effluent of this protein was a light red colour, though the image I took of the protein while it was running through the gel showed no difference in colour from the yellow-cream colour of the gel. On the other hand, the fact that there's supposed to be a second band tells me that there probably was a red band briefly and that I just wasn't paying enough attention at the time to notice it before it washed through. That's the only explanation I have.

[Babcock_Hall]

If I understand what you did, the first band was ferritin and the second band was cytochrome c.  How dark the band of cytochrome c is depends upon its concentration, as one can appreciate from Beer's law.  However, the second issue is pH.  At neutral pH cytochrome c has an intense red color that is not very different from the color of hemoglobin or myoglobin.  If you initially prepared the protein mixture at neutral pH and applied it to the column, you should have seen the red color.  The chromatography would have had two effects potentially.  One is that the final concentration of the protein changed relative to its starting concentration, and the other is pH.  Once the pH was increased, it is possible that a side-chain in close proximity to the heme group was deprotonated (there is one candidate that comes to mind--look up the groups that coordinate to the iron) and that this deprotonation altered the spectrum in some way.