Hello,I have been trying to use this assay and have had a few...

1. So , what's in your protein buffer? Imidazole, DTT, TCEP, anything that could be binding to the heme? That's the best guess I have. Something might be out-competing the pyridine for binding to the heme. And have you scanned it before you add the dithionite to reduce the heme? Is the band there originally?
2. From the spectrum, it looks like you have heme c in your protein. Mass spectrometry might be one way to show this. There is also an old paper I found to separate heme c from your protein. KG Paul (Acta Chemica Scandinavica (1950) 4, 239-240 ) for how to do this; essentially, 0.2 volumes of glacial acetic acid and 1 volume 25 mM siver sulfate, and they keep it at 60 C for 4 hours. You could then use HPLC or something similar to identify it. I've never used it though. If you have any cytochrome c lying around, give that a try; the spectra should be the same.