I'm studying a protein that degrades heme. I'm interested in using the active site's tryptophan fluorescence as a measure of binding affinity for heme, to obtain a Kd. Using an experiment in the literature as my basis, I designed an experiment where I titrate heme, in 8 nM increments, into my protein at 80 nM. I do this until I've exceeded 2:1 molar ratio of heme to protein, to ensure full quenching.

I've successfully measured nanomolar affinity using this experiment. However, recently, I've ran into a confounding series of results. Using the same means of determining protein concentration (UV-Vis absorbance at 280 nm) and dilution strategy, I've found that my initial measurement, sans heme, is showing almost no fluorescence whatsoever (100 or so arb. units, compared to the 5000 I was measuring previously).

I've now experienced this with a freshly purified protein batch and a previously used batch that had been frozen in a glycerol storage buffer. The previously used, older batch is showing a similar low intensity to the fresh batch now, which makes me think that I'm at fault here. I suspected that my measuring protein concentration might be the culprit, or perhaps my dilutions. However, I have several experiments worth of data that seems to suggest otherwise, given that my measurements were as expected for these.

Steps I've taken to troubleshoot this include:

1.) Running both the old and new batches on an SDS gel to compare band intensity by eye - they were perfectly comparable.

2.) Trying a different preparation of buffer. No variation in intensity.

3.) Repeating dilutions/checking math/re-spec'ing protein concentration too many times to count.

4.) Added an excess of protein to ensure tryptophan fluorescence is still present at increased concentrations - it was.

5.) Checked instrument/scan parameters to ensure they're consistent. I'm one of only three or so people that uses this fluorimeter, and the only to use it recently, and moreover, I was able to confirm per my notebook that the parameters are consistent.

I have some other ideas for troubleshooting, but this is just so frustrating, as I've run this experiment many times without this problem, recently even! Is there something obvious I'm missing here? I'm really frustrated, as both myself and other members of my lab group have checked my work, and no one can seem to determine what the issue is.