Dear Woong Young So,

From your question what i can understand is, your ligand act as donor and acceptor is some metal or molecule. so i would suggest keep the concentration of donor same and increase or decrease concentration of accceptor only to observe any kind of behavior of ligand towards acceptor. now i could not understand one thing in your question is, weather you are taking only ligand-acceptor system or you are making some complex of that and than again some third participant is adding to the system and checking the behavior of the complex towards that participant.

if only ligand and acceptor is taking part and on increasing concentration of ligand you are getting enhancement in fluorescence than "YES", it is because of increasing density of donating state.

but i am still nor sure about your system.

Best of luck.

Cheers!!!

I, too, am unclear on the exact nature of the experiment, but I wonder whether you might be seeing resonance energy transfer causing increased acceptor fluorescence.

From the description, it's difficult to tell what may be going wrong in your experiment, but if you are exciting at 264 nm and looking at emissions at much longer wavelengths, your ligand donor has a large Stokes shift, which may account for unexpected increases in fluorescence upon complex formation, unrelated to FRET. There are lots of ways to understand this, and it is worth considering, for a non-optimal D/A pair, whether FRET is the best way to measure whatever you are trying to measure using fluorescence.