I am reacting NBD-CO-Hz with oxalate. When reacted (condensed) with a single carboxylic acid-containing molecule the fluorescence spectrum of NBD-CO-Hz does not normally change much. It is normally approximately: Excitation 475 nm, Emission 530 nm.
I have been reacting it with oxalate, which is a dicarboxylate. Does anyone know if a dual labeling of the oxalate would result in a significant change in the fluorescence spectrum?
Well, NBD is sensitive to environment, so in principle you could see a shift of several nanometers and change in intensity because of the nearby charge. Since oxalate can act as a reducing agent, a potential problem is that it might reduce the nitro moiety of NBD into amino moiety resulting in non-fluorescent ABD. I am not sure, though, if oxalate is strong enough reducing agent. Nevertheless, this property of NBD has been used a lot in work with membranes, because it enables one to estimate the exposed fraction of labelled lipid or protein by using a reducing agent that permeates through membrane very slowly such as dithionite and by recording the kinetic trace where the fastly disappearing fluorescence is on the exposed side of the lipid bilayer. With stopped-flow fluorescence recordings even more detailed picture of NBD exposure to reducing agent can be seen.
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