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Proton expulsion upon dUMP binding. Phenol red was added to unbuffered FDTS (10 μM in FAD) in 50 mM NaCl, pH 8.0. Aliquots of concentrated dUMP at pH 8.0 was added and spectra were recorded. Proton release upon dUMP binding was evident by the decrease in absorbance of deprotonated phenol red. The inset shows that proton releases ceases at ~1:1 stoichiometry of dUMP:FAD.
