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In the reverse titration approach, the fluorescing ligand is titrated with the nonfluorescing macromolecule and SYi decreases throughout the titration (Bujalowski and Jezewska, 2000; Jezewska and Bujalowski, 1997; Lohman and Bujalowski, 1991). Although both types of titration curves can be quantitatively analyzed, we will limit our discussion to the normal titration method, as more commonly occurring in the experimental practice. , 2010). For multiple binding of nucleotides to the protein, it is rather obvious that the nucleotide cofactor is the ligand and the protein is the macromolecule.
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