Specific Interactions of Xanthene Dyes with Nucleotide-Binding Sites of Membrane Energy-Transducing Enzymes and Carriers

This chapter discusses specific interactions of xanthene dyes with nucleotide-binding sites of membrane energy-transducing enzymes and carriers. It investigates the interaction of a wide number of xanthene and related dyes with a variety of (di) nucleotide-dependent enzymes, including mitochondrial F₁- adenosine triphosphatase (ATPase) and several kinases and dehydrogenases. The chapter presents data from inhibition studies employing over 45 different dyes. Two of the xanthene dyes, tetraiodofluorescein (TIF) and tetraiodotetrachlorofluorescein (Rose bengal), were found to be potent inhibitors of both adenosine triphosphate (ATP)/ adenosine diphosphate (ADP) and NAD⁺/NADH-dependent enzymes. On the basis of a number of criteria including availability; ease of purification; high molar absorptivity; ease of modification and conversion into affinity and photoaffinity active site labeling reagents; synthesis of the radiolabeled derivative; and inherent photochemical and photophysical properties, TIF was selected as the choice nucleotide site probe. The study found that there were variations in the modes and stoichiometries of TIF binding with different F₁preparations, enzyme preparations, which were otherwise indistinguishable on the basis of sedimentation coefficient, specific activity, or subunit composition. Model enzyme studies employing native nucleotide displacement of nucleotide site-bound TIF demonstrated the potential utility of the approach toward a facile examination of the cooperativity associated with nucleotide binding to the more complex, multisite oligomeric F₁. The site stoichiometries obtained for TIF binding and native nucleotide replacement of bound TIF are in precise agreement with the known nucleotide binding capacity and subunit stoichiometry of F₁.