The interactions of Rose Bengal and other aromatic anionic dyes with mannitol-1-phosphate dehydrogenase fromEscherichia coli

Abstract

The interaction of Rose Bengal with mannitol-1-phosphate dehydrogenase has been investigated. Binding of this aromatic anionic dye causes a quenching of the protein fluorescence and various changes in the spectral properties of the dye. As is the case with other dehydrogenases, the titration of the enzyme with Rose Bengal, monitoring enhancement in the dye fluorescence at 590 nm, or quenching of the protein fluorescence, can be described by a simple binding model: one dye binding site per enzyme subunit with a dissociation constant of ∼2 µM. However, kinetic studies indicate a more complex scheme, since Rose Bengal induces a biphasic time-dependent inhibition of the enzyme. The first phase is over in 1–5 min and is partially reversible, while the second phase is essentially irreversible and continues beyond 1 h. The dyes 8-anilino-1-naphthalene sulfonate and 2-p-toluidinylnaphthalene-6-sulfonate also cause biphasic time-dependent inhibitions of the enzyme. Only mannitol-1-phosphate, and fructose-6-phosphate in the presence of NAD+, show high levels of protection against these inhibitory processes. The different effects of coenzymes and substrates on the dye-induced inhibitions support earlier observations from fluorescence studies (preceding paper). A binding scheme describing the interactions of Rose Bengal with the enzyme that is consistent with the experimental results is presented.