Interaction of a fluorescent reagent, fluorescein mercuric acetate, with nucleic acids

Abstract

Fluorescein mercuric acetate (fluorescein Hg Ac), which is a fluorescent thiol reagent, was shown to bind to various nucleic acids by measuring the changes in its absorption and fluorescence properties. Up to a critical concentration of free fluorescein Hg Ac (1 · 10 −7 M for calf thymus DNA, with 42% GC, and 2 · 10 −7 M for Micrococcus lysodeikticus DNA, with 72% GC) this reagent appears to bind selectively to single-stranded sections in DNA. Above this critical concentration, cooperative binding to double helical DNA occurs, and denatured DNA is obtained after removal of bound fluorescein Hg Ac by dialysis against 1 M KCl. These facts indicate that fluorescein Hg Ac causes the denaturation of double helical DNA prior to binding as has been shown in the case of methylmercuric hydroxide. The binding of fluorescein Hg Ac to DNA is much stronger than that of methylmercuric hydroxide. The number of total binding sites for fluorescein Hg Ac is close to the number of base pairs for both calf thymus DNA and M. lysodeikticus DNA. Furthermore, it was shown that fluorescein Hg Ac binds to thymidine, deoxyguanosine, poly(U) and poly(G). Since fluorescence quenching of fluorescein Hg Ac accompanies its complex formation with DNA and the affinity is markedly high as indicated by the association constant of 6.8 · 10 7 M −1 for single-stranded calf thymus DNA, fluorescein Hg Ac can be used for the structural studies of small amounts of nucleic acids.