Molecular spectroscopic study of DNA binding with neutral red and application to assay of nucleic acids

Abstract

Under acidic conditions, neutral red (NR) binds with DNA. The Scatchard plots for the reaction were constructed from the fluorescence spectral data which are best fitted by the neighbour exclusion model with an intrinsic binding constant of 4.5×105M−1 and an exclusion parameter of about 1 base pair. Upon binding to DNA, broadening was observed; in addition there is a hypochromic effect and small blue shift at low concentrations of DNA. In contrast, there is a hyperchromic effect and red shifts in the absorption spectrum for high concentrations of DNA. Fluorescence of NR is efficiently quenched by DNA at low concentrations and is greatly enhanced by DNA at high concentrations. The Stern–Volmer quenching constant was obtained from the linear quenching plots. Resonance light scattering (RLS), using a common spectrofluorometer, was applied for determination of nucleic acids with NR. At pH 2.3, the weak light scattering of NR is enhanced greatly at 330nm and 590nm. The enhanced light scattering is proportional to the concentration of nucleic acids in the range 0.048–5.25μgml−1 for calf thymus DNA, 0.035–4.30μgml−1 for fish sperm DNA and 0.205–5.80μgml−1 for yeast DNA. The detection limits were 48.2ngml−1 for calf thymus DNA, 35.2ngml−1 for sperm fish DNA and 205ngml−1 for yeast RNA. The relative standard deviations (8 replicates) were within 4.3% in the middle of the linear response range.