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

Under acidic conditions, neutral red (NR) binds with DNA. The Scatchard plo ts for the reaction were constructed from the fluorescence spectral data wh ich are best fitted by the neighbour exclusion model with an intrinsic bind ing constant of 4.5 x 10(5) M-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 co ntrast, there is a hyperchromic effect and red shifts in the absorption spe ctrum for high concentrations of DNA. Fluorescence of NR is efficiently que nched by DNA at low concentrations and is greatly enhanced by DNA at high c oncentrations. The Stern-Volmer quenching constant was obtained from the li near quenching plots. Resonance light scattering (RLS), using a common spec trofluorometer, was applied for determination of nucleic acids with NR. At pH 2.3, the weak light scattering of NR is enhanced greatly at 330 nm and 5 90 nm. The enhanced light scattering is proportional to the concentration o f nucleic acids in the range 0.048-5.25 mu g ml(-1) for calf thymus DNA, 0. 035-4.30 mu g ml(-1) for fish sperm DNA and 0.205-5.80 mu g ml(-1) for yeas t DNA. The detection limits were 48.2 ng ml(-1) for calf thymus DNA, 35.2 n g ml(-1) for sperm fish DNA and 205 ng ml(-1) for yeast RNA. The relative s tandard deviations (8 replicates) were within 4.3% in the middle of the lin ear response range. (C) 1999 Elsevier Science B.V. All rights reserved.