Equilibrium studies on neutral red–DNA binding

Spectrophotometric binding studies were undertaken on the interaction of neutral red with native and heat‐denatured, sonicated, calf thymus DNA in a 0.2 M ionic strength buffer containing Tris–sodium acetate–potassium chloride at 25°C. The p K A of neutral red was found to be 6.81. At pH 5 the binding of protonated neutral red was complicated even at low concentration ratios of dye to DNA. In the pH range 7.5–8.5 the tight binding process could be studied and it was found that both protonated and free base species of neutral red significantly bind with DNA having association constants (in terms of polynucleotide phosphate) of 5.99 × 10 3 M −1 and 0.136 × 10 3 M −1 , respectively, for native DNA and 7.48 × 10 3 M −1 and 0.938 × 10 3 M −1 , respectively, for denatured DNA. The p K A value of the neutral red–DNA complexes were 8.46 for native DNA and 7.72 for denatured DNA. These results are discussed in terms of possible binding mechanisms.