By means of ultraviolet–visible (UV-Vis) and fluorescence spectra, the binding ratio between vitamin K<sub>3</sub> and herring-sperm DNA in a physiological pH environment (pH = 7.40) was determined as <i>n</i><sub>K3</sub>:<i>n</i><sub>DNA</sub> = 2:1, and the binding constants of vitamin K<sub>3</sub> binding to DNA at different temperatures were determined as <i>K</i><sup>θ</sup><sub>298K</sub> = 1.28 × 10<sup>5</sup> L·mol<sup>−1</sup> and <i>K</i><sup>θ</sup><sub>310K</sub> = 7.19 × 10<sup>4</sup> L·mol<sup>−1</sup>, which were confirmed using the double reciprocal method are Δ<sub>r</sub><i>H</i><sub>m</sub><sup>θ</sup> = −3.57 × 10<sup>4</sup> J·mol<sup>−1</sup>, Δ<sub>r</sub><i>G</i><sub>m</sub><sup>θ</sup> = −2.92 × 10<sup>4</sup> J·mol<sup>−1</sup>, and Δ<sub>r</sub><i>S</i><sub>m</sub><sup>θ</sup> = 217.67 J·mol<sup>−1</sup>K<sup>−1</sup>. The driving power of this process was enthalpy. An intercalation binding of the vitamin K<sub>3</sub> with DNA was supported by a competitive experiment using acridine orange (AO) as a spectral probe. By combination analysis of the Scatchard method and cyclic voltammetry, we suggested that the interaction mode between vitamin K<sub>3</sub> and herring-sperm DNA would be a mixed mode. The quinonoid, duality fused-ring of vitamin K<sub>3</sub> can intercalate into the base pairs of DNA, and there is an electrostatic binding along with intercalation binding.

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