Stokke T, Steen H B
J Histochem Cytochem. 1985 Apr;33(4):333-8. doi: 10.1177/33.4.2579998.
Two binding modes for the bisbenzimidazole Hoechst 33258 to native DNA at physiological conditions have been distinguished. Type 1 binding, which dominated at low dye/phosphate ratios (D/P less than 0.05) or low dye concentrations, had a high quantum yield of fluorescence with maximum emission at 460 nm. Binding of the dye at type 2 sites (0.05 less than D/P less than 0.4) lead to quenching of fluorescence from type 1 bound dye, presumably by nonradiative energy transfer. Fluorescence quantum yield of type 2 bound dye was low (phi = 0.05-0.1) and it peaked around 490 nm. At D/P greater than 0.4, the dye/DNA complex precipitated. This was caused by an additional dye-DNA interaction that was strongly cooperative. The anomalous dispersion of the refractive index of the complex changed abruptly around D/P = 0.4, indicating that the precipitating dye-DNA interaction involved strong electronic interaction between dye molecules. Hoechst 33258 precipitated polynucleotides irrespective of strandedness and base composition when dye concentration was raised above 1 X 10(-5) M. In the presence of 25% ethanol, type 2 binding to DNA did not occur, whereas the binding constant for type 1 binding (kappa = 2 X 10(3) M-1) was about two orders of magnitude smaller than in physiological buffer. DNA was not precipitated by high concentrations of Hoechst 33258 in 25% ethanol.
在生理条件下，已区分出双苯并咪唑Hoechst 33258与天然DNA的两种结合模式。1型结合在低染料/磷酸盐比率（D/P小于0.05）或低染料浓度时占主导，具有高荧光量子产率，最大发射波长为460nm。染料在2型位点结合（0.05小于D/P小于0.4）会导致1型结合染料的荧光猝灭，推测是通过非辐射能量转移。2型结合染料的荧光量子产率较低（φ = 0.05 - 0.1），且在490nm左右达到峰值。当D/P大于0.4时，染料/DNA复合物沉淀。这是由一种强烈协同的额外染料 - DNA相互作用引起的。复合物折射率的反常色散在D/P = 0.4左右突然变化，表明沉淀的染料 - DNA相互作用涉及染料分子之间的强电子相互作用。当染料浓度提高到1×10⁻⁵M以上时，Hoechst 33258会使多核苷酸沉淀，与链的类型和碱基组成无关。在25%乙醇存在下，不会发生染料与DNA的2型结合，而1型结合的结合常数（κ = 2×10³M⁻¹）比在生理缓冲液中小约两个数量级。在25%乙醇中，高浓度的Hoechst 33258不会使DNA沉淀。
