Binding of Hoechst 33258 to chromatin in situ

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Abstract
The binding of Hoechst 33258 to rat thymocytes, human lymphocytes, and NHIK 3025 tissue culture cells was studied by measuring the fluorescence and light scattering of the cells as functions of dye concentration using flow cytometry. The results indicated that there were two different modes of binding of Hoechst 33258 to chromatin in situ at physiological pH. Type 1 binding, which dominated at total dye/phosphate ratios below 0.1 (0.15, M), was characterized by a binding constant of the order 107 M−1 and fluorescence with high quantum yield. Further binding of the dye resulted in a reduced blue/green fluorescence ratio, indicating that secondary sites were occupied. Binding at secondary sites above a certain density (0.1 ≤ bound dye/phosphate ≤0.2) induced strong quenching of fluorescence and precipitation of chromatin. Precipitation was quantitated by measuring the large-angle (≥ 15°) light scattering of the cells above 400 nm, i.e., outside the Hoechst 33258/DNA absorption spectrum, as a function of dye concentration. In contrast, the light scattering at 365 nm, i.e., within the absorption spectrum of Hoechst 33258/DNA, was independent of the total dye/phosphate ratio. The coefficient of variation of the light-scattering (≥ 400 nm) histograms decreased with Hoechst 33258 concentration. Type 2 binding to histone-depleted chromatin was cooperative (Hill-coefficient ≈ 2) and the apparent binding constant was 2–3 · 105 M−1 as determined from quenching and precipitation data. The final stoichiometry in the dye/chromatin complex calculated from quenching data was q = 0.70 (bound dye/phosphate ratio), while light-scattering data yielded q = 1.35. The dye-induced precipitation of intact chromatin of human lymphocytes was not cooperative (Hill-coefficient ≈ 1.0). The precipitation of DNase 1 digested chromatin, however, was described by two-component kinetics. The components had Hill-coefficients of 1.0 and 2.4, respectively; the appearance of the latter was interpreted as reflecting precipitation of relaxed chromatin. Histone-depletion increased the number of Hoechst 33258 type 1 binding sites in chromatin by about 30%, while the dye-induced increase in light scattering decreased by about 40%.