Latt S A, Wohlleb J C
Chromosoma. 1975 Nov 11;52(4):297-316. doi: 10.1007/BF00364015.
The interaction of the bisbenzimidazole dye 33258 Hoechst with DNA and chromatin is characterized by changes in absorption, fluorescence, and circular dichroism measurements. At low dye/phosphate ratios, dye binding is accompanied by intense fluorescence and circular dichroism and exhibits little sensitivity to ionic strength. At higher dye/phosphate ratios, additional dye binding can be detected by further changes in absorptivity. This secondary binding is suppressed by increasing the ionic strength. A-T rich DNA sequences enhance both dye binding and fluorescence quantum yield, while chromosomal proteins apparently exclude the dye from approximately half of the sites available with DNA. Fluorescence of the free dye is sensitive to pH and, below pH 8, to quenching by iodide ion. Substitution of 5-bromodeoxyuridine (BrdU) for thymidine in synthetic polynucleotides, DNA, or unfixed chromatin quenches the fluorescence of bound dye. This suppression of dye fluorescence permits optical detection of BrdU incorporation associated with DNA synthesis in cytological chromosome preparations. Quenching of 33258 Hoechst fluorescence by BrdU can be abolished by appropriate alterations in solvent conditions, thereby revealing changes in dye fluorescence of microscopic specimens specifically due to BrdU incorporation.
双苯并咪唑染料33258 Hoechst与DNA和染色质的相互作用通过吸收、荧光和圆二色性测量的变化来表征。在低染料/磷酸盐比率下，染料结合伴随着强烈的荧光和圆二色性，并且对离子强度的敏感性很小。在较高的染料/磷酸盐比率下，可以通过吸光度的进一步变化检测到额外的染料结合。这种二次结合通过增加离子强度而受到抑制。富含A-T的DNA序列增强了染料结合和荧光量子产率，而染色体蛋白质显然将染料排除在大约一半可与DNA结合的位点之外。游离染料的荧光对pH敏感，在pH 8以下，对碘离子的猝灭敏感。在合成多核苷酸、DNA或未固定的染色质中用5-溴脱氧尿苷（BrdU）替代胸腺嘧啶会猝灭结合染料的荧光。这种染料荧光的抑制允许在细胞学染色体制备中对与DNA合成相关的BrdU掺入进行光学检测。通过适当改变溶剂条件可以消除BrdU对33258 Hoechst荧光的猝灭，从而揭示由于BrdU掺入而导致的微观标本中染料荧光的变化。
