Woody A Y, Reisbig R R, Woody R W
Biochim Biophys Acta. 1981 Aug 27;655(1):82-8. doi: 10.1016/0005-2787(81)90069-1.
The azo dye Congo Red has a high affinity for nucleotide-binding enzymes. We have studied the binding of Congo Red to RNA polymerase by circular dichroism (CD) and difference absorption spectroscopy, steady-state kinetics, and nitrocellulose filter-binding. Induced CD shows that a large number of Congo Red molecules bind to the holoenzyme. CD also demonstrates that the core enzyme at low ionic strengths has a distinctive Congo Red binding site which is not present in the holoenzyme, nor in the core enzyme at higher ionic strengths or in the presence of poly(dT). CD studies indicate that Congo Red can readily displace double-stranded polynucleotides (T7 DNA or poly[d(A-T)] from RNA polymerase. Single-stranded DNA (poly(dT) and T7 DNA in open complexes) is not displaced from RNA polymerase except at high Congo Red concentrations. Both kinetics and nitrocellulose filter-binding measurements support this conclusion. Difference spectra indicate that the bound Congo Red molecules undergo stacking. We postulate that RNA polymerase binds Congo Red in a region with which a segment of DNA normally interacts, and that Congo Red is a potent inhibitor because the stacked dye has a polyanionic character.
偶氮染料刚果红对核苷酸结合酶具有高亲和力。我们通过圆二色性（CD）、差示吸收光谱、稳态动力学和硝酸纤维素滤膜结合法研究了刚果红与RNA聚合酶的结合。诱导CD表明大量刚果红分子与全酶结合。CD还证明，在低离子强度下，核心酶具有一个独特的刚果红结合位点，该位点在全酶中不存在，在高离子强度下的核心酶中或存在聚（dT）时也不存在。CD研究表明，刚果红能轻易地从RNA聚合酶上取代双链多核苷酸（T7 DNA或聚[d（A-T）]）。单链DNA（开放复合物中的聚（dT）和T7 DNA）除非在高刚果红浓度下，否则不会从RNA聚合酶上被取代。动力学和硝酸纤维素滤膜结合测量均支持这一结论。差示光谱表明结合的刚果红分子发生了堆积。我们推测RNA聚合酶在一个通常与一段DNA相互作用的区域结合刚果红，并且刚果红是一种强效抑制剂，因为堆积的染料具有聚阴离子特性。
