MECHANISM OF ETHIDIUM-BROMIDE FLUORESCENCE ENHANCEMENT ON BINDING TO NUCLEIC-ACIDS
MECHANISM OF ETHIDIUM-BROMIDE FLUORESCENCE ENHANCEMENT ON BINDING TO NUCLEIC-ACIDS
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DOI：
10.1021/bi00635a022
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发表时间：
1977-01-01
期刊：
影响因子：
2.9
通讯作者：
KEARNS, DR
中科院分区：
文献类型：
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作者：
OLMSTED, J;KEARNS, DR
约翰·奥姆斯特德三世和大卫·R·卡恩斯*摘要：研究了溴化乙锭与双螺旋RNA和DNA结合的荧光增强机理。通过考察不同溶剂对荧光寿命的影响，质子受体对荧光的猝灭，以及在不同介质中观察到的氨基质子氢化态寿命的亚态延长，我们得出结论：在大多数极性溶剂中，激发单重态的质子转移是导致低荧光产率的主要过程。嵌入时的荧光增强溴化乙二胺是一种阳离子染料(结构I)，它与双螺旋RNA和DNA强烈而特异地相互作用，由于它在结合时表现出显著的荧光增强而被广泛用于荧光研究(Wering，1965；LePecq和Paoletti，1967；Bittman，1969；Burns，1969,1971；陶氏等人，1970；LePecq，1971；Angerer等人，1974；Bontemps和Fredericq，1974；Genest等人，1974；Gatti等人，1975)。人们普遍认为，强烈的荧光增强伴随着染料插入到核酸的双螺旋构象中，但也有证据表明，存在额外的非插层、较少的荧光增强位点，这些位点被认为涉及静电结合(Wering，1965；LePecq和Paoletti，1967；Bittman，1969)。最近，溴化乙锭已被用于探测tRNA结构(Bittman，1969；Urbanke等人，1973)、5S RNA(Gray和Saunders，1971；Feunteun等人，1975)、环状DNA(Hudson等人，1969)、染色质结构(Ide和Baserga，1976)、核糖体RNA(Lawrence和Da une，1976)、合成DNA(Aktipis和Martz，1974)、tRNA蛋白质相互作用(Rigler等人，1971)，并确定DNA的分子量(Weissman等人，1976)。溴化乙锭还会引起广泛的生化效应(Lurquin和Buchet-Mahieu，1971；Avadhani等人，1973；Kramer等人，1974；Griddle等人，1976)。尽管对溴化乙锭与多核苷酸的络合物进行了广泛的研究，但解释结合时的高度荧光增强的令人满意的机制还没有
John Olmsted III and David R. Kearns* abstract: The mechanism of the enhancement of the fluo-rescence of ethidiumbromide on binding to double helical RNA and DNA has been investigated. From an examination of the effect of different solvents on the fluorescence lifetime, quenching of fluorescence by proton acceptors, and the sub-stantial lengthening of lifetime observed upon deuteration of the amino protons, regardless of the medium, we conclude that proton transfer from the excited singlet state is the process primarily responsible for the low fluorescence yield in most polar solvents. Enhancement of fluorescence upon intercalationEzthidium bromide, a cationic dye (structure I) which in-teracts strongly and specificallywith double helical RNAs and DNAs, is widely used in spectrofluorimetric studies because of the striking fluorescence enhancement it displays upon binding (Waring, 1965; LePecq and Paoletti, 1967; Bittman, 1969; Burns, 1969, 1971; Tao et al., 1970; LePecq, 1971; Angerer et al., 1974; Bontemps and Fredericq, 1974; Genest et al., 1974; Gatti et al., 1975). It is generally agreed that strong fluorescence enhancement accompanies intercalation of the dye into the double helix conformation of the nucleic acid but there is alsoevidence for additional nonintercalative, less flu-orescence-enhanced sites which are presumed to involve electrostatic binding (Waring, 1965; LePecq and Paoletti, 1967; Bittman, 1969). Recently, ethidium bromide has been used toprobe tRNA structure (Bittman, 1969; Urbanke et al., 1973), 5S RNA (Gray and Saunders, 1971; Feunteun et al., 1975), circular DNA (Hudson et al., 1969), chromatin structure (Ide and Baserga, 1976), ribosomal RNA (Lawrence and Da une, 1976), synthetic DNA (Aktipis and Martz, 1974), tRNA protein interactions (Rigler et al., 1971), and to de-termine the molecular weight of DNA (Weissman et al., 1976). Ethidium bromide also elicits a wide range of biochemical effects (Lurquin and Buchet-Mahieu, 1971; Avadhani et al., 1973; Kramer et al., 1974; Griddle et al., 1976). Despite extensive studies of ethidium bromide complexes with polynucleotides, a satisfactory mechanism explaining the high degree of fluorescence enhancement upon binding has not