Sjöback R, Nygren J, Kubista M
Department of Biochemistry and Biophysics, Chalmers University of Technology, Göteborg, Sweden.
Biopolymers. 1998 Dec;46(7):445-53. doi: 10.1002/(SICI)1097-0282(199812)46:7<445::AID-BIP2>3.0.CO;2-5.
The properties of fluorescein are substantially altered upon conjugation to nucleic acids, affecting not only the molar absorptivities and fluorescence quantum yields but also the protolytic equilibrium constant and fluorescence lifetimes. Around neutral pH, the fluorescein moiety is present as both mono- and dianion, and the pKa relating them is increased from 6.43 for free fluorescein to about 6.90 for fluorescein attached to both single- and double-stranded oligonucleotides of at least 12 bases/base pairs. This difference reflects the local electrostatic potential around the nucleic acid, which is calculated to -28 mV. The molar absorptivities and spectral responses of the conjugated fluorescein protolytic species are also determined, from which the concentrations of fluorescein-oligonucleotide conjugates can be calculated by assuming: epsilon 494 = 62000/[1 + 10-(pH-6.90)] + 12000/[1 + 10(pH-6.90)] (M-1 cm-1). The fluorescence quantum yield of the conjugates depends, in a complex way, on temperature, environment and oligonucleotide length, sequence and conformation, and must be determined for each experimental situation.
与核酸结合后，荧光素的性质会发生显著改变，这不仅会影响其摩尔吸光系数和荧光量子产率，还会影响其质子解离平衡常数和荧光寿命。在中性pH值附近，荧光素部分以单阴离子和双阴离子形式存在，连接它们的pKa值从游离荧光素的6.43增加到与至少12个碱基/碱基对的单链和双链寡核苷酸相连的荧光素的约6.90。这种差异反映了核酸周围的局部静电势，经计算为-28 mV。还测定了共轭荧光素质子解离物种的摩尔吸光系数和光谱响应，据此可通过假设：ε494 = 62000/[1 + 10-(pH-6.90)] + 12000/[1 + 10(pH-6.90)]（M-1 cm-1）来计算荧光素-寡核苷酸缀合物的浓度。缀合物的荧光量子产率以复杂的方式取决于温度、环境以及寡核苷酸的长度、序列和构象，必须针对每种实验情况进行测定。
