Reid S L, Parry D, Liu H H, Connolly B A
Department of Biochemistry and Genetics, The University of Newcastle, Newcastle upon Tyne NE2 4HH, UK.
Biochemistry. 2001 Feb 27;40(8):2484-94. doi: 10.1021/bi001956p.
Oligonucleotides labeled with hexachlorofluorescein (hex) have enabled the interaction of the restriction endonuclease EcoRV with DNA to be evaluated using fluorescence anisotropy. The sensitivity of hex allowed measurements at oligonucleotide concentrations as low as 1 nM, enabling K(D) values in the low nanomolar range to be measured. Both direct titration, i.e., addition of increasing amounts of the endonuclease to hex-labeled oligonucleotides, and displacement titration, i.e., addition of unlabeled oligonucleotide to preformed hex-oligonucleotide/EcoRV endonuclease complexes, have been used for K(D) determination. Displacement titration is the method of choice; artifacts due to any direct interaction of the enzyme with the dye are eliminated, and higher fluorescent-labeled oligonucleotide concentrations may be used, improving signal-to-noise ratio. Using this approach (with three different oligonucleotides) we found that the EcoRV restriction endonuclease showed a preference of between 1.5 and 6.5 for its GATATC target sequence at pH 7.5 and 100 mM NaCl, when the divalent cation Ca2+ is absent. As expected, both the presence of Ca2+ and a decrease in pH value stimulated the binding of specific sequences but had much less effect on nonspecific ones.
用六氯荧光素（hex）标记的寡核苷酸能够利用荧光各向异性来评估限制性内切酶EcoRV与DNA的相互作用。hex的高灵敏度使得能够在低至1 nM的寡核苷酸浓度下进行测量，从而能够测量低纳摩尔范围内的解离常数（K(D)）值。直接滴定法，即向hex标记的寡核苷酸中加入逐渐增加量的内切酶，以及置换滴定法，即向预先形成的hex-寡核苷酸/EcoRV内切酶复合物中加入未标记的寡核苷酸，都已用于K(D)的测定。置换滴定法是首选方法；消除了由于酶与染料的任何直接相互作用而产生的假象，并且可以使用更高浓度的荧光标记寡核苷酸，从而提高信噪比。使用这种方法（使用三种不同的寡核苷酸），我们发现，在pH 7.5和100 mM NaCl条件下，当不存在二价阳离子Ca2+时，EcoRV限制性内切酶对其GATATC靶序列的偏好性在1.5至6.5之间。正如预期的那样，Ca2+的存在和pH值的降低都刺激了特定序列的结合，但对非特异性序列的影响要小得多。
