Chou W Y, Bieber C, Matthews K S
Department of Biochemistry, Rice University, Houston, Texas 77251.
J Biol Chem. 1989 Nov 5;264(31):18309-13.
Anilinonaphthalenesulfonate (ANS) and tryptophan compete for binding to the trp repressor protein; thus, the fluorescence decrease associated with ANS dissociation can be used as a fluorometric marker for tryptophan binding to the protein. Using this approach, the tryptophan equilibrium dissociation constant was measured at 25 degrees C to be 3.7 (+/- 1.2) X 10(-5) M, a value which compares favorably with that obtained by other methods for determining the affinity of this ligand. The presence of nonspecific DNA had no effect on the binding affinity, whereas addition of trp operator DNA yielded a 6-fold increase in affinity of tryptophan binding. The kinetics of tryptophan binding to the aporepressor were monitored directly and by ANS displacement at 4 degrees C. The association rate constant was approximately 4 X 10(6) M-1 s-1, and the dissociation rate constant was approximately 60 s-1. The ratio of these values agrees with the binding constant determined by equilibrium dialysis at this temperature. Using the gel retardation method (Carey, J. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 975-979), the dissociation rate constant for the 40-base pair operator fragment was estimated to be 2 X 10(-2) s-1, which combines with the measured Kd of 0.3 nM to yield an association rate constant comparable to other DNA binding proteins (approximately 10(8) M-1 s-1).
苯胺基萘磺酸盐（ANS）和色氨酸竞争与色氨酸阻遏蛋白的结合；因此，与ANS解离相关的荧光下降可作为色氨酸与该蛋白结合的荧光标记。采用这种方法，在25℃下测得色氨酸的平衡解离常数为3.7（±1.2）×10⁻⁵ M，该值与通过其他方法测定该配体亲和力所得到的值相当。非特异性DNA的存在对结合亲和力没有影响，而添加色氨酸操纵基因DNA则使色氨酸结合亲和力提高了6倍。在4℃下直接监测色氨酸与无辅阻遏物的结合动力学，并通过ANS置换法进行监测。缔合速率常数约为4×10⁶ M⁻¹ s⁻¹，解离速率常数约为60 s⁻¹。这些值的比值与在此温度下通过平衡透析测定的结合常数一致。使用凝胶阻滞法（凯里，J.（1988年）美国国家科学院院刊85，975 - 979），估计40个碱基对的操纵基因片段的解离速率常数为2×10⁻² s⁻¹，它与测得的0.3 nM的解离常数相结合，得到的缔合速率常数与其他DNA结合蛋白相当（约为10⁸ M⁻¹ s⁻¹）。
