York S S, Lawson R C, Worah D M
Biochemistry. 1978 Oct 17;17(21):4480-6. doi: 10.1021/bi00614a019.
8-Anilion-1-naphthalenesulfonate (Ans), recrystallized from water as the magnesium salt, contains a fluorescent impurity representing 0.3% of the absorbance at 351 nm. This impurity can be removed by Sephadex LH-20 chromatography. The chromatographic and spectral properties of this impurity suggest that it is bis(Ans), a dimer of Ans. This bis(Ans) impurity makes a significant contribution to the fluorescence increment observed when lac repressor is added to recrystallized Ans. This occurs because bis(Ans) binds much more tightly to this protein than does Ans. The dissociation constant divided by the number of binding sites per subunit is 3.1 X 10(-6) M for bis(Ans); the corresponding value for Ans is greater than 1 X 10(-4) M. Because of their differing absorption spectra, the impact of this bis(Ans) impurity is especially large with excitation wavelengths above 400 nm. Users of recrystallized Ans should consider the potential consequences of this impurity whenever working with a protein to which Ans binds weakly.
8-苯胺基-1-萘磺酸盐（Ans）以镁盐形式从水中重结晶，含有一种荧光杂质，其在351nm处的吸光度占比为0.3%。这种杂质可通过葡聚糖凝胶LH-20色谱法去除。该杂质的色谱和光谱性质表明它是双（Ans），即Ans的二聚体。当将乳糖阻遏物添加到重结晶的Ans中时，这种双（Ans）杂质对观察到的荧光增量有显著贡献。出现这种情况是因为双（Ans）与该蛋白质的结合比Ans紧密得多。双（Ans）的解离常数除以每个亚基的结合位点数为3.1×10⁻⁶M；Ans的相应值大于1×10⁻⁴M。由于它们不同的吸收光谱，当激发波长高于400nm时，这种双（Ans）杂质的影响尤为显著。每当使用与Ans弱结合的蛋白质时，重结晶Ans的使用者都应考虑这种杂质的潜在后果。
