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.

SYNOPSIS A total of 177 consecutive patients with either tension or migraine headaches were examined with unenhanced CT Scans. For the overall group 28% were abnormal while 26% of the patients with migraine and 33% of those with tension headaches had abnormal scans. Our findings were similar to several smaller studies in more seriously disabled patients. Follow‐up studies are planned to determine if our CT Scan results are indicative of a progressive disorder.

The stoichiometry of lac repressor binding to nonspecific DNA was investigated by three different techniques. Four molecules of the fluorescent probe 5,5'-bis(8-anilino-1-naphthalenesulfonate) [bis(ANS)] bind to each repressor subunit with an average dissociation constant of 20 microM. Nonspecific DNA displaces most of this bound bis(ANS), reducing the fluorescence. Titrations of repressor with nonspecific DNA monitored with high [bis(ANS)] (5-15 microM) had end points at 8 base pairs per repressor. Lower [bis(ANS)] (0.1-1 microM) resulted in end points at either 15 or 26 base pairs per repressor, depending on the ionic strength. These end points correspond to complexes containing approximately one, two, or four repressors per 28 base pairs. Boundary sedimentation velocity experiments with saturating amounts of repressor revealed that five repressors can bind to 28 base pairs. By monitoring the circular dichroism as DNA was added to repressor, the sequential appearance of complexes containing approximately four, two, and one repressors per 28 base pairs was observed. The inability of repressor cores or iodinated repressor to bind to complexes containing one or two repressors per 28 base pairs implies that all of the repressors directly contact the DNA in the complex containing four repressors per 28 base pairs. It is proposed that while two subunits of each repressor contact the DNA in complexes containing one or two repressors per 28 base pairs, only one subunit of each repressor contacts the DNA in the complex with four repressors per 28 base pairs. These results suggest a novel mechanism for the one-dimensional diffusion of repressor along DNA.

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