Equilibrium binding experiments using fluorescence and absorption techniques have been performed throughout a wide concentration range (1 nM to 30 microM) of the dye Hoechst 33258 and several DNAs. The most stable complexes found with calf thymus DNA, poly[d(A-T)], d(CCGGAATTCCGG), and d(CGCGAATTCGCG) all have dissociation constants in the range (1-3) X 10(-9) M-1. Such complexes on calf thymus DNA occur with a frequency of about 1 binding site per 100 base pairs, and evidence is presented indicating a spectrum of sequence-dependent affinities with dissociation constants extending into the micromolar range. In addition to these sequence-specific binding sites on the DNA, the continuous-variation method of Job reveals distinct stoichiometries of dye-poly[d(A-T)] complexes corresponding to 1, 2, 3, 4, and 6 dyes per 5 A-T base pairs and even up to 1 and 2 (and possibly more) dyes per backbone phosphate. Models are suggested to account for these stoichiometries. With poly[d(G-C)] the stoichiometries are 1-2 dyes per 5 G-C pairs in addition to 1 and 2 dyes per backbone phosphate. Thermodynamic parameters for formation of the tightest binding complex between Hoechst 33258 and poly[d(A-T)] or d-(CCGGAATTCCGG) are determined. Hoechst 33258 binding to calf thymus DNA, chicken erythrocyte DNA, and poly[d(A-T)] exhibits an ionic strength dependence similar to that expected for a singly-charged positive ion. This ionic strength dependence remains unchanged in the presence of 25% ethanol, which decreases the affinity by 2 orders of magnitude. In addition, due to its strong binding, Hoechst 33258 easily displaces several intercalators from DNA.

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Fluorescence titrations have been carried out to determine the association constants (Ka) for binding of the dyes Hoechst 33258 and DAPI to the self-complementary decamer d(CTGAATTCAG) and nine duplex derivatives with exocyclic substituent changes in the six central base pairs. Many Ka values are in the range (2-5) x 10(8) (duplex M)-1 at 5.5 degrees C. Replacement of the leftmost adenine by 2-aminopurine in the sequence decreases Ka for Hoechst 33258 by a factor of 170. When the centermost adenine is replaced by 2-aminopurine, Ka for Hoechst 33258 and DAPI is too small to be evaluated. When the centermost adenine is replaced by purine, Ka for both dyes increases, but this very stable duplex-Hoechst 33258 complex is nonfluorescent. The measured affinities are compared to expectations derived from X-ray studies with dodecamer-dye complexes having an identical central binding sequence (Pjura et al., 1987; Teng et al., 1988; Larsen et al., 1989).

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The binding of 4-methylumbelliferyl per-N-acetyl-chitooligosaccharides, MeUmbGlcNAc (I), MeUmb(GlcNAc)z (11) and MeUmb(GlcNAc)s (111) to wheat germ agglutinin, was studied by equilibrium dialysis, difference absorption spectrometry and extrinsic fluorescence quenching [Privat, J. P., FEBS Lett. 46, 229-2321 titration at a fixed wavelength or above 370 nm. The change in optical properties of the MeUmb group upon binding of the glycosides to the agglutinin depends on the length of the carbohydrate chain. An intense and carbohydrate-specific difference absorption spectrum was observed with I (-Ae at 316 nm equal to 2770 M-' cm-I), a weaker one with I1 ( -A s at 316 nm = 1070 M-' cm-'), but none was observed with 111; the fluorescence quenching is 100% for compounds I and I1 and 44% for compound 111. As determined with I, using equilibrium dialysis and difference absorption spectrometry, there are two identical and independent binding sites per subunit. For I the value of the association constant is independent of the experimental method or the saturation range used. It has long been suggested [1,2] (for a recent review see [3]) that wheat germ agglutinin possesses an extended binding site, possibly accomodating two or three GlcNAc residues. As determined [4,5] by the enhancement of intrinsic tryptophan fluorescence, the value of the association constants for the (GlcNAc), series increases with the length of these oligosaccharides. With the corresponding fluorescent 4-methylumbelliferyl glycosides MeUmb(GlcNAc),, which were