Biomedical subjects
Publications and source records attributed to F Tjerneld.
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The interaction between B-form DNA and twelve cationic triaryl-methane dyes was studied with respect to optical properties and stabilities, using linear dichroism (LD) and aqueous two-phase partition techniques. Monovalent dyes derived from crystal violet as a rule form a single strong complex (K1 ca 10(5) M-1; site density per nucleotide base n1 ca 0.1 at 0.1M ionic strength) in which the plane of the dye is at an angle of less than 50 degrees to the local DNA helix axis. The complex with fuchsin is weaker (10(4) M-1) but can be explained by a similar orientation. For some of the dyes (those with pseudo-C2v symmetry) the angular orientations of two molecule-fixed axes can be obtained. For the divalent methyl green a second complex appears to be formed at low ionic strength. Methyl green (and to some extent 2-thiophene green and malachite green) show exciton splitting in the LD spectrum and circular dichroism assignable to exciton coupling between transition dipoles roughly parallel to the helical strands, indicating a dye-dye interaction. The optical data, supported by fitting experiments with space-filling models, suggests a general structure for the binding site. The dye is not intercalated but is bound to exposed hydrophobic regions in the major groove. The ligand is in part (the charged amino groups) in contact with the phosphoribose chain but its main surface lies against the hydrophobic base-pair stack. For a diphenylmethane dye, Michler's hydro blue, a perpendicular orientation was observed, possibly due to intercalation.
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A stoichiometrical application of a sensitive method for linear dichroism (LD) detection is suggested for biochemical purposes. The complex formation between a binding site on a polynucleotide and a ligand may be studied with high precision if the following conditions are fulfilled: (1) The polymer can be given a fixed degree of orientation. (2) The site has a specific orientation with respect to the orientation axis of the polymer (e.g., intercalation). (3) The ligand has an anisotropic optical absorption property. The method was applied to studying the complex between DNA and ethidiumbromide, which was detected by LD with precision of +/- 0.5 X 10(-7) M in a 4 X 10(-4) M DNA solution, i.e., 0.1% occupation of the total site concentration can be detected. The complexation could be explained by a single type of site (n = 0.14 +/- 0.01 sites per nucleotide residue) and a stability constant K1 = (2.5 +/- 1) X 10(5) M-1 at 0.2 M ionic strength. From the specific LD an average angle 60 degrees was concluded between the helix axis and the long axis of the ethidiumbromide molecule. This value formally contradicts the Watson-Crick model or the intercalation model but may be explained by extension and deformation effects on the xhain by the flow.