DNA unwinding induced by photoaddition of psoralen derivatives and determination of dark-binding equilibrium constants by gel electrophoresis

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Abstract
Derivatives of furo[3,2-g]coumarin (psoralen) can bind to the DNA double helix and, in the presence of long-wavelength UV light, the bound psoralen may react covalently with pyrimidine residues on 1 or both strands of the helix. By using agarose gel electrophoresis, the unwinding angle associated with each of 4 different psoralen derivatives was determined to be 28.degree. .+-. 4.degree.. For 4,5'',8-trimethylpsoralen (trioxsalen) the unwinding angle was independent of the initial DNA superhelix density in the range that is accessible to agarose gel electrophoresis. Also by using agarose gel electrophoresis, the unwinding angle for ethidium intercalation was determined. This was done by the total relaxation of supercoiled DNA in the presence of a series of ethidium concentrations. By using published values for the association constant for ethidium binding to DNA and evaluating the final superhelix density (after removal of ethidium) of the DNA on gels, an unwinding angle of 29.degree. .+-. 3.degree. was calculated. Assuming an unwinding angle of 28.degree. for the noncovalent intercalation of psoralen derivatives, the same procedure was used to determine intercalation binding constants. The association constants for 4''-aminomethyltrioxsalen were 300-1400 M-1 in NaCl at 0.2-0.05 M and 300-2500 M-1 in Mg2+ at 4-0.5 mM. The association constant for 4''-hydroymethyltrioxsalen in 0.5 mM Mg2+ was 70 M-1.