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

Derivatives of furo3,2-gcoumarin (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 one or both strands of the helix. By using agarose gel electrophoresis, we have determined the unwinding angle associated with each of four different psoralen derivatives to be 28 degrees +/- 4 degrees . For 4,5',8-trimethylpsoralen (trioxsalen) the unwinding angle was found to be independent of the initial DNA superhelix density in the range that is accessible to agarose gel electrophoresis. Also by using agarose gel electrophoresis, we have determined the unwinding angle for ethidium intercalation. 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, we calculated an unwinding angle of 29 degrees +/- 3 degrees . Assuming an unwinding angle of 28 degrees for the noncovalent intercalation of psoralen derivatives, we used the same procedure 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 Mg(2+) at 4-0.5 mM. The association constant for 4'-hydroxymethyltrioxsalen in 0.5 mM Mg(2+) was determined to be 70 M(-1).