Immunological Evidence for the Identity of a Photoproduct Formed during Photooxidation of DNA with Methylene Blue, Rose Bengal, Thionin, and Acridine Orange

Abstract

Irradiation of DNA with visible light, in the presence of oxygen and a photosensitizing dye such as methylene blue, is known to destroy specifically the guanine residues in nucleic acids (Simon and Van Vunakis, 1962). The chemistry of the photooxidation reaction is being studied actively in several laboratories (cf. Bellin and Grossman, 1965) but it is not yet understood. It is known that the photoproducts may vary depending upon the composition of the reaction mixture. Immunological experiments provided the first evidence that, during the photooxidation of DNA, a reactive photointermediate condensed with the available amino groups of the buffer, Tris(hydroxymethyl)amino methane (Tris) to yield an adduct (Van Vunakis et al., 1966). The evidence that established the existence of this adduct will be reviewed only briefly. DNA was photooxidized in Tris buffer at pH 8.5 using methylene blue as the photosensitizing dye. DNA thus treated (DNA_Tris-MB) was complexed with methylated bovine serum albumin (Plescia et al., 1964) and injected into rabbits. The serologic activities of native and denatured DNAs with the anti-DNA_Tris-MB became apparent as the reaction mixture containing DNA, Tris buffer and methylene blue was irradiated with visible light.

Aided by grants from the NIH (AI-01940 and AI-02792) and the Charles Simon Memorial Grant for Cancer Research from the American Cancer Society (E.222). L. L. is an American Cancer Society Professor of Biochemistry and H. V. V. is a recipient of a Public Health Service Research Career Award K6-AI-2372. Publication No. 566 from the Graduate Department of Biochemistry, Brandeis University.