Further fluorospectrophotometric studies on the binding of acridine orange with DNA. Effects of thermal denaturation of DNA and additions of spermine, kanamycin, dihydrostreptomycin, methylene blue and chlorpromazine

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Abstract

Fluorospectrophotometric studies on the binding of acridine orange (AO) with calf thymus DNA showed that the thermal denaturation of DNA reduced markedly the fluorescence of Complex II and the extent of this decrease depended on the temperature to which the DNA solutions were heated. The denaturation was carried out in the absence and presence of AO (methods A and B, respectively), and then fluorescence measurements of solutions were carried out at 23 °C. The fluorescence intensity-heating temperature curves obtained by methods A and B were similar in shape to the usual melting curves of DNA and AO-DNA solutions, respectively. The higher midpoint value obtained with method B indicates the stabilizing activity of AO against denaturation. These findings support an intercalation model for Complex II and an external self-association binding model for Complex I.

A high concentration of ethylene diamine (EDA) restored the fluorescence of denatured Complex II to about 80% of the intensity value of native Complex II. The effects of spermine, kanamycin and dihydrostreptomycin were much stronger than that of EDA.

Methylene blue (MB) and chlorpromazine (CP) reduced the fluorescence of native Complex II markedly. Since the analysis of the difference absorption spectra declared that MB and CP were intercalated without release of bound AO, the interacting MB and CP were considered to weaken the interaction between AO and DNA bases, that made AO more fluorescent. Free radical (CP·) of CP was prepared by a new method using H2O2, peroxidase, and ascorbic acid. Intercalated CP· showed a much stronger quenching effect on Complex II, indicating that unpaired electron spin contained in the costacking unit between CP· and DNA bases might affect the fluorescence of the adjacent AO molecule by paramagnetic perturbation.

References (38)

  • R.F. Steiner et al.

    Arch. Biochem. Biophys

    (1959)
  • L.S. Lerman

    J. Mol. Biol

    (1961)
  • A. Orgel et al.

    J. Mol. Biol

    (1961)
  • V. Luzzati et al.

    J. Mol. Biol

    (1961)
  • R.E. Boyle et al.

    Arch. Biochem. Biophys

    (1962)
  • L.S. Lerman

    J. Mol. Biol

    (1964)
  • D.M. Neville et al.

    J. Mol. Biol

    (1966)
  • H.J. Li et al.

    J. Mol. Biol

    (1969)
  • S. Yamabe

    Arch. Biochem. Biophys

    (1969)
  • J. Marmur et al.

    J. Mol. Biol

    (1962)
  • D. Freifelder et al.

    Biophys. J

    (1961)
  • V. Kleinwächter et al.

    Biochim. Biophys. Acta

    (1964)
  • I.O. Walker

    Biochim. Biophys. Acta

    (1965)
  • N.F. Gersch et al.

    J. Mol. Biol

    (1965)
  • A.R. Peacocke et al.

    Trans. Faraday Soc

    (1952)
  • D.F. Bradley et al.
  • G. Weill et al.

    Biopolymers

    (1963)
  • L.S. Lerman
  • J. Chambron et al.

    C.R. Acad. Sci. Paris

    (1964)
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