Studies on the mechanism of indirubin action in the treatment of chronic granulocytic leukemia. V. Binding between indirubin and DNA and identification of the type of binding.

Abstract

The binding between indirubin and calf thymus DNA in vitro has been verified by means of the isotope labelling method, spectrophotometric method and thermal denaturation measurements. The lambda max 207 nm of indirubin shifted toward longer wave length with decrease of absorbance after the incubation of indirubin with DNA. The escalation of Tm value of DNA induced by indirubin was about 2.4 degrees C and it was reproducible. The binding force between them was rather weak, as indirubin molecules were easily released during the precipitation with alcohol or the gel filtration. The binding was not affected by sodium chloride even at high concentration but greatly decreased (to 20-30% of the control) in the presence of 8 M urea. These results showed that the binding between indirubin and DNA might be of hydrogen bond rather than ionic. The amount of bound 3H-indirubin was directly proportional to the concentration of indirubin. However, it increased abruptly when the concentration of indirubin reached 1.5 x 10(-4) M. This suggested another binding force in the latter instance. By using spectrophotometric analysis and Scatchard plot it was found that calf thymus DNA bound 46 indirubin molecules/1000 nucleotides. The association constant (K) was 5.7 x 10(6).