The use of low concentrations of heavy metals in the isolation of rat liver nuclei

Abstract

Methods have been developed for isolating rat liver nuclei in the presence of very low concentrations (Ca 0.0002 m) of lead acetate, cadmium sulfate, or indium trichloride. The nuclei are sufficiently stabilized by the metals so that they can be isolated without pH control or the addition of other stabilizers such as Ca 2+ or Mg 2+. The use of lead or cadmium in the isolation procedure overcomes the loss of the easily extractable histone fraction which occurs when CaCl 2 or MgCl 2 are used as nuclear stabilizers without pH control. If lead acetate is used in the first steps of the isolation, MgCl 2 can be used in the subsequent steps (in the absence of more lead) without causing loss of the easily extractable histone. The use of lead acetate has made it possible to recover the easily extractable histone fraction from nuclei isolated at pH 5.8. This was not possible previously. The recovered fraction was shown to exhibit disc-gel electrophoretic patterns typical of histones. Indium has been found useful in the isolation of nuclei but apparently it should be present in the form of partially hydrolized InCl 3 to be of real advantage. Disc-gel electrophoresis has shown that lead apparently is not combined with histones at least at relatively low pH but is probably present as Pb 2+ which can easily be removed through precipitation as PbS in acid solution. The electrophoretic patterns of the total histone extract were essentially the same in the cases of nuclei isolated with or without the use of lead acetate; the patterns were not changed whether the lead was left in the histone extract or removed before electrophoresis. All samples of nuclei isolated in the presence of lead cadmium or indium at pH 5.8 or without pH control formed strong gels at pH 8 to 9, indicating that the macromolecular structure of the chromatin had not been degraded through DNAase or protease action. This observation testifies to the effectiveness of these metals in blocking macromolecular degradation, since it is practically impossible, owing to autolytic degradation, even to isolate nuclei in sucrose homogenates without controlling the pH or adding metals such as Ca 2+ and Mg 2+ for purposes of stabilization.