Studies on nucleic acid metachromasy. I. The effect of certain fixatives on the dye stacking properties of nucleic acids in solution.

The stacking coefficients (K's) of nucleic acids have been thought to influence the color contrast between DNA and RNA in tissue sections stained with metachromatic dyes. This idea was tested by titrating toluidine blue (TB) and acridine orange (AO) in solution against DNA and RNA, native or treated with formaldehyde, acrolein, or Carnoy's fluid. Absorption spectra at varying polymer-dye ratios were used to compute K values by the methods of Bradley and colleagues. Results with both dyes fit Bradley's stacking equations. Fixatives did not block dye-binding sites but markedly altered K values. K of DNA was low, unaffected by aldehyde fixative, increased by Carnoy's fluid or heat denaturation. K of RNA was higher than that of DNA and was increased greatly by formaldehyde, almost as much by acrolein, considerably less by Carnoy's fluid. Aldehyde effects were partially reversed upon removal of aldehyde by dialysis. These observations accord with known effects of aldehydes and denaturation upon nucleic acid conformation. Differences between K's of DNA and RNA were greater after aldehyde treatment than after Carnoy's, and were greater with AO than with TB. This is generally consistent with the magnitude of the color contrasts observed in tissues. Additional factors must contribute to the intense color contrast observed in acrolein-fixed tissues stained with TB.