[On the quantitative fluorometric determination of cellular DNA by ethidium bromide and the constancy of the quantum yield of the ethidium DNA-complex in the biological environment (author's transl)]

Abstract

The fluorescent staining of fixed A9 cells (mouse fibroblasts) with ethidium bromide E has been investigated qualitatively and quantitatively. E is bound to DNA, RNA and protein. After enzymatic RNA digestion and staining at pH = 4.0 the dyestuff is bound to DNA specifically. The formed E-DNA-complex is stoichiometric if DNA will be saturated by highdye concentration, CF = 1.0 x 10(-2) M. The stoichiometric factor n = 0.21 was determined from Scatchard binding isotherms and by spectrophotometric titration. The binding of E to DNA and RNA has been investigated by binding isotherms. E is bound to DNA by intercalation and by ionic interaction. It is bound to RNA by ionic interaction only. The ionically bound dyestuff can be replaced by salts like LiCl from DNA and RNA. Only intercalated E remains. The competitive salt effect can be used to avoid the enzymatic RNA digestion in the specific staining of DNA. Within the cell the E-DNA-complex fluoresces strongly. Using a microspectrophotometer we determined the fluorescence intensity J and the extinction E in cell sections of q = 1 mu 2 area. The fluorescence was excited in the minimum of the absorbance of the E-DNA complex at lambda 1 = 365 nm. The extinction E2 was measured in the short wavelength band lambda 2 = 260 nm. Under these conditions we received a linear relation J = J (E2) up to high extinctions E2. The quantum yield Q of the E-DNA-complex is nearly constant and independent of the biological environment. Q is also unaltered by LiCl. Within the limits of error intercalated and ionic bound dye have the same quantum yeild. Under the conditions of stoichiometric staining the amount of E-DNA-complex and therefore DNA in the cell can be determined by J. The extinction coefficient epsilon 2 = 45 200 M-1 cm-1 of the E-DNA-complex at 260 nm was calculated from the concentration of dependence of the absorbance spectra using the law of mass action. By epsilon 2 the amount of DNA in the cell is accessible from J. According to our measurements with A9 cells the superficial density of DNA has the order of magnitude rho N = mN/q = 10(1) nmol cm-2, the amount mN of DNA per section 1 = 1 mu 2, mN = 10(-1) fmol, and the DNA concentration in the cell nucleus CN = 10(-1) M. The ray pass and diagram of electronics of the based microspectrophotometer are described.