| | 1 | Author
| Jürgen Pauluhn, Annem Arie, N. Aujok, H. Erbert, W. Zim, Erm Ann | Requires cookie* | | | Title
| Über die quantitative fluorometrische Bestimmung von DNA mit Ethidiumbromid in der Zelle und die Konstanz der Quantenausbeute des Ethidium-DNA-Komplexes im biologischen Milieu On the Q uantitative Fluorom etric D eterm ination of Cellular DNA by Ethidium Bromide and the Constancy o f the Quantum Yield of the Ethidium -D N A -Com plex in the Biological Environment  | | | | Abstract
| The fluorescent staining o f fixed A 9 cells (m ouse fibroblasts) with ethidium brom ide E has been investigated qualitatively and quantitatively. E is bound to D N A , R N A and protein. After enzymatic R N A digestion and staining at pH = 4.0 the dyestuff is bound to D N A specifically. The formed E -D N A -com plex is stoichiom etric if D N A will be saturated by high dye concentra tion, CF = 1.0 x 10~2 M. The stoichiom etric factor n = 0.21 was determined from Scatchard bind ing isotherms and by spectrophotometric titration. The binding o f E to D N A and R N A has been investigated by binding isotherms. E is bound to D N A by intercalation and by ionic interaction. It is bound to R N A by ionic interaction only. The ionically bound dyestuff can be replaced by salts like LiCl from D N A and RNA. Only inter calated E remains. The com petitive salt effect can be used to avoid the enzym atic R N A digestion in the specific staining o f D N A . W ithin the cell the E-D N A -com plex fluoresces strongly. U sing a microspectrophotom eter we determined the fluorescence intensity J and the extinction E in cell sections o f q = 1 |a2 area. The fluorescence was excited in the m inimum o f the absorbance o f the E -D N A -com plex at = 365 nm. The extinction E 2 was measured in the short wavelength band at X2 = 260 nm. Under these conditions we received a linear relation J = J (E ?) up to high extinctions E 2. The quantum yield Q o f the E -D N A -com plex is nearly constant and independent o f the biological environment. Q is also unaltered by LiCl. W ithin the lim its o f error intercalated and ionic bound dye have the same quantum yield. Under the conditions o f stoichiom etric staining the am ount o f E -D N A -complex and therefore D N A in the cell can be determined by J. The extinction coefficient e2 = 45200 M-1 cm -1 o f the E -D N A -com plex at 260 nm was calcu lated from the concentration dependence o f the absorbance spectra using the law o f mass action. By e2 the am ount o f D N A in the cell is accessible from J. According to our measurements with A 9 cells the superficial density o f D N A has the order o f m agnitude £>n = m ^ /q = 101 nmol cm -2, the amount raN o f D N A per section q = 1 jj.2, mN = 10_l fmol, and the D N A concentration in the cell nucleus CN = 10-1 M. The ray pass and diagram o f electronics o f the used m icrospectrophotom eter are described. | | | |
Reference
| Z. Naturforsch. 35c, 585—5 (1980); eingegangen am 14. Januar/21. April 1980 | | | |
Published
| 1980 | | | |
Keywords
| D N A -determ ination, Ethidium Bromide, M ouse Fibroblasts, Specific Fluorescent Staining, Quantum Y ield, M icrospectrophotometer | | | |
Similar Items
| Find | | | DEBUG INFO
| | | | | TEI-XML for
| default:Reihe_C/35/ZNC-1980-35c-0585.pdf | | | | Identifier
| ZNC-1980-35c-0585 | | | | Volume
| 35 | |
|
