Ethidium bromide

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Structural formula
Structure of the ethidium ion     Structure of the Br ion
General
Surname Ethidium bromide
other names
  • Homidium bromide ( INN )
  • Homidium
  • Ethidium ®
  • Novidium ®
  • 3,8-diamino-5-ethyl-6-phenyl-phenanthridinium bromide
Molecular formula C 21 H 20 BrN 3
Brief description

red solid

External identifiers / databases
CAS number 1239-45-8
EC number 214-984-6
ECHA InfoCard 100.013.622
PubChem 14710
Wikidata Q408634
properties
Molar mass 394.32 g · mol -1
Physical state

firmly

density

1.01 g cm −3

Melting point

261-264 ° C 

solubility

soluble in water (40 g l −1 at 25 ° C) 

safety instructions
GHS hazard labeling from  Regulation (EC) No. 1272/2008 (CLP) , expanded if necessary
06 - Toxic or very toxic 08 - Dangerous to health

danger

H and P phrases H: 302-330-341
P: 260-281-284-310
Toxicological data
As far as possible and customary, SI units are used. Unless otherwise noted, the data given apply to standard conditions .

Ethidium bromide (often abbreviated as EtBr , not to be confused with ethyl bromide ), even ethidium or Homidium called, is a red phenanthridine - dye , in the molecular biology for the detection of nucleic acids , DNA and RNA , is used. Ethidium bromide is the homologous ethyl derivative of dimidium bromide .

history

In search of new active substances against trypanosomal diseases, ethidium bromide was synthesized by Carl Hamilton Browning in 1938 (substance no. 897). Ethidium bromide showed antitrypanosomal activity against Trypanosoma congolense infections in mice and cattle. Ethidium bromide was launched in 1952 under the trade name Ethidium ® and was used extensively in the 1960s and 1970s for the treatment and prophylaxis of trypanosomal infections in cattle. Ethidium bromide is still used for this purpose today; a dose of 1 mg / kg body weight is used both for the treatment of infected animals and for infection prophylaxis, which lasts up to 3 months.

In 1965 it was discovered that ethidium bromide binds to DNA and that its absorption spectrum changes in the process. In 1972 ethidium bromide was used for the first time to stain DNA in gel electrophoresis .

properties

Absorption spectrum of ethidium bromide in water.

The absorption spectrum of ethidium bromide in water shows two absorption bands in the UV range (λ = 210 nm, ε = 40000; λ = 285, ε = 50000) with two shoulders (λ = 316 nm, ε = 5000–10000; λ = 343 nm , ε = 200–500) and one in the visible range (λ = 480 nm, ε = 5300).

Ethidium bromide intercalates in nucleic acids and thereby changes its absorption spectrum. The emission spectrum remains almost unchanged. The intercalation of ethidium bromide in nucleic acids increases the intensity of the fluorescence emission by a factor of 50–100.

use

DNA bands under UV light in an agarose gel stained with ethidium bromide.

Ethidium bromide is used in molecular biology for staining nucleic acids in gel electrophoresis . While the negatively charged nucleic acid migrates to the positively charged anode, the positively charged ethidium bromide migrates in the opposite direction. Thereby intercalate single ethidium bromide molecules between the bases of DNA or RNA (incorporation at a distance of 10 base pairs), the excitation spectrum of ethidium bromide thereby changing and so the fluorescence of the substance when excited by ultraviolet light is greatly increased. In this way, the areas in the agarose gel where nucleic acids are located light up brightly, while areas without nucleic acids appear dark. The light intensity is proportional to the existing DNA / RNA concentration and the length of the nucleic acid. The ethidium bromide concentration used in the agarose gel is usually 0.1–0.5 µg / ml.

In flow cytometry , ethidium bromide is used to determine cell viability .

Ethidium bromide specifically inhibits the replication of mitochondrial DNA (mtDNA) in eukaryotes in low concentrations (0.1–2 µg / ml) . It is therefore used to generate mtDNA-deficient cells ( rho zero cells , ρ 0 ).

Alternatives

In order not to expose yourself to the dangers of ethidium bromide, alternatives such as SYBR Green I or SYBR Gold can be used. However, these are very expensive and their toxicity and mutagenicity are unknown, so that ethidium bromide is still used as a colorant in many laboratories.

Further alternatives are methylene blue , Nile blue A , acridine orange .

Risks

In its 2005 annual report, the Senate Commission of the German Research Foundation for the testing of harmful substances classified ethidium bromide as a suspect substance in the carcinogen category 3B. The suspicion of a mutagenic effect on germline cells is classified as “well founded”. The National Toxicology Program (Executive Summary) of the National Institutes of Health notes that no case is known where ethidium bromide actually caused a tumor. In-vivo studies carried out by the National Toxicology Program on rats and mice could not demonstrate any carcinogenic effects. Instead, there were some studies in the early 1970s that described ethidium bromide as having anti-tumor effects. In a more recent study it could be shown that EtBr acts as a topoisomerase 1 poison, comparable to various cytostatic drugs that are used to treat cancer in humans. A subchronic carcinogenicity study in mice, which was carried out at the University of Düsseldorf, could not prove any mutagenic effects either.

Ethidium bromide only has an acutely toxic effect in very high concentrations. The LD 50 of ethidium bromide for rats after oral administration is given as 1503 mg / kg body weight.

safety

Ethidium bromide is possibly mutagenic. The use of gloves when handling ethidium bromide or ethidium bromide-stained gels is urgently indicated, as ethidium bromide is absorbed through the skin. Studies by the work safety department at the University of Freiburg have shown that the thin-walled disposable gloves made of latex commonly used in laboratories are penetrated by ethidium bromide after a very short time (1% solution: approx. 30 s; 0.1% solution: approx. 60 s ). Nitrile gloves showed no detectable penetration when tested under the same conditions and are therefore preferred. Latex gloves should only be used as short-term splash protection.

The use of ready-to-use solutions, for example a concentration of 10 mg / ml in water, is recommended to avoid dust formation.

No MAK value has been specified for ethidium bromide, as no action limits can be determined for carcinogenic substances, substances suspected of being cancerous and mutagens.

disposal

Waste containing ethidium bromide must not enter the sewage system and must be inactivated before disposal, e.g. B. by adsorption of ethidium bromide on activated carbon (1 mg activated carbon adsorbs approx. 50 μl of a freshly prepared 1% EtBr solution). The ethidium bromide-free eluate is usually disposed of via the sink. The activated carbon is either annealed in the muffle furnace (pyrolysis) or collected separately as solid hazardous waste. Ready-made adsorbers are commercially available.

Others

Note on the abbreviation: the abbreviation EtBr for ethidium bromide, which is regularly used in the biosciences, can lead to confusion in a chemical context. In organic chemistry, Et is the abbreviation for the ethyl group –CH 2 –CH 3 , which is also recognized in international publications , so EtBr stands for ethyl bromide in chemistry .

Web links

Individual evidence

  1. ↑ Entry on homidium bromide. In: Römpp Online . Georg Thieme Verlag, accessed on November 10, 2014.
  2. ↑ a b c d e Entry on ethidium bromide in the GESTIS substance database of the IFA , accessed on July 23, 2016(JavaScript required) .
  3. ↑ Entry on 3,8-diamino-1-ethyl-6-phenylphenantridinium bromide in the Classification and Labeling Inventory of the European Chemicals Agency (ECHA), accessed on February 1, 2016. Manufacturers or distributors can expand the harmonized classification and labeling .
  4. ↑ Entry on ethidium bromide in the ChemIDplus database of the United States National Library of Medicine (NLM), accessed on December 7, 2016.
  5. ↑ a b ChemDAT . The Merck chemistry database.
  6. ↑ Microbiological characterization of aquatic semen: Collection of methods , pp. 47–58, Oldenbourg Verlag, ISBN 3-486-26435-4 .
  7. ↑ Browning, CH et al. (1938): The trypanocidal action of certain phenanthridinium compounds . In: J. Path. Bact. Vol. 46, pp. 203-204. doi : 10.1002 / path.1700460121
  8. ↑ Waring, MJ (1965): Complex formation between ethidium bromide and nucleic acids . In: J. Mol. Biol. Vol. 13, pp. 269-282, PMID 5859041 .
  9. ↑ Le Pecq, JB and Paoletti, C. (1966): A new fluorometric method for RNA and DNA determination . Anal. Biochem. Vol. 17, pp. 100-107, PMID 6008008 .
  10. ↑ Borst, P. (2005): Ethidium DNA agarose gel electrophoresis: how it started . In: IUBMB Life . Vol. 57, pp. 745-747, PMID 16511967 .
  11. ↑ Aaij, C. and Borst, P. (1972): The gel electrophoresis of DNA . In: Biochim. Biophys. Acta . Vol. 269, pp. 192-200, PMID 5063906 .
  12. ↑ Sharp, PA, Sugden, B., Sambrook, J. (1973): Detection of two restriction endonuclease activities in Haemophilus parainfluenzae using analytical agarose - ethidium bromide electrophoresis . In: Biochemistry . Vol. 12, pp. 3055-3063, PMID 4354250 .
  13. ↑ Bruce Hudson, Russell Jacobs: The ultraviolet transitions of the ethidium cation . In: Biopolymers . tape 14 , no. 6 , 1975, p. 1309-1312 , doi : 10.1002 / gdp . 1975.360140619 .
  14. ↑ Entry on Ethidium Bromide in the Hazardous Substances Data Bank , accessed on November 18, 2014.
  15. ↑ M. Holtzhauer: Biochemical Laboratory Methods , p. 51, 3rd edition, Springer Verlag, Berlin, 2009, ISBN 3-540-62435-X .
  16. ↑ Huang, Q. and Fu, WL (2005): Comparative analysis of the DNA staining efficiencies of different fluorescent dyes in preparative agarose gel electrophoresis . In: Clin. Chem. Lab. Med. Vol. 43, pp. 841-842, PMID 16201894 .
  17. ↑ Senate Commission of the German Research Foundation for the testing of harmful substances, annual report 2005. PDF
  18. ^ National Toxicology Program: Testing Status: Ethidium bromide
  19. ^ National Toxicology Program: Executive Summary: Ethidium Bromide .
  20. ↑ Gentry AC, Juul S, Veigaard C, Knudsen BR, Osheroff N .: The geometry of DNA supercoils modulates the DNA cleavage activity of human topoisomerase I. . In: Nucleic Acids Research . 39, No. 3, 2011, pp. 1014-1022. doi : 10.1093 / nar / gkq822 .
  21. ↑ Marossek V: Identification and characterization of molecular biological changes using the example of the tumor suppressor p53 in tamoxifen- or bromodeoxyuridine-induced carcinogenesis in laboratory rodents. . December 18, 2001. Retrieved September 8, 2011.